Analytical methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations by liquid chromatography: a review

REVIEW

Analytical methods for determination of new fluoroquinolonesin biological matrices and pharmaceutical formulationsby liquid chromatography: a review

Joana Sousa & Gilberto Alves & Ana Fortuna &

Amílcar Falcão

Received: 6 November 2011 /Revised: 24 December 2011 /Accepted: 29 December 2011 /Published online: 10 February 2012# Springer-Verlag 2012

Abstract Fluoroquinolones are one of the most promisingand intensively studied drugs of contemporary anti-infectivechemotherapy. New fluoroquinolone antibacterials with im-proved pharmacokinetic properties and a broad spectrum ofactivity have been developed, opening new windows ofopportunity for clinical use. To our knowledge, no compre-hensive and critical review of the analytical methods for thedetermination of these agents, which correspond to thethird- and fourth-generation quinolones, has yet been pub-lished. This work summarizes for the first time most of theliquid chromatographic methods reported in the literaturefor the separation and quantification of the new fluoroqui-nolones in biological matrices and pharmaceutical formula-tions. A systematic and detailed survey of physicochemicalproperties, sample preparation procedures, and chromato-graphic and detection conditions is presented herein. In thecourse of this review several liquid chromatographic methodsare discussed: reversed-phase high-performance liquid chro-matography (RP-HPLC), ion-exchange high-performanceliquid chromatography (IEX-HPLC), hydrophilic interactionliquid chromatography (HILIC), high-performance thin-layer

chromatography (HPTLC) and other chiral chromatographicmethods. Their advantages, applicability and limitations arealso examined.

Keywords Fluoroquinolones . Analytical methods .

Bioanalysis . Pharmaceuticals . Liquid chromatography

Introduction

The first quinolone, nalidixic acid, was accidentally discov-ered in 1962 as a by-product of the synthesis of the antima-larial drug chloroquine. The introduction of nalidixic acidfor clinical use was limited to the treatment of uncomplicat-ed urinary tract infections and its usefulness was hinderedby its short half-life. Since then, several modifications havebeen made to its chemical structure in an attempt to improvethe antibacterial activity and pharmacological properties,leading to the development of numerous quinolone com-pounds [1–4]. This class of antibacterial agents is currentlyclassified into four generations. The first-generation quino-lones (e.g. nalidixic acid and oxolinic acid) have moderateactivity against aerobic gram-negative bacteria and verylittle or no activity against aerobic gram-positive bacteria,anaerobes or atypical pathogens. These agents also haveminimal systemic distribution and are less used nowadays[1–4]. Second-generation agents correspond to the originalfluoroquinolones that resulted from the addition of a fluo-rine atom at position 6 of the basic molecular nucleus(Fig. 1), including norfloxacin, ciprofloxacin, ofloxacinand lomefloxacin [1–5]. These drugs offer expanded activityagainst gram-negative bacteria compared with the first-generation quinolones, atypical pathogen coverage andmoderately improved gram-positive activity, but they stilllack activity against anaerobes. With the appearance of

J. Sousa :A. Fortuna :A. Falcão (*)Pharmacology Department, Faculty of Pharmacy, University ofCoimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba,3000-548 Coimbra, Portugale-mail: [email protected]

J. Sousa :G. Alves :A. Fortuna :A. FalcãoCNC – Centre for Neurosciences and Cell Biology,University of Coimbra,3004-517 Coimbra, Portugal

G. AlvesCICS-UBI – Health Sciences Research Centre,University of Beira Interior,Av. Infante D. Henrique,6200-506 Covilhã, Portugal

Anal Bioanal Chem (2012) 403:93–129DOI 10.1007/s00216-011-5706-8

fluoroquinolones, this class of antibacterial agents becamean important and effective group of drugs with considerableinterest in clinical practice; new-generation analoguesemerged then as a result of multiple structural modifications.Third-generation agents (e.g. sparfloxacin, grepafloxacin,gatifloxacin and temafloxacin) have improved activityagainst gram-positive bacteria, in particular pneumococci,and good activity against anaerobes, while retaining theexpanded gram-negative and atypical pathogen activity.These compounds of the third-generation are known as therespiratory fluoroquinolones because they are characterizedby great activity against pathogens that cause respiratoryinfections, such as Streptococcus pneumoniae, Haemophilusinfluenzae and Moraxella catarrhalis. Finally, the fourth-generation agents (e.g. trovafloxacin, gemifloxacin, clina-floxacin and sitafloxacin) are fluoroquinolones similar to theprevious generation but with superior coverage and in-creased activity against pneumococci and anaerobes [2–4,6, 7]. The last two generations of quinolones have severaladvantages over the earlier ones; these fluoroquinoloneshave not only a broader spectrum of antibacterial activitybut also improved pharmacokinetic properties, includinghigh oral bioavailability, long elimination half-lives andgood tissue penetration that provides, in some circumstan-ces, higher tissue drug concentrations than those attained inplasma. All these features capture the attention of research-ers and clinicians to the third- and fourth-generation quino-lones, named herein as the new fluoroquinolones. In fact,clinical applications of the new fluoroquinolones extend farbeyond the urinary tract infections. They are indicated forthe treatment of a large variety of other infections, such asrespiratory, gastrointestinal and gynaecologic infections,sexually transmitted diseases, prostatitis, as well as skin,soft tissue, bone and joint infections [3, 4, 6]. The appropri-ate use of the new fluoroquinolones is an important issuethat remains a difficult challenge. Despite the remarkableproperties of these new agents, the pharmacokinetic behav-iour and activity in changing therapeutic environments mustbe continuously monitored in order to select the optimal

agent, dose and duration of treatment, avoiding therapeuticfailure and development of antibacterial resistance [2, 8].Hence, in this context, the availability of fast, selective,sensitive, precise and accurate analytical methods for thequantitative determination of fluoroquinolones is imperativein order to establish appropriate pharmacokinetic/pharma-codynamic relationships.

Taking into account the value of the review by Carlucci [9]published in 1998, discussing a number of liquid chromatog-raphy methods previously developed to measure severalsecond-generation quinolones, and the lack of a broad reviewof the analytical methods concerning new fluoroquinolones,we decided to present this scientific contribution. This work isa comprehensive and critical review of the liquid chromato-graphic methods reported in the literature for the determina-tion of third-generation quinolones, namely levofloxacin(LEV), gatifloxacin (GAT), grepafloxacin (GRE), sparfloxa-cin (SPA), temafloxacin (TEM), tosufloxacin (TOS) and pazu-floxacin (PAZ), and fourth-generation quinolones, namelyclinafloxacin (CLI), gemifloxacin (GEM), moxifloxacin(MOX), sitafloxacin (SIT), alatrofloxacin (ALA), trovafloxa-cin (TRO), prulifloxacin (PRU) and ulifloxacin (ULI), inbiological matrices and pharmaceutical formulations(Fig. 2). These methods constitute useful tools for pharmaco-kinetic and toxicological studies or for quality control tests.Moreover, some of them may support the routine therapeuticdrug monitoring of these new fluoroquinolones in clinicalpractice.

Pharmacokinetic and pharmacodynamic properties

The main pharmacokinetic parameters of the new fluoroqui-nolones are summarized in Table 1. These fluoroquinolonesare well absorbed following oral administration and showhigh bioavailability (at least 70%). Systemic drug exposureachieved after oral administration is comparable to thatattained by intravenous administration. These antibacterialagents are also quickly absorbed from the gastrointestinaltract, with the maximum plasma concentration (Cmax) beingusually achieved within 1 to 2 h. Although food intake maydecrease the rate of fluoroquinolones absorption, prolongingthe time to reach the Cmax (tmax), this does not significantlyaffect the extent of their oral absorption or overall bioavail-ability. However, absorption of fluoroquinolones is impairedby chelation with di- and trivalent cations, such as alumin-ium, magnesium, calcium, iron and zinc that are present inantacids and other medications (e.g. sucralfate, nutritional,multivitamin and mineral supplements). This kind of inter-action in the gastrointestinal tract reduces oral absorption offluoroquinolones and their bioavailability which may result insub-therapeutic levels and potential clinical failure. Therefore,co-administration of fluoroquinolones and antacids or other

Fig. 1 General structure of fluoroquinolones, using the accepted num-bering scheme for positions on the molecule. The radicals R1, R2, R5,R7 and R8 indicate possible positions for structural modification; Xusually corresponds to a C or N atom

94 J. Sousa et al.

products containing those cations must be avoided [6, 14, 17].New fluoroquinolones vary to some extent with respect totheir plasma protein binding ability, ranging from 20 to 70%.This pharmacokinetic parameter may have a significant im-pact on clinical efficacy, because only the unbound drug exertsits antibacterial activity. These antibacterial agents are welldistributed throughout the body, although their distribution isnot uniform in all tissues and fluids. The new fluoroquino-lones have increased tissue penetration, as reflected by theirvalues of apparent volume of distribution, with tissue andfluid concentrations usually being higher than plasma concen-trations. Aminimanizani et al. [13] and Zhanel and Noreddin[14] tabulated some data related to the penetration character-istics of several fluoroquinolones into selected tissues andfluids of the body. According to published information, newfluoroquinolones show excellent distribution to respiratorytissues and fluids with concentrations exceeding those foundin plasma, which accounts for the success of these drugs in thetreatment of respiratory infections. Remarkable levels of fluo-roquinolones are also achieved in the prostate gland, neutro-phils and macrophages, whereas penetration into thecerebrospinal fluid is poor and therefore drug concentrationsare not above plasma drug levels [4, 6, 13, 14, 17, 32].Another important advantage of the new fluoroquinolones istheir long elimination half-lives, allowing once-daily dosing;however, there could be some exceptions such as CLI whichhas a shorter half-life and must be administrated twice daily.Fluoroquinolones are eliminated via renal and hepatobiliaryroutes. The degree to which they are eliminated by hepaticmetabolism or by renal excretion differs greatly amongst

fluoroquinolone agents. The majority of fluoroquinolonesare primarily excreted in urine as the parent compound,through glomerular filtration or tubular secretion (e.g. GAT,LEV and SIT). In contrast, some agents are eliminated pre-dominantly by metabolism in the liver (e.g. GRE, MOX andTRO), whereas others undergo modest metabolism and sig-nificant renal elimination (e.g. GEM). Moreover, when hepat-ic metabolism is involved, fluoroquinolones are primarilyinactivated by glucuronide conjugation at the carboxylicgroup of the molecule nucleus [4, 6, 14, 17, 33]. PRU andALA are prodrugs of the active metabolites ULI and TRO,respectively. The antibacterial agent PRU, which wasdesigned to improve oral absorption of ULI, is immediatelymetabolized after oral administration, partly in the intestinalmembrane but mostly in the portal blood and liver. ALA is theprodrug of TRO and is suitable for intravenous administrationowing to its enhanced solubility in water; in this case, theconversion of ALA into TRO rapidly takes place in plasmaafter intravenous administration. Both prodrugs (PRU andALA) are undetectable in systemic circulation after adminis-tration or, in the particular case of ALA, 5 min after theinfusion [31, 34, 35]. Because fluoroquinolones are not highlyprotein-bound and their metabolism by the cytochromeP450 (CYP) system is limited to the CYP1A2 isoenzyme,fluoroquinolone interactions with other drugs are somewhatminimized [7].

From a pharmacodynamic viewpoint, fluoroquinolonesexert their antibacterial action by inhibiting two enzymesinvolved in the replication and transcription of bacterialDNA. These enzymes are the DNA topoisomerases—DNA

Fig. 2 Chemical structures of several new fluoroquinolones: levoflox-acin (LEV), gatifloxacin (GAT), grepafloxacin (GRE), sparfloxacin(SPA), temafloxacin (TEM), tosufloxacin (TOS), pazufloxacin (PAZ),

clinafloxacin (CLI), gemifloxacin (GEM), moxifloxacin (MOX), sita-floxacin (SIT), alatrofloxacin (ALA), trovafloxacin (TRO), pruliflox-acin (PRU) and ulifloxacin (ULI). Each asterisk denotes a chiral centre

Determination of new fluoroquinolones by liquid chromatography 95

Tab

le1

Mainph

armacok

inetic

parametersof

thenew

fluo

roqu

inolon

es

FQ/references

Dose(m

g)F(%

)Cmax

(mg/L)

t max

(h)

AUC∞(m

ghL−1)

t 1/2

el(h)

Vd(L/kg)

PPB(%

)Metabolites

UE(%

)

CLI

Wiseet

al.[10]

200PO

od80

–98

1.34

1.78

9.9

5.7

2.38

50–6

0Not

described

NR

Randinitis

etal.[11]

200PO

od80.6

0.89

1.2

7.08

5.9

NR

NR

53.4

(48h)

200IV

–1.61

NR

8.82

6.0

NR

NR

68.8

(48h)

GAT

Nakashimaet

al.[12]

400PO

odNR

3.3

1.97

32.4

8.4

2.2

20GATglucuronide

83

Aminim

anizaniet

al.[13]

400PO

od96

3.8

1.0

33.0

7.8

NR

NR

Open-ring

metabolitesa

72

400IV

–5.5

NR

35.1

7.4

1.5

NR

62

GEM

Aminim

anizaniet

al.[13]

320PO

odNR

2.0

0.8

9.3

8.2

NR

NR

E-isomer

33

ZhanelandNoreddin[14]

320PO

od70

1.19

1.2

7.3

8.0

3.5

60GEM

acyl

glucuronide

27N-A

cetyl-GEM

b

GRE

Efthymiopoulos

[16]

400PO

od72

0.93

2.49

11.35

11.68

8.11

NR

GREglucuronide

NR

Turnidge[17]

400PO

od90

0.9

NR

11.4

11.7

7.5

50Sulphateconjugates

8.47

Oxidisedmetabolitesc

LEV

Aminim

anizaniet

al.[13]

500IV

–6.3

NR

55.3

6.6

1.2

NR

Desmethyl-LEV

61


500PO

od99

5.08

1.7

48.0

6.9

1.1

31LEV-N-oxide

d83

MOX

Aminim

anizaniet

al.[13]

400IV

–3.6

NR

34.6

15.4

2.1

NR

N-Sulphate-MOX

22.1


400PO

od86

3.34

1.7

33.8

12.1

3.3

48MOX

acyl

glucuronidee

19

PAZ

Yam

akiet

al.[19]

200PO

od≈70

2.98

<1.0

NR

NR

≈1.43

20PA

Zglucuronide

>80

PAZFLO

leaflet[20]

500IV

–11.0

0.5

21.7

1.88

NR

NR

90

SIT

Nakashimaet

al.[21]

100PO

odNR

1.0

1.2

5.55

5.02

1.84

NR

SIT

acyl

glucuronide

NR

O’G

rady

etal.[22]

500PO

od89

4.65

1.25

28.1

7.0

≈2.57

NR

N-A

cetyl-SIT

61(48h)

Deaminated/ketonereduced

metabolitesf

SPA

Ritz

atal.[23]

200PO

odNR

0.61

≈3.5

18.3

18.3

4.3

56SPA

glucuronided

6.69

(24h)

Montay[24]

400PO

odNR

1.18

5.0

32.73

18.0

NR

459.53

(96h)

TEM

Grannem

anet

al.[25]

400PO

odNR

2.43

2.50

29.69

7.9

≈2.27

26Ethylenediamine-substituted

TEM

analogues

59.2

Aminoquinolone

analogue

g

TOS

Minam

iet

al.[26]

204PO

odNR

0.88

2.3

5.57

NR

NR

NR

Not

described

34

96 J. Sousa et al.

gyrase and topoisomerase IV—which are responsible formaintaining the integrity of the supercoiled DNA double-helix during replication and transcription events. Hence, theinterference with their normal action culminates in rapid bac-terial death. DNA gyrase is the target of fluoroquinoloneagents in gram-negative bacteria, whereas topoisomerase IVis typically the primary target in gram-positive bacteria. It isgenerally accepted that the fourth-generation fluoroquino-lones are likely to have a dual activity, inhibiting both DNAgyrase and topoisomerase IV, thus limiting the emergence offluoroquinolone resistance [6, 33, 36].

Fluoroquinolones have a concentration-dependent anti-bacterial activity and a post-antibiotic effect (PAE), twoimportant pharmacodynamic properties. Given the differentantibacterial susceptibility, expressed by the minimum in-hibitory concentration (MIC), and the pharmacokinetics offluoroquinolones, some parameters incorporating pharma-cokinetic and pharmacodynamic data must be determined toevaluate the various therapeutic options, selecting the bestagent and dosing. The parameters used to predict fluoroqui-nolone efficacy are the Cmax/MIC ratio and the 24 h areaunder the plasma concentration versus time curve (AUC24)to MIC ratio (AUC24/MIC). These parameters are also im-portant in preventing the development of bacterial resistanceto fluoroquinolone agents. Several in vitro, animal modelsor human studies have been performed to correlate theseparameters with bacteriological and clinical outcomes. Clin-ical data indicate that a Cmax/MIC ratio of at least 10 andAUC24/MIC ratios in the range of 100–125 were associatedwith maximum bacterial eradication and clinical cure incritically ill patients with nosocomial lower respiratory tractinfection caused by gram-negative bacteria, such as Pseu-domonas aeruginosa. Other studies have not validated thesame thresholds for Cmax/MIC and AUC24/MIC, suggestingthat these values depend on the disease state and on thetarget pathogen. For example, in outpatients withcommunity-acquired respiratory infections caused by Strep-tococcus pneumoniae, an AUC24/MIC ratio greater than 25produced the best rates of bacterial eradication in vitro, aswell as in animal and clinical studies [7, 14, 17]. Morerecently, some scientists examined whether pharmacody-namic parameters correlate with the emergence of bacterialresistance to fluoroquinolones [7, 14, 37]. Falagas et al. [37]investigated whether the use of high doses of fluoroquino-lones may reduce the development of antimicrobial resis-tance. Despite the fact that laboratory studies show a benefitfrom using high daily doses, the clinical trials could notsupport or reject this hypothesis, pointing to the need forfurther comparative clinical studies [37]. The new fluoro-quinolones exhibit a PAE of 1 to 6 h (or longer), dependingon the drug and pathogen involved. The probability ofresumption of bacterial growth during the periods of plasmaor tissue drug concentrations inferior to MIC decreases withT

able

1(con

tinued)

FQ/references

Dose(m

g)F(%

)Cmax

(mg/L)

t max

(h)

AUC∞(m

ghL−1)

t 1/2

el(h)

Vd(L/kg)

PPB(%

)Metabolites

UE(%

)

TRO

Tenget

al.[27

]200PO

od87.6

2.2

2.3

30.4

11.3

NR

73TRO

glucuronide

NR

Vincent

etal.[28]

200IV

(ALA)

–2.3

1.0

31.2

12.3

1.3

NR

N-Sulphate-TRO

≈10

N-A

cetyl-TROh

ULI

Keam

andPerry

[30]

600PO

od(PRU)

NR

1.6

1.0

7.3

10.7

≈17.58

≈45

ULI(activemetabolite)

17–2

3(48h)

ULIacyl

glucuronide,

ethylenediam

ino,

diol,

aminoandoxoform

si

ALAalatroflox

acin,C

LIclinafloxacin,GATgatifloxacin,GEM

gemifloxacin,GREgrepafloxacin,LEVlevo

flox

acin,M

OXmox

ifloxacin,PA

Zpazuflox

acin,P

RUprulifloxacin,SITsitaflox

acin,SPA

sparflox

acin,T

EM

temafloxacin,

TOStosuflox

acin,T

ROtrov

afloxacin,

ULIulifloxacin,

AUCarea

undertheconcentration–

timecurve,Cmaxmaxim

umplasmaconcentration,

Foralbioavailability,

FQ

fluo

roqu

inolon

e,od

once

daily,IVintravenou

sadministration,

NRno

trepo

rted,P

Ooraladministration,

PPBplasmaproteinbind

ing,

t maxtim

eto

reachmaxim

umplasmaconcentration,

t 1/2

el

elim

inationhalf-lifetim

e,UEurinaryexcretionof

unchangeddrug

,Vdapparent

volumeof

distribu

tion

aNakashimaet

al.[12];bYoo

etal.[15];cEfthy

miopo

ulos

[16];dMartin

etal.[18];eAminim

anizaniet

al.[13];fO’G

rady

etal.[22];gGrann

eman

etal.[25];hVincent

etal.[29];iMatera[31]


increasing magnitude of PAE. The degree of this persistenteffect is also dose-dependent and may have an importantimpact on the design of dosing regimens. Furthermore,the PAE is another pharmacodynamic parameter to beconsidered in preventing the emergence of fluoroquinoloneresistance [7, 14, 33].

Another aspect that appears to be dose related is the rateof adverse events (AEs) associated with both oral and intra-venous administration of fluoroquinolones. These agents arewell tolerated and relatively safe, but some adverse drugreactions have been reported in patients taking fluoroquino-lones. Gastrointestinal disturbances (nausea and diarrhoea)are the most frequent AEs, followed by central nervoussymptoms such as headache, drowsiness or dizziness. Skinreactions are fairly uncommon and correspond to rash andpruritus effects. Other AEs include tendinopathy with in-creased risk of tendinitis and tendon rupture; dysglycaemias(hypoglycaemia and hyperglycaemia), particularly inducedby GAT; and phototoxicity, typically induced by fluoroqui-nolones and manifested as an intensive sunburn with expo-sure to ultraviolet (UV) light. This last drug reaction isstrongly associated with dihalogenated agents, like the caseof SIT and CLI, both with a halogen atom at position 8 (notcommercially available at the moment). Prolongation of QTinterval is a serious adverse event of fluoroquinolones as itprovokes torsades de pointes or other ventricular arrhyth-mias (GRE and SPAwere removed from the market becauseof increased risk of arrhythmia). Therefore, fluoroquino-lones must be avoided in patients with predisposition toarrhythmias, uncorrected hypokalaemia and in patients re-ceiving antiarrhythmic drugs or other medications thatmight prolong QT interval. Severe hepatotoxicity is associ-ated with TRO and haemolytic anaemia with TEM, conse-quently both drugs were withdrawn from the market. Thus,fluoroquinolones should not be regarded as innocuous orequally tolerable and safe agents and, therefore, therapeuticswitching must be carefully performed. In spite of unfore-seen adverse effects during the evolution of fluoroquino-lones, recent reports indicate that their window ofopportunities persists and research in this field is still fullyjustified [3, 6, 7, 38, 39]. Hence, taking into considerationthe broad spectrum of antibacterial activity of these newfluoroquinolones associated with the development of phar-macometric analyses, the clinical reintroduction of some ofthese fluoroquinolones incorporated in new pharmaceuticalformulations is expected in the next few years; their appli-cation will induce less systemic drug exposure and, conse-quently, acceptable toxicity profiles.

Bearing in mind all the aspects discussed above, it is ofthe utmost importance to understand in depth the pharma-cokinetic/pharmacodynamic relationships of these new flu-oroquinolones. Hence, there is no doubt that the availabilityof appropriate analytical methods is the main determinant.

Therefore, in addition to some general pharmacokinetic andpharmacodynamic considerations, a comprehensive andcritical survey of the physicochemical properties and ana-lytical aspects underlying the liquid chromatographic anal-ysis of the new fluoroquinolones is given herein for the firsttime. Such information may provide the suitable back-ground for the development of novel and improved quanti-tative analytical methods involving these agents.

Physicochemical properties and stabilityof fluoroquinolones

Fluoroquinolones are bicyclic heterocyclic aromatic com-pounds with a ketone group at position 4, a carboxylic groupat position 3 and a fluorine atom at position 6 [39, 40]. Thisbasic structure of fluoroquinolone molecules (pharmaco-phore) is represented in Fig. 1. Decades of fluoroquinolonedevelopment gave rise to several structurally related com-pounds with some molecular differences in order to enhancetheir antibacterial activity and pharmacological properties[7]. The new fluoroquinolones mentioned herein are 6-fluoroquinolone derivatives (Fig. 2) that result from struc-tural modifications around the 6-fluoroquinolone nucleus(Fig. 1). In general, these modifications can include theintroduction of cyclopropyl (GAT, GRE, SPA, CLI, GEM,MOX and SIT) or difluorophenyl (TEM, TOS and TRO)groups at position 1 and amine substituents at position 7,such as piperazine (LEV, GAT, GRE, SPA, TEM, PRU andULI) or pyrrolidine (TOS, CLI and GEM) rings or azabicy-clo (e.g. MOX and TRO) groups. There are also fluoroqui-nolones with a third ring fused to the basic structure,producing a tricyclic benzoxazine nucleus as in the case ofLEV and PAZ, or with a four-membered ring in the 1,2-position including a sulphur atom, like ULI and its prodrug(PRU). Other fluoroquinolones have a second nitrogen atomat position 8 of the bicyclic ring; these compounds, namelyTRO and TOS, are technically naphthyridones, however theirring system falls under the general class name of quinolones.Differentiation between these fluoroquinolone molecules issometimes achieved or reinforced by inclusion of small sub-stituents, commonly amine, alkyl, methoxy and halogengroups [34]. Substituents groups play an active role in deter-mining the physicochemical properties of fluoroquinolones.

The fluoroquinolones are compounds with molecularweights usually between 300 and 500 Da (Table 2). Owingto presence of the carboxylic group of the nucleus and theamine substituent at position 7, the new fluoroquinolonesare amphoteric molecules. Consequently, they may exist insolution as four different chemical species, corresponding topositive, zwitterionic, neutral and negative forms; their rel-ative abundance depends on the dissociation constants of thefluoroquinolone (Table 2) and on the pH of the solution.

98 J. Sousa et al.

These different molecular species have distinct physico-chemical properties, for example, with respect to watersolubility and lipophilicity. It is well known that the ionizedforms are usually more water soluble, whereas neutral formsare more lipophilic and have higher membrane permeability.Moreover, the knowledge of the dissociation constants offluoroquinolones is particularly important when applyingliquid chromatography because retention times depend onthe molecular species present in solution [40]. The acid-basebehaviour of fluoroquinolones in solutions with different pHvalues was studied by some authors and their ionizationpattern demonstrated that the zwitterionic form predominatesat their isoelectric point, which is near to neutral pH [51, 52].The influence of the pH of the medium on the lipophilicitywas also evaluated by measuring the apparent partition coef-ficient between 1-octanol and water (Poctanol/water) as a func-tion of pH. The profile of the curve obtained (on a log scale)has a parabolic shape with a maximum at the isoelectric point,which reflects the fact that the predominant species at theisoelectric point are the most lipophilic (zwitterionic andneutral species). The decrease of Poctanol/water on either sideof the maximum is due to the increased contribution of thecharged, less lipophilic, species [49, 51, 52]. The structure offluoroquinolone molecules influences their lipophilicity. Afew examples are given by Takács-Novák et al. [51] andAndersson et al. [53]. The introduction of a second fluorineatom at position 8 or the presence of an oxazine ring attachedto the fluoroquinolone nucleus decrease their lipophilicity.Pyrrolidine rings, which are common substituents at position

7, induce low water solubility; similarly azabicyclo groupsresult in increased lipophilicity. The lipophilicity parameterlog P octanol/water is shown in Table 2 for the new fluoroquino-lones reported in this review.

Physicochemical properties of the fluoroquinolones arealso expected to have some influence on drug stability.Indeed, the stability of the analytes in stock and workingsolutions and also in biological samples is an importantissue for bioanalysis. In this field it is indispensable to knowthe stability of compounds in all storage and sample han-dling conditions in order to ensure the reliability of theanalytical results. Therefore, stability conditions of newfluoroquinolones will be discussed below.

The stock solutions of the new fluoroquinolones, used forthe validation of liquid chromatographic methods, havebeen prepared in several solvents: water, methanol, acetoni-trile and a mixture of water/methanol or water/acetonitrile[54–58]. In some cases, to deviate from neutral pH and thusenhance solubility in water, a basic solution (NaOH) wasadded in the preparation of LEV and SPA stock solutions,whereas for GAT, MOX, TRO and ULI an acid (HCl, formicacid, orthophophoric acid) or a phosphate buffer at pH 3were also used [59–65]. In addition, to facilitate the dissolvingprocess some solutions were placed in an ultrasound bath orheated in a boiling water bath [64, 66]. Stock solutions areusually stored at 4 °C or −20 °C and kept in the dark; underthese conditions they were found to be stable for periods of2 weeks to 3 months [67, 68]. Because of the light sensitivityof the fluoroquinolones, calibration standards and quality

Table 2 Physicochemical properties of the new fluoroquinolones

Fluoroquinolone Molecularweight (Da)

Dissociation constantspKa1; pKa2

Lipophilicitya

(Log Poctanol/water)Lipophilicitya

(Log Poctanol/water at pH 6.98)

CLI 365.8 6.5b; 9.7a 1.44 1.44

GAT 375.4 5.56; 9.00c 2.31 1.20

GEM 389.4 5.76; 8.40c −0.3 −0.305

GRE 359.4 7.1; 8.80d 2.28 2.27

LEV 361.4 5.5; 8.0d 0.97 0.61

MOX 401.4 6.4; 9.5e 2.44 2.49

PAZ 318.3 5.5–6.3f 0.27 0.26

SIT 409.8 5.7; 9.0g 1.34 1.34

SPA 392.4 6.27; 8.80h 4.56 4.55

TEM 417.4 5.61; 8.75i 3.36 3.35

TOS 404.3 9.46a 1.36 1.63

TRO (ALA) 416.4 (558.5) 5.87; 8.09j 1.33 0.53

ULI (PRU) 349.4 (461.5) NA NA NA

ALA alatrofloxacin, CLI clinafloxacin, GAT gatifloxacin, GEM gemifloxacin, GRE grepafloxacin, LEV levofloxacin, MOX moxifloxacin, PAZpazufloxacin, PRU prulifloxacin, SIT sitafloxacin, SPA sparfloxacin, TEM temafloxacin, TOS tosufloxacin, TRO trovafloxacin, ULI ulifloxacin, NAnot availablea Hu et al. [41]; b Robinson et al. [42]; c Singh et al. [43]; d Hirota et al. [44]; e Cárceles et al. [45]; f Phapale et al. [46]; g Araki et al. [47]; h Kamberiet al. [48]; i Ross et al. [49]; j Belal et al. [50]


control samples are freshly prepared daily or stored and pro-tected from light until assay, in amber flasks or/and coveredwith aluminium foil [69–71]. In fact, the stability of drugs is akey factor that requires thorough investigation. All the proce-dures prior to chromatographic analysis and the storage con-ditions may cause degradation of drugs either in solutions orin biological matrices. According to the US Food and DrugAdministration (FDA) Guidance for Industry: BioanalyticalMethod Validation, the drug stability in biological samples is afundamental parameter that is a function of the chemicalproperties of the drug and the matrix itself and must bedetermined together with the evaluation of drug stability instock solutions [72]. The conditions used in stability experi-ments should reflect those occurring in sample handling,analytical process and sample storage of the developedmethod. Therefore, short-term, long-term and freeze-thawcycles stability studies must be performed. For short-term teststhe biological sample is kept at room temperature during aperiod of time corresponding to the expected duration ofsample handling before analysis. Long-term stability shouldtest the maximum time of sample storage at freezing temper-atures before drug degradation; this should exceed the timeelapsed between sample collection and the end of its analysis.For the fluoroquinolones of interest in this review, long-termstability times were found to vary between 2 weeks and12 months [60, 62]. Concerning the freeze-thaw stability,those fluoroquinolones showed no significant degradationfor at least three freeze-thaw cycles. Complementary stabilitytests are required to establish the effect of time on the drug inthe processed sample before injection, also called autosamplerstability. Forced degradation studies are also described toidentify possible degradation products and assess the abilityof the analytical method to quantify the drug analyte in thepresence of its degradants; these studies demonstrate thestability-indicating power of the developed method. The stressconditions used in this context were acid and base hydrolysis,oxidation and reduction, exposure to light and thermal stress.In the case of LEV, Gupta et al. [58] found minimal drugdegradation in 1 N HCl and considerable drug degradationunder oxidation with 30% H2O2 and under reduction condi-tions with 10% of Na2S2O5, always at room temperature.Gupta et al. [73] achieved similar results for SPA althoughthe samples were submitted to the above conditions for dif-ferent times (8 h as opposed to 12 h in the case of LEV). Underphotolytic conditions (4-min exposure to UVA radiation) andthermal stress (60 °C for 64 h) significant degradation of GEMwas found. One of the major drawbacks of fluoroquinolones istheir photosensitivity. It has been proposed that a high degreeof halogenation may lead to low photostability and, conse-quently, to the formation of photoproducts that can induceadverse effects and toxicity [47, 74, 75]. Therefore, a consid-erable interest has arisen in the investigation of the process ofdegradation. Forced degradation studies of CLI which was

exposed to sunlight for 2 h reveal two mechanisms of photo-degradation: loss of a chlorine atom at position 8 followed byreactions involving the fluoroquinolone nucleus, and degra-dation of the pyrrolidine side chain [75]. Photodegradation ofSPA was observed after irradiation with UV light for 8 h,yielding a photoproduct that is likely to have lost the fluorineatom at position 8 and another involving the loss of thatfluorine atom and the cyclopropyl group at position 1; nophotodegradation of the piperazine side chain was observed[74]. SIT decompose rapidly in aqueous solution by photo-irradiation with a fluorescent lamp (that emits visible and UVlight). The two major photoproducts obtained show the dis-sociation of the chlorine atom from position 8. In the samestudy, the effect of pH and the influence of wavelength on theSPA photostability were analysed. It was found that SPAwasmore sensitive to light in the zwitterionic form andmore stablein the acidic region. With respect to wavelength dependencethe conclusion was that SPAwas more labile at the maximumabsorption wavelength [47]. Overall, these results demon-strate that dehalogenation at position 8 is a significant photo-degradation mechanism for these fluoroquinolones. However,the photodegradation of LEV, which does not have halogenatoms in position 8, arises from modifications to the pipera-zine group [76]. In fact, the photoreactivity of fluoroquino-lones depends on their structure and medium. It is suggestedthat light irradiation generates toxic photoproducts intermedi-ates (e.g. carbenes) and reactive oxygen species that mayattack cell components and cause tissue damage, leading toremarkable phototoxicity of fluoroquinolones [47, 74, 75, 77,78]. An increased incidence of phototoxicity was observed indihalogenated fluoroquinolones and also in molecules thatcontain a methoxy group at position 5 and molecules with acyclopropyl or ethyl group at position 1 [39].

Hence, a better insight into the chemistry and structure ofnew fluoroquinolones is a major step for the manipulation ofthese compounds and the success of the optimization ofanalytical methods.

Analysis of new fluoroquinolones by liquidchromatography

For a variety of fluoroquinolones, clinical and molecularstudies of pharmacological profiles have demonstrated abetter correlation between the observed effects of a fluoro-quinolone and its plasma concentration than that obtainedbetween the observed effect and total daily drug dosage [9].This idea has prompted the development and validation ofnumerous liquid chromatography methods for routine deter-mination of these antimicrobial agents in body fluids over thelast few years. However, liquid chromatography has becomean important tool not only to support the therapeutic drugmonitoring of fluoroquinolones, but also to support the non-

100 J. Sousa et al.

clinical and clinical drug development and the quality controlof pharmaceutical formulations. Actually, the analysis of flu-oroquinolones was traditionally performed using microbio-logical assays, which have poor precision and selectivity [9].These disadvantages are overcome by the use of liquid chro-matography methods that enable the detection, separation andquantification of the parent compounds and metabolic degra-dation products in a short period of time with good selectivity,sensitivity, precision and accuracy. Nevertheless, it is impor-tant to note that the development of novel liquid chromato-graphic methods can progress in a number of ways. At oneextreme, it may simply involve adapting an existing method,making minor changes so that it is suitable for a new applica-tion; at the other extreme, the analyst may start out with a fewsketchy ideas and apply expertise and experience to develop asuitable method [79].

During the last two decades, several liquid chromatograph-ic methods have been developed for the quantification of themost interesting new fluoroquinolones (LEV, GAT, GRE,SPA, TEM, TOS, PAZ, CLI, GEM, MOX, SIT, ALA, TRO,PRU and ULI; Fig. 2) in a variety of matrices. Therefore, thisreview focuses on almost all such liquid chromatographymethods available in the literature, providing an appropriatebasis for the development of improved or new analyticalmethods for the determination of either the new fluoroquino-lones herein considered or of new fluoroquinolone-relatedcompounds under development as drug candidates. A specialemphasis will be placed on the critical steps to be consideredduring the process of analytical method development, namelythe sample preparation as well as the chromatographic anddetection conditions.

Sample preparation

Sample preparation, also known as sample pre-treatment,sample clean-up or sample extraction, is a fundamental andcritical part of any bioanalytical method. It has a high impacton the quality of data and it is usually the most time-consuming step of drug analysis in biological matrices,accounting for 80% of the total analysis time. Biologicalmatrices (e.g. blood, plasma/serum, tissue, urine, saliva)which contain the drug/analyte under investigation havedifferent degrees of complexity and comprise several com-ponents, such as salts, acids, basis, proteins, cells and lipids.Sample preparation aims at selective isolation of the analytefrom the matrix by removing the aforementioned endoge-nous compounds. This minimizes possible chromatographicinterferences and avoids the clogging and deterioration ofthe column, enabling bioanalysis by a chromatographicmethod [80–82]. The selection of a sample preparationmethodology depends on the specific goal of the investiga-tion and on the biological matrix and chemical properties of theanalyte. The sample preparation techniques most commonly

found in the literature for extraction of new fluoroquinolonesfrom biological matrices and subsequent chromatographicanalysis are protein precipitation (PP), liquid–liquid extraction(LLE), solid-phase extraction (SPE), ultrafiltration and dilution(Tables 3 and 4).

PP is the fastest and simplest sample preparation tech-nique and has been widely used for fluoroquinolones anal-ysis in plasma/serum matrices. PP consists in denaturationof proteins present in the matrix by addition of organicsolvents, acidification or by heating the sample. Duringdenaturation, proteins lose their secondary and tertiary struc-tures and drugs/analytes bound to these proteins becomefreely soluble in the medium. Centrifugation is an importantprocedure that follows protein denaturation because it sep-arates protein precipitate, leading to the formation of aprotein pellet and a clear supernatant containing the analytesusually ready for chromatographic analysis [82]. This su-pernatant can be directly injected into the chromatographicsystem (most common practice) or injected after dilution orconcentration of the supernatant [125]. The precipitatingagents most commonly used for the extraction of the newfluoroquinolones are acetonitrile, methanol and perchloricacid (Table 3). The type and amount of the precipitatingagent determines the efficiency of protein removal andinfluences the extraction efficiency of the analytes. Severalauthors have investigated different organic solvents andacids for PP. The acetonitrile appears to be more effectivein removing plasma proteins when compared with metha-nol; however, methanol is equally a good option in manycases, because it yields sufficiently clear supernatants andpossibly lower drug degradation [107]. Moreover, higherpeaks and increased peak areas were obtained after methanolprecipitation compared with acetonitrile precipitation. Forinstance, the peak areas of LEV in spiked plasma sampleswere approximately two- and three-fold higher after methanolprecipitation. This finding was also observed by analysingLEV in Ringer’s solution mixed with methanol or acetonitrile;thus, the lower peak areas obtained using acetonitrile cannotbe attributed to a different degree of co-precipitation of LEVwith plasma proteins [107]. Therefore, methanol wasemployed by Neckel et al. [107] to achieve a good sensitivityof the method. The explanation for these phenomena is notfully clarified; however, differences between the effect ofacetonitrile and methanol (present in the injected supernatant)in the chromatographic elution or in the intensity of thefluoroquinolone fluorescence signal appear to be plausiblecauses. On the other hand, protein-precipitating acids causean almost immediate protein precipitation of samples and,therefore, are frequently used; however, sometimes, analyterecovery is not totally predictable, particularly when strongacids are used, probably because of analyte co-precipitation[125]. In fact,Watabe et al. [87] found that recovery was lowerwith perchloric acid, and by adding an organic solvent


Tab

le3

Sam

plepreparationprocedures

appliedfortheanalysisof

new

fluo

roqu

inolon

esfrom

severalbiolog

ical

fluids

FQs

Matrix

Volum

e(m

L)

Sam

plepreparation

Solvent

Recov

ery(%

)Year

Reference

ULI

Rat

andrabb

itplasma

0.25

LLE

Chloroform/isoamyl

alcoho

l(85:15

,v/v)≈2.5mL

96.90–

99.01

2011

[83]

LEV

Mou

seplasma

0.15

PP

Methano

l4mL

85.9–90

.820

10[84]

LEV

Rabbitaqueou

shu

mou

r0.00

045

Directinjection

NA

98.2–10

1.4

2010

[58]

MOX

Rabbitaqueou

shu

mou

r0.05

PP

Acetonitrile

0.1mL

NR

2010

[85]

SPA

Rat

plasma

0.10

PP

Acetonitrile

0.2mL

>90

.020

10[86]

PAZ

Hum

anplasma

0.50

PP+dilutio

nAcetonitrile

0.4mL+20

mM

citric

acid,5mM

1-octane

sulpho

nicacid

salt(pH

5,NaO

H)0.4mL

94.2–99

.620

10[46]

Urine

0.02

Dilu

tion

20mM

citric

acid,5mM

1-octane

sulpho

nicacid

salt

(pH

5,NaO

H)0.4mL

105.2–

101.0

GEM

Rat

serum

0.20

LLE

Chloroform/isoamyl

alcoho

l(9:1,v/v)

3mL

79.32–

88.21

2010

[57]

PAZ

Hum

anserum

0.20

PP+PP

6.0%

(w/v)perchloric

acid

0.1mL+methano

l0.1mL

97(PAZ),88

(CIP),

90(LEV)

2010

[87]

CIP

LEV

LEV

Hum

anplasma

0.30

PP+evap/recon

stMethano

l0.30

mL

75.9

2009

[68]

MOX

(and

other

compo

unds)

OFL

Hum

anplasma

0.40

PP+evap/recon

stAcetonitrile

1mL

95(O

FL),86

.4(CIP),

94.2

(MOX)

2009

[88]

CIP

MOX

MOX

Hum

anplasma

0.10

PP

7%perchloric

acid

0.05

mL

96–10

520

09[65]

LEV

Hum

anurine

NR

Dilu

tion1:25

0.05

MSDSpH

3.0

96–10

320

09[89]

CIP

OFL

LOM

MOX

GEM

mesylate

Hum

anplasma

NR

LLE

Chloroform

5.4mL+acetic

acid

0.1mL(H

PLC)

80.06–

84.88(H

PLC)

2009

[90]

Chloroform

5.9mL+acetic

acid

0.1mL(H

PTLC)

80.01–86.17(HPT

LC)

CIP

Hum

anaqueou

shu

mou

r0.02

Dilu

tion1:6

Acetonitrile/85%

phosph

oric

acid

(15:85

,v/v)

0.1mL

NR

2008

[64]

ULI

GEM

(and

other

compo

unds)

Hum

anserum

0.10

PP

Acetonitrile

0.15

mL

97.41–

104.23

2008

[91]

GAT

Hum

anplasma

0.50

LLE

Ethyl

acetate3mL

56.39–57.75(G

AT),

74.27–79.79(SPA

),62.50–67.85(M

OX)

2008

[56]

SPA

MOX

102 J. Sousa et al.

Tab

le3

(con

tinued)

FQs

Matrix

Volum

e(m

L)

Sam

plepreparation

Solvent

Recov

ery(%

)Year

Reference

LEV

Hum

anplasma

0.50

PP

0.6M

perchloric

acid

0.1mL

86.9–95

.020

07[92]

ENO

Hum

anplasma

and

amniotic

fluid

0.50

SPE

1%trifluoroacetic

acid/acetonitrile

(25:75

,v/v)

2mL

94.7–99

.620

07[63]

LEV

PEF

CIP

LOM

MOX

OXO

LEV

Hum

anplasma

0.25

LLE

Dichlorom

ethane

5mL

NR

2007

[93]

GAT

Rat

plasma

0.10

Autom

ated

on-lineSPE

2.5mM

phosph

oric

acid/m

ethano

l/acetonitrile/TEA

(pH

2.8)

(64.8:15

:20:0.2,

v/v/v/v)

95.6–99

.720

07[94]

MOX

Hum

anplasma

0.50

PPandderivatization

Methano

l1mL

95.73

2007

[95]

ULI

Hum

anplasma

0.20

PP

10%

perchloric

acid

0.1mL

78.3–84

.320

07[96]

LEV

Hum

anplasma

0.10

LLE

Dichlorom

ethane

1mL

86–89

2007

[97]

GAT

Hum

anplasma

0.50

Ultrafiltratio

nNR

85–90

2006

[61]

ULI

Hum

anplasma

0.20

PP+evap/recon

stMethano

l0.4mL

92.1–98

.220

06[62]

LEV

Hum

anplasma

0.02

LLE

Dichlorom

ethane

0.8mL

55.2

2006

[98]

MOX

Hum

anplasma

NR

Directinjection

Previou

sadditio

nof

10mM

SDS

92.5

2006

[99]

GAT

Sem

en0.50

PP+PP+dilutio

nAcetonitrile

0.5mL(×2)+10

mM

TBA

phosph

ate,

25mM

citric

acid/acetonitrile

(90:10

,v/v)

0.8mL

9520

06[100

]

LEV

Hum

anplasma

0.40

LLE

Acetonitrile

0.8mL

106(LEV),97

(MOX)

2006

[101

]MOX

LEV

Hum

anplasma

0.05

PP

50%

trifluoroacetic

acid

0.02

5mL

97.2–10

4.7

2006

[102

]Dialysate

Directinjection

NA

LEV

Hum

anplasma

0.50

LLE

Dichlorom

ethane

7mL

NR

2005

[32]

MOX

LEV

Hum

anurine

0.50

Dilu

tion1:20

+microfiltration

Water

NR

2005

[103

]MOX

GAR

LEV

Hum

anplasma

NR

Autom

ated

SPE

Methano

l/trifluo

roacetic

acid

(99.9:0.1,

v/v)

1mL

92.93

2004

[104

]BAL

0.5

82.45

LEV

Hum

anserum

0.00

5Colum

n-sw

itching

direct

injection

10mM

KH2PO4bu

ffer,2mM

TBA

brom

ide

(pH

2.5)/acetonitrile

(88:12

,v/v)

83.9–98

.820

04[55]

GAT

MOX

GRE

Hum

anurine

NR

Microfiltration

NA

94.7–10

0.5

2004

[67]

GAT

Hum

anserum

0.45

Ultrafiltratio

nPho

sphate

buffer

pH7.5,

0.5%

SDS/acetonitrile

(4:1,v/v)

0.45

mL–displacing

reagent

95–99

2003

[71]


Tab

le3

(con

tinued)

FQs

Matrix

Volum

e(m

L)

Sam

plepreparation

Solvent

Recov

ery(%

)Year

Reference

Urine

0.05

Dilu

tion

10mM

SDS,10

mM

TBA

acetate,25

mM

citric

acid/acetonitrile

(50:50

,v/v)

1mL

NR

LEV

Hum

anplasma

0.25

LLE

Dichlorom

ethane

5mL

NR

2003

[105

]Urine

0.10

MOX

Hum

anplasma

0.25

PP

Acetonitrile/0.1

Mph

osph

atebu

ffer

pH4(9:1,v/v)

NR

2002

[106

]

LEV

Hum

anplasma

0.50

Ultrafiltratio

nPho

sphate

buffer,0.5%

SDS/acetonitrile

(4:1)–

displacing

agent

>95

2002

[70]

CIP

GAT

MOX

TRO

LEV

Hum

anplasma

0.20

PP

Methano

l0.40

mL

NR

2002

[107

]Microdialysates

0.01

Dilu

tion

Ringer’ssolutio

n0.04

mL

NR

LEV

(and

metabolites)

Serum

,bile

0.20

PP

Methano

l0.8mL

>98

2001

[108

]

SPA

Hum

anurine

2.50

Dilu

tionandderivatization

0.1M

H2SO43mL

91.5–65

.720

01[109

]

CLI

Hum

anplasma

0.20

PP

Acetonitrile/perchloricacid

(4:1,v/v)

NR

2001

[11]

Urine

0.50

Dilu

tion

Water

5mL+aceton

itrile/perchloricacid

(4:1,v/v)

NR

GAT

Hum

anplasma

0.10

SPE

Methano

l/trifluo

roacetic

acid

(99.9:0.1,

v/v)

94.93–

98.89

2001

[110]

GAT

Hum

anplasma

0.20

PP+dilutio

nAcetonitrile

0.4mL+10

mM

TBA

phosph

ate

0.8mL

92.2–10

2.0

2000

[111]

Urine

0.01

Dilu

tion

10mM

TBA

phosph

ate0.99

mL

97.1–97

.8

GEM

Hum

anplasma

0.05

PP

Acetonitrile

0.25

mL

NR

2000

[112]

GRE

Hum

anplasma

0.50

PP+dilutio

nN,N-D

imethy

lformam

ide0.5mL+water

95.79–

98.64

2000

[54]

Urine

NR

Dilu

tion1:4

Water

MOX

Hum

anplasma

1.2

Autom

ated

SPE

Methano

l/trifluo

roacetic

acid

(99.9:0.1,

v/v)

1mL

97.40

2000

[113]

GRE

Rabbitplasma

0.20

LLE

Trichloromethane

6mL

9020

00[114]

TRO

Serum

0.20

PP+dilutio

nAcetonitrile/perchloricacid

(99.75

:0.25,

v/v)

0.4mL+10

mM

TBA

hydrog

ensulphate

(pH

3.68

)0.8mL

98–10

819

99[115]

Urine

0.05

Dilu

tion

Sod

ium

hydrox

ide/ph

osph

oric

acid

(pH

3.60

)0.95

mL

99.6–10

7.2

GRE

Hum

anplasma

1.0

LLE

Dichlorom

ethane

5mL

90.08–

96.34

1999

[116]

CIP

Urine

92.34–

96.99

SPA

Plasm

a0.20

PP

20%

perchloric

acid

0.04

mL

96.7–97

.919

99[48]

Urine

0.05

Dilu

tion1:4

Distilledwater

CLI

Hum

anplasma

1.0

SPE

Methano

l/water

(40:60

,v/v)

1mL

107

1998

[117]

0.2

PP

Acetonitrile/perchloricacid

(4:1,v/v)

0.05

mL

67.4

SPA

Hum

anplasma

1.0

LLE

Dichlorom

ethane

3mL(2×)

94.9

1998

[118]

MOX

Hum

anplasma

0.25

PP

Acetonitrile/0.1

Mph

osph

oric

acid

(9:1,v/v)

0.75

mL

9319

97[119]

104 J. Sousa et al.

Tab

le3

(con

tinued)

FQs

Matrix

Volum

e(m

L)

Sam

plepreparation

Solvent

Recov

ery(%

)Year

Reference

Urine

NR

Dilu

tion1:20

0.1M

dihy

drog

enph

osph

atepH

4

Saliva

NR

LE

Acetonitrile/0.1

Mph

osph

atebu

ffer

pH4/0.1M

phosph

oricacid

(45:50

:5)

0.5mL(2×)

LEV

Hum

anplasma

0.25

LLE

Dichlorom

ethane

4mL

88–98

1997

[120

]Urine

0.01

87–95

TEM

Hum

anserum

1.0

LLEandderivatization

Dichlorom

ethane

5mL

NR

1996

[121

]Urine

0.5

TRO

Hum

anserum

0.20

SPE

Methano

l2mL

71.8–80

.919

96[60]

Urine

0.50

Potassium

phosph

atemon

obasic-sod

ium

hydrox

ide

buffer

pH9/aceton

itrile

(25:75

,v/v)

3mL

71.9–78

.9

SIT

Hum

anserum

0.20

SPE

Tetrahy

drofuran/0.15%

H3PO4(50:50

,v/v)

3mL

96.1

1994

[122

]Urine

105.5

SPA

Hum

anserum

0.50

PP+evap/recon

stAcetonitrile

1mL

99.5–10

019

92[59]

Urine

NR

Dilu

tion1:4

0.1M

phosph

oric

acid

97.0–97

.8

TEM

(and

metabolites)

Plasm

aNR

Ultrafiltratio

n0.07

5M

phosph

atebu

ffer

pH7.4,

0.5%

SDS/

aceton

itrile

(70:30

,v/v)

–displacing

reagent

NR

1991

[123

]

Plasm

a0.04

LLE

Dichlorom

ethane,10

%ethano

l6mL

Urine

NR

Dilu

tion

Acetonitrile/0.04M

H3PO4,0.01

MNaH

2PO4,

0.2%

SDS,n-acetylhy

drox

amic

acid

(53:47

,v/v)

FLE

Serum

0.50

LLE+LLE

Dichlorom

ethane

3.2mL

82.8–10

0.0(FLE,

TEM),

40.8–56

.8(TOS)

1989

[66]

TEM

Urine

(1:20)

+0.1M

sodium

hydrox

ide0.2mL(FLE,TEM)

TOS

Bile

(1:10)

+Acetic

acid

pH2.5,

0.2mL(TOS)

CIP

ciprofloxacin,

CLIclinafloxacin,

ENO

enox

acin,FLEflerox

acin,GARgareno

xacin,

GATgatifloxacin,

GEM

gemifloxacin,

GREgrepafloxacin,

LEVlevo

flox

acin,LOM

lomefloxacin,

MOX

mox

ifloxacin,

OFLofloxacin,

OXOox

olinicacid,PAZpazuflox

acin,P

EFpeflox

acin,S

ITsitaflox

acin,S

PAsparflox

acin,T

EM

temafloxacin,

TOStosuflox

acin,T

ROtrov

afloxacin,

ULIulifloxacin,

HPLChigh

-perform

ance

liquidchromatog

raph

y,HPTLChigh

-perform

ance

thin-layer

chromatog

raph

y,LEliq

uidextractio

n,LLEliq

uid–

liquidextractio

n,PPproteinprecipitatio

n,SP

Esolid

-phase

extractio

n,BALbron

choalveolarlavage,FQsfluo

roqu

inolon

es,SD

Ssodium

dodecylsulphate,TBAtetrabutylam

mon

ium,TEAtriethylam

ine,NAno

tapplied,

NRno

trepo

rted


Tab

le4

Sam

plepreparationprocedures

appliedfortheanalysisof

new

fluo

roqu

inolon

esfrom

severalbiolog

ical

tissues

FQs

Matrix

Tissueweigh

t(m

g)Sam

plepreparation

Solvent

Recov

ery(%

)Year

Reference

LEV

Mou

setissues:brain,

lung

,liv

er,kidn

ey,sm

allintestine

100

Hom

ogenization+PP

10%

methano

l0.6mL+methano

l0.6mL

92.6–95

.520

10[84]

GEM

Rat

tissues:lung

,liv

er,kidn

ey,

heart,testis,stom

ach,

brain,

adiposetissue

300

Hom

ogenization+LLE

Pho

sphate-bufferedsalin

e1mL

(2×)+

chloroform

/isoamyl

alcoho

l(9:1,v/v)

3mL

84.96–

93.48(lun

g,liv

er,kidn

ey)

2010

[57]

79.63–

94.20(heart,testis,stom

ach)

78.66–

84.02(brain,adiposetissue)

LEV

Cathetersegm

entstissue

50SPE

Methano

l,2μg

/mLascorbic

acid/formic

acid

(100

:0.1,v/v)

80.0–87

.220

08[124

]

LEV

Hum

anparanasalsinu

smucosa

30–60

LE

Pho

sphate

buffer

0.25

mLand

dichloromethane

1mL

NR

2007

[93]

LEV

Neutrop

hils

NR

Son

ication

Acetonitrile/pho

sphate-buffered

salin

e,5mM

TBA

brom

ide,5%

TEA

(pH

2.2)

(85:15

,v/v)

0.1mL

NR

2005

[32]

MOX

LEV

Bon

etissues

200

Hom

ogenization+

ultrafiltratio

nPho


e4mL

89.85

2004

[104

]

MOX

Hum

ansinu

smucosa

>10

0LE

Acetonitrile/0.1

Mph

osph

oric

acid

(1:1,v/v)

2mL

94–99

2002

[106

]

LEV

(and

metabolites)

Bon

e,softtissue

200

Hom

ogenization

Water/m

ethano

l/70%

perchloric

acid/pho

spho

ricacid

(500

:500

:10:1,

v/v/v/v)

2mL

>90

2001

[108

]

MOX

Lun

gtissue

250

Hom

ogenization+

automated

SPE

Pho


e2.5mL+methano

l/trifluo

roacetic

acid

(99.9:0.1,

v/v)

1mL

94.57

2000

[113]

SPA

Stool

100

LE(3×)

Acetonitrile/0.1

Mph

osph

oric

acid,23

mM

NaO

H(pH

3.82

)(75:25

,v/v)

NR

1992

[59]

GATgatifloxacin,

GEM

gemifloxacin,

LEVlevo

flox

acin,M

OXmox

ifloxacin,

SPAsparflox

acin,F

Qsfluo

roqu

inolon

es,L

Eliq

uidextractio

n,LLEliq

uid–

liquidextractio

n,PPproteinprecipitatio

n,SP

Esolid

-phase

extractio

n,TBAtetrabutylam

mon

ium,TEAtriethylam

ine

106 J. Sousa et al.

(methanol) co-precipitated analytes were extracted from thepellet to the supernatant leading to higher recovery of fluoro-quinolones. PP is expected to be more efficient by combina-tion of an acid and an organic solvent, because they bothreduce protein solubility in aqueous media. However, themajority of authors used only one organic solvent, acetonitrileor methanol (Table 3). In overall terms, the selection of theappropriate precipitating agent(s) must take into account theamount of supernatant obtained, its clearness and analyterecovery [88]. It is worth pointing out that the proportion ofprecipitating agent/sample volumes differs whether organicsolvents or acids are used as precipitants. In the first case thevolume proportion is higher or equal to 1:1, whereas in thelatter the volume proportion was found to vary between 1:2and 1:5. Furthermore, chromatography is sensitive to theamount of the organic solvent used in sample preparation;errors may arise from band broadening and shifting retentiontimes, especially when large volumes of organic solvents areused. The technique of on-column focusing minimizes theseeffects because it concentrates the sample on the top of thecolumn and strips off the high amount of the organic solventadded for sample preparation [119]. Although PP is the sim-plest and a non-selective sample preparation method, it is theleast time-consuming and found to give sufficiently low limitsof quantification for the intended purpose of many bioanalyt-ical methods developed for the analysis of fluoroquinolones.

One of the first sample preparation techniques developedwas LLE that involves the transfer of the analyte from theaqueous sample to a water-immiscible organic solvent. Thistechnique is more labour-intensive but affords cleaner chro-matograms. Nevertheless, LLE has several limitations suchas the need for large sample volumes, high organic solventconsumption, variable recovery, unsuitable for hydrophilicanalytes and the difficulty in extracting a wide variety ofanalytes with different lipophilicities. Despite this, LLE isstill used in the sample preparation of biological fluids [80,82]. Fluoroquinolones have an amphoteric character andtheir LLE requires adjustment of the pH of the sample to thatcorresponding to the isoelectric point of the compound. At theisoelectric point, the dominant form is the zwitterion, which isthe least hydrophilic and so the one with the highest affinity forthe organic phase. If two or more fluoroquinolones are to beextracted, it may be impossible to have the right pH for both[88]. A phosphate buffer is almost always added to the sampleto achieve a neutral pH (near to the isoelectric pH) and theorganic solvent most commonly used is dichloromethane asshown in Table 3. Whereas the recovery obtained with PP is atleast 67.4%, in the case of LLE it may be as low as 40.8%. Thedevelopment of LLE procedures for fluoroquinolones takesconsiderable effort and its application is time-consuming.

SPE has gained more popularity with time and is nowa-days considered the leading sample preparation method.This technique provides a better extraction of analytes, more

efficiently eliminates the interfering endogenous compoundsand is, therefore, the sample preparation method with higherrecovery values. SPE is based on the partitioning of analytesbetween a solid phase (sorbent) and a liquid phase (sample)governed by the different affinities for the two phases. Theprinciple of this sample preparation method is similar to theseparation mechanisms of liquid chromatography [80, 82,125]. In general, SPE involves the following steps: sorbentconditioning/equilibration, sample loading with retention ofthe analytes in the sorbent, sorbent washing to eliminateimpurities and subsequent elution of the analytes. The reten-tion of analytes can be established through nonpolar, polar orionic interactions and there is a wide variety of currentlyavailable SPE sorbents that offer the bioanalyst severaloptions of extraction capacity and selectivity [80]. Therefore,the choice of the sorbent is a key factor for the success ofsample preparation and strongly depends on the physicochem-ical properties of the target analyte and on the matrix compo-sition [63, 80]. In SPE techniques for the extraction of the newfluoroquinolones, two types of sorbent have been used, name-ly silica-based reversed-phase cartridges and polymericreversed-phase sorbents. The latter was chosen in most casesbecause it gives the best results. Vishwanathan et al. [110]studied C18, C8, C2 and silica cartridges and found that therecoveries were low (< 60%) or that a few interferences wereobserved; on the contrary, the polymeric Oasis HLB cartridges(that exhibit a large specific area) were demonstrated to be themost efficient extraction sorbent. The polymeric sorbents usedfor fluoroquinolones extraction are predominantly hydropho-bic, so the dominant retention mechanism is based on hydro-phobic interactions; however, they have some hydrophilicfunctional groups, an ideal situation for amphoteric com-pounds like fluoroquinolones that have an aromatic core andalso ionisable groups. Another advantage of polymeric sorb-ents is the absence of secondary silanol interactions, which areresponsible for complicated retention and elution of analytesand are typically observed in silica-based sorbents [104, 113].Automated SPE systems were recently introduced for thestudy of fluoroquinolones [94, 104, 113]. They reduce poten-tially dangerous sample handling, improve reproducibility byeliminating manual variability and, most importantly, enablehigh-throughput analysis. Technological advances have per-mitted the integration of sample preparation and chromato-graphic separation into a single system leading to fullyautomated on-line SPE. This labour-saving procedure hasbeen used for direct injection of plasma samples [55, 94].For this purpose two types of on-line SPE columns weredeveloped, namely restricted access media (RAM) and turbu-lent flow chromatography (TFC) columns [82, 99]. Both canbe used as pre-columns in combination with an analyticalcolumn by column switching or function as analytical col-umns by themselves. Such strategies for direct injection ofbiological samples into the chromatographic system will be


detailed in the next section. Despite the advantages of SPE,this sample preparation technique is expensive and has its ownlimitations; many new approaches have been designed, devel-oped and validated to overcome those limitations and toimprove the performance of the SPE technique [82]. Forexample, Morales-Cid et al. [126] described a microextractionby packed sorbents (MEPS) sample treatment coupled withthe analytical equipment to determine some fluoroquinolones.MEPS is a miniaturized SPE bed in a microvolume syringethat reduces the sample preparation time and the organicsolvent consumption.

Some authors also used ultrafiltration for plasma samplepreparation [61, 70, 71, 123]. This technique combines sim-plicity and precision for analyte extraction, but to be success-fully applied plasma samples must be treated with a displacingreagent before ultrafiltration [123]. Sodium dodecyl sulphate(SDS) enhances the solubility of proteins, releasing the drugfrom plasma protein binding [99]; thus it has been used as adisplacing reagent. In ultrafiltration techniques found in theliterature for bioanalysis of new fluoroquinolones, the displac-ing reagent most commonly used was a mixture of SDS andacetonitrile (Table 3). The proportion of acetonitrile is animportant aspect to take in consideration because a concentra-tion greater than 30% causes plasma protein precipitation andcompromises the integrity of ultrafiltration membrane [123].

Biological fluids such as urine, aqueous humour andmicrodialysates are easy to prepare owing to their matrixsimplicity. Urine is usually diluted to yield solutions withanalyte concentrations feasible for quantification analysis[119]. Compared with plasma and urine, aqueous humour isrelatively poor of salts and proteins, being constituted byapproximately 99% of water [64]. Therefore, this fluid canbe directly injected into the chromatographic system or afterbeing diluted [58, 64]. Microdialysates are collected directlyfrom the interstitial space fluid of the target tissues by recentmicrodialysis techniques; their sample volumes are small andthey consist of an isotonic aqueous medium free of proteins.For analysis of microdialysates, the samples can be alsodirectly injected into the chromatographic system; however,it is usually necessary to first dilute the microdialysates toobtain appropriate sample volumes [107].

To quantify analytes in biological tissues, the solid samplesmust be disrupted by a mechanical/physical method for thecomplete liberation of analytes from all cellular structures[127], as shown in Table 4. The homogenization procedurealso contributes to the extraction of the analytes from thetissue into a solvent that can be further treated by any of thetechniques mentioned above [57, 84, 113]. In some cases thehomogenization was carried out with a buffer, namelyphosphate-buffered saline solution, but in other cases it wasused in combination with a chemical disrupting reagent suchas an organic solvent or an acid [84, 106, 108]. For thequantification of LEV from infected tissues within Teflon

catheter segments, Bao et al. [124] found that 5 min ofvortexing was sufficient to give a homogeneous tissue sus-pension owing to the relatively soft matrix of the infectedtissue inside the catheters subcutaneously implanted. For in-homogeneous samples such as soft tissue and bone, bloodcontamination and variable moisture content can lead to highvalues of standard deviation, forcing the bioanalyst to extendthe limits of the acceptance criteria. The effect of bloodcontamination is an important aspect that should be carefullyaccounted for. In fact, for drugs with a low volume of distri-bution and consequently with a large difference betweenblood and tissue concentrations, the blood contaminationleads to errors in quantitative drug analysis, forcing the appli-cation of a correction factor to the tissue concentration. Con-trarily, when drugs are well distributed, the presence of bloodin tissues has only a minor effect [108]. Fluoroquinoloneshave an excellent tissue penetration and are used for thetreatment of device-related infections. In these infections, thepathogen is confined to extravascular tissue sites, which areseparated from blood by biological barriers, hindering theaccess of fluoroquinolones to the tissue [113]. Moreover,tissue penetration may be also affected by the inflammatoryprocess [107]. To determine the real concentration of MOX inlung tissue, Lemoine et al. [113] wiped off all external tracesof blood. Djabarouti et al. [104] applied a correction factor bycalculation of the amount of blood contamination in bone andbronchoalveolar lavage fluid samples.

The determination of fluoroquinolones in pharmaceuticalformulations requires a few simple sample preparation steps.In the case of tablets, these are individually weighed andpulverized to obtain a homogeneous mixture and an appro-priate amount of the resultant powder is thereafter dissolvedin a selected solvent and sonicated to facilitate solubilisa-tion. Finally, each sample is filtered through a suitable filterand injected into the chromatographic system [58, 73,128–131]. For eye drops and injectable solutions, dilutionis the only procedure that is performed [58, 73, 129].

Finally, another important feature is the addition of aninternal standard (IS) to the sample prior to initiation of thesample pre-treatment procedures. The use of IS can compen-sate the loss of analytes during sample preparation and there-fore improves the accuracy and precision of the method. Thiscompound must have similar physicochemical properties tothe analytes, must be stable and unreactive with sample com-ponents, column and mobile phase and must not be present inthe original sample [90]. The selection of the appropriate IS isguided by its extraction recovery value, chromatographic res-olution, retention time and potential interference with analytes.

Chromatographic separation

High-performance liquid chromatography (HPLC) has beenwidely used in the last few decades for the analysis of a

108 J. Sousa et al.

variety of compounds, including drugs and their metabo-lites. As a result of considerable efforts devoted to theoptimization of the chromatographic steps involved, it isnowadays a proven and well-established technique [73].

In particular, reversed-phase high-performance liquidchromatography (RP-HPLC) is the most frequent method-ology found in the literature for the determination of the newfluoroquinolones considered in this review (Table 5). Somedifficulties may however be identified in their separation onreversed-phase columns. Fluoroquinolones are weak hetero-cyclic amino acids with two protonation sites: an aminegroup which can be protonated and a carboxyl group whichcan be deprotonated [85, 88, 99]. Because of this amphotericcharacter, fluoroquinolones may exist in solution in cationic,neutral, zwitterionic and anionic forms. The difference in theirhydrophobicity explains their distinct rates of migrationthrough the chromatographic column, resulting in differentretentions enhancing fluoquinolone peak bandwidth. This,together with other factors such as column particle size, col-umn packing technology, mobile phase organic solvents, gra-dient elution and sample nature may influence resolution andbe responsible for poor peak shape and decreased sensitivity[85]. The relative percentage of each form depends on the pHof the solution. The neutral and zwitterionic forms are prefer-entially retained in the stationary phase of a reversed-phasecolumn. However, in aqueous solutions fluoroquinolones arelikely to exist in the ionic form; therefore, adjustment of pH ofthe mobile phase or addition of ion-pair reagents is a commonprocedure in RP-HPLC methods [88, 99]. Hence, to optimizethe mobile phase whilst achieving a compromise between agood resolution and a reasonable analysis time, several issueshave to be taken into account: the percentage of the organicmodifier, the pH, the type and concentration of buffers andion-pairing reagents.

The type and percentage of organic modifiers in themobile phase are important parameters that determine theeluting strength of the solution. Methanol and acetonitrilewere found to be the organic solvents most commonly usedin RP-HPLC methods; the selection of the proportion oforganic/aqueous polar phase is known to be a crucial stepand must be investigated in each case. Laban-Djurdjević etal. [99] and Nemutlu et al. [63] described a response surfacemapping technique to find the optimal composition of mo-bile phase. Surface responses were visualized as three-dimensional plots of volume percentage of acetonitrile, pHand one of the relevant parameters: resolution or retentiontime. For determination of MOX in plasma samples, Laban-Djurdjević et al. [99] observed that the response surface forresolution showed a relatively flat maximum in the range of10–15% for acetonitrile and 3.0–4.5 for pH values. Similarresults were obtained for retention time: the response surfaceshowed a broad minimum at the same location as before.Using the same technique, Nemutlu et al. [63] concluded that

for simultaneous separation of seven quinolones, includingLEV and MOX, the optimum resolution and retention timewere achieved with 9% of acetonitrile and pH 3.2 in themobile phase.

In line with the above results, the present review of RP-HPLC methods shows that in most cases an acidic mobilephase was selected (Table 5). In order to optimize thechromatographic separation of LEV, GAT and MOX,Nguyen et al. [55] chose a mobile phase at pH 2.5 to reversefluoroquinolone carboxylic function ionization; they evalu-ated the influence of pH in the range 2.0–4.0 and concludedthat the resolution of fluoroquinolones did not vary signif-icantly within this range. Ocaña González et al. [67] inves-tigated the retention time of GRE using acetonitrile/0.1 Mphosphoric acid–sodium hydroxide buffer/0.01 M n-octyl-amine as mobile phase over the pH range 2.5–6.0 and foundthat it remained almost constant in the range 2.5–3.5, where-as it increases approximately 50% at pH 6.0; therefore, thevalue of pH 3.0 was selected for the mobile phase. To keepthe correct pH of mobile phase constant a number of buffersolutions are commonly used; the most frequently used arevarious forms of phosphate buffer although formic, citricand trifluoroacetic acid amongst others are also possiblebuffer solutions (Table 5). Only a few authors investigatedthe influence of the buffer nature on the separation offluoroquinolones. Liang et al. [70] and Nemutlu et al. [63]studied the effect of phosphate buffer and citric acid; theyobserved that resolution of fluoroquinolones was much bet-ter using citric acid than phosphate buffer; furthermore, thebuffer capacity of citric acid is wider than that of phosphateat low pH. Therefore in those techniques citric acid wasselected as buffer in the mobile phase. Nevertheless,Nguyen et al. [55] found no differences in the resolutionof fluoroquinolones when using citric acid instead of phos-phate buffer and the latter was chosen for the final mobilephase.

Another approach used for the chromatographic separa-tion of ionizable compounds is the addition of ion-pairreagents. Some of the RP-HPLC methods found in theliterature use a mobile phase supplemented with SDS asion-pair reagent [69, 70]. This molecule has a hydrophobicregion that interacts with the stationary phase and a polarhead with a negatively charged sulphonate group. Thisgroup interacts electrostatically with the positively chargedamine group of fluoroquinolones forming ion pairs and thusimproving retention [99]. For the separation of LEV, cipro-floxacin, GAT, MOX, TRO and cinoxacin by HPLC, Lianget al. [70] developed a technique with a mobile phase con-taining SDS. During the optimization of mobile phase com-position, they observed that at pH 3.0 fluoroquinolones wereoverlapped; this was attributed to the fact that they were notretained because they were completely ionized. To increaseretention and selectivity of those compounds the effect of


Tab

le5

Liquidchromatog

raph

ymetho

dsfordeterm

inationof

new

fluo

roqu

inolon

es

FQs

Technique

Detectio

n(m

/z;nm

)Sam

ple

ISColum

nElutio

nMobile

phase

Flow

rate

(mL/m

in)

Run

time

(min)

Calibratio

nrange

(μg/mL,μg

/g)

LLOQ

(μg/mL,μg

/g)

Year

Ref.

BES

LC

MS/MS

m/z394.2→

376.2

(BES)

m/z376.2→

358.2

(GAT)

m/z402.2→

384.2

(MOX)

Hum

anaqueous

humour

No

XBridge

C18(3.5

μm)

Isocratic

0.2%

form

icacid/

acetonitrile

(80:20,v/v)

NR

NR

0.001–1.000

0.001

2011

[132]

GAT

MOX

GEM

mesylate

HPLC

PDA

λ0287nm

Bulkdrug

No

InertsilODS3V

C18(5

μm)

Gradient

27°C

0.1%

trifluoroacetic

acid

(pH

2.5,

liquid

ammonia)/methanol

(55:45,v/v)

or(20:80,v/v)

1.0

150.1–

200

1.0

2011

[133]

ULI

HPLC

MS/M

Sm/z350.2→

248.5

(ULI),

m/z332.4→

231.4

(IS)

Rat

andrabbit

plasma

CIP

PeerlessBasic

C18(3

μm)

Isocratic

r.t.

Methanol/0

.5%

form

icacid

(9:1,v/v)

0.5

3.0

0.01

–2.50

0.01

2011

[83]

LEV

HPLC

MS/M

SMouse

tissues

andplasma

GAT

Welch

materialsC4

(5μm

)Gradient

25°C

0.05%

form

icacid/

methanol

1.0

150.13

–26.2

(tissues)

0.13

(tissues)

2010

[84]

m/z362.1→

318.1

(LEV)

(93:7,

v/v)

m/z376.2→

332.2

(IS)

(88:12,v/v)

0.09

–4.53

(plasm

a)0.0218

(plasm

a)

(10:90,v/v)

LEV

UPLC

UV λ0288nm

Rabbitaqueous

humour

No

WatersAcquity

HSS

T-3C18(1.8

μm)

Gradient

50°C

0.1%

trifluoroacetic

acid/acetonitrile

0.45

5.0

20–45

0.05

2010

[58]

Pharm

aceutical

form

ulations

(92:08,v/v)

(80:20,v/v)

SPA

UPLC

UV λ0290nm

Pharm

aceutical

form

ulations

Bulkdrug

No

WatersAcquity

HSS

T-3C18(1.8

μm)

Gradient

50°C

0.1%

orthophophoric

acid/acetonitrile

(90:10,v/v)

or(75:25,v/v)

0.5

5.0

80–120

0.6

2010

[73]

MOX

UPLC

UVλ0296nm

Rabbitaqueous

humour

No

WatersAcquity

BEH

C18(1.7

μm)

Gradient

50°C

0.1%

trifluoroacetic

acid/

acetonitrile

(80:20,v/v)

or(70:30,v/v)

0.4

4.0

0.01

–1.00

0.0025

2010

[85]

SPA

HPLC

MS/M

SRat

plasma

Sotalol

AtlantisC18(3

μm)

Isocratic

10mM

ammonium

acetate(pH

4,acetic

acid)/acetonitrile

(20:80,v/v)

0.3

3.5

0.01

–1.00

0.01

2010

[86]

m/z392.9→

272.9

(SPA

)

m/z348.7→

254.9

(IS)

PAZ

HPLC

UV λ0330nm

Hum

anplasma

Urine

ENO

Agilent

Zorbax

Eclipse

XDB-C

18

(5μm

)

Isocratic

30°C

20mM

citric

acid,

5mM

1-octane

sulfonic

acid

salt

(pH

5.0,

NaO

H)/

acetonitrile

(81:19,v/v)

1.0

15(plasm

a)0.02

–30

(plasm

a)0.02

(plasm

a)2010

[46]

FL λex/em0330/

394nm

17(urine)

0.5–

200(urine)

0.5(urine)

GEM

LC

MS/M

SRat

tissues

and

serum

CIP

PeerlessBasic

C18(3

μm)

Isocratic

35°C

Methanol/1

%form

icacid

(9:1,v/v)

0.6

12.5

2.5×10

−4 –2.0×10

−2

(lung,liver,kidney)

NR

2010

[57]

m/z390.1→

272.1

(GEM)

1.25

×10

−4 –0.5×10

−2

(heart,testis,stomach)

m/z332.1→

314.2

(IS)

0.75×10

−4 –0.05×10

−2

(adiposetissue,brain,

serum)

110 J. Sousa et al.

Tab

le5

(con

tinued)

FQs

Technique

Detectio

n(m

/z;nm

)Sam

ple

ISColum

nElutio

nMobile

phase

Flow

rate

(mL/m

in)

Run

time

(min)

Calibratio

nrange

(μg/mL,μg

/g)

LLOQ

(μg/mL,μg

/g)

Year

Ref.

PAZ

HPLC

FL λex/em0300/

450nm

Hum

anserum

No

InertsilC8-3

column

(5μm

)Isocratic

r.t.

1%TEA

(pH

3.0,

phosphoric

acid)/

acetonitrile

(86:14,v/v)

1.0

180.1–20

0.1

2010

[87]

CIP

LEV

LEV

HPLC

UV

(LEV,MOX)

λ0280nm

Hum

anplasma

Quinoxalin

eAtlantisC18(3

μm)

Gradient

35°C

50mM

dihydrogen

phosphatebuffer/

acetonitrile

1.0

280.23–15.0

(LEV)

0.156(M

OX)

2009

[68]

MOX

(and

other

compounds)

UV

(IS)

λ0270nm

0.16–10.0

(MOX)

0.234(LEV)

OFL

HPLC

FL(CIP)

λ ex/em0279/442nm

Hum

anplasma

SAR

WatersXBridge™

C18(3.5μm

)Gradient

Acetonitrile/m

ethanol/

0.025M

TBA

chloride/

trifluoroacetic

acid

(pH

3.5)

(75:25:899:1,

v/v/v/v)

or(150:50:799:1,

v/v/v/v)

0.25

170.02–7.5

0.02

2009

[88]

CIP

FL(M

OX,O

FL,IS)

λex/em0290/500nm

MOX

MOX

HPLC

FL λex/em0290/

460nm

Hum

anplasma

OFL

Lichrospher

100

Isocratic

r.t.

50mM

phosphatebuffer

(pH

2.6,

1N

HCl)/

acetonitrile

(80:20,v/v)

1.5

8.0

0.125–10.0

0.09

2009

[65]

RP-18e

(5μm

)

LEVa

MLC

FL(CIP,LEV)

λ ex/em0295/465nm

Hum

anurine

No

KromasilC18

(5μm

)Isocratic

r.t.

a 0.15M

SDS,12.5%

propanol,0.5%

TEA

(pH

3)

1.0

10a

0.001–1.000

0.001

2009

[89]

CIP

a

OFLb

FL(OFL

,LOM,

MOX)

λ ex/em0285/465nm

b0.05

MSDS,12.5%

propanol,0.5%

TEA

(pH

3)

22b

LOM

b

MOXb

GEM

mesylate

HPLC

UV λ0263nm

Hum

anplasma

Hydrochlorothiazide

Eurosphere-100

C18(5

μm)

Isocratic

25°C

Methanol/1

%sodium

acetate/orthophosphoric

acid

(pH

2.1)

(65:35:0.5,v/v/v)

0.8

8.0

0.03–0.60

0.03

2009

[90]

GEM

mesylate

HPTLC

UV

densito

metry

λ0254nm

Hum

anplasma

Linezolide

Silica

gelplate60

F254

NR

Ethyl

acetate/methanol/

ammonia

(8.0:4.0:3.0,

v/v/v)

NA

NA

0.05–0.60

0.05

2009

[90]

LEV

HPLC

MS/M

SCatheter

segm

ents

tissue

GAT

Sym

metry

C18(3.5

μm)

Gradientr.t.

Acetonitrile/water/formic

acid

(3:97:0.2,

v/v/v)

0.4

120.02–2

0.02

2008

[124]

m/z362→

318

(LEV)

Acetonitrile/formic

acid

(100:0.2,v/v)

m/z376→

332(IS)

LEV

HPLC

PDA

λ0295nm

Pharm

aceutical

form

ulation

No

Phenomenex

C18(5

μm)

Isocratic

r.t.

KH2PO4pH

6.8/methanol/

acetonitrile

(70:15:15,v/v)

1.5

NR

1–10

0.94

2008

[131]

CIP

HPLC

UV λ0278nm

Hum

anaqueous

humour

No

ZORBAX

Eclipse

XDB-C

8(5

μm)

Isocratic

r.t.

Acetonitrile/85%

aqueous

phosphoric

acid

(15:85,v/v)

0.5

100.005–0.600(U

LI)

0.006(U

LI)

2008

[64]

ULI

GEM

(and

other

compounds)

LC

ESI-MS/M

SSerum

Diazepam

LunaC18(5

μm)

Gradient

Methanol/w

ater/formic

acid/trifluoroacetic

acid

0.5

200.0035–0.0456

0.0028

2008

[91]

m/z390→

372

(GEM)

(20:80:0.1:0.1,v/v/v/v)

m/z285→

257(IS)

(90:10:0.1:0.1,v/v/v/v)

GAT

HPLC

UV λ0293nm

Hum

anplasma

LEV

Kromasil100

C18(5

μm)

Isocratic

Phosphate

buffer

pH2.5/

acetonitrile

(80:20,v/v)

1.0

180.100–1.000

0.1

2008

[56]

SPA

MOX

LEV

HPLC

UV λ0294nm

Hum

anplasma

CIP

KromasilC18

(5μm

)Isocratic

r.t.

Acetonitrile/water/

phosphoric

acid/TEA

(14:86:0.6:0.3,v/v/v/v)

1.0

100.05–5.0

0.05

2007

[92]


Tab

le5

(con

tinued)

FQs

Technique

Detectio

n(m

/z;nm

)Sam

ple

ISColum

nElutio

nMobile

phase

Flow

rate

(mL/m

in)

Run

time

(min)

Calibratio

nrange

(μg/mL,μg

/g)

LLOQ

(μg/mL,μg

/g)

Year

Ref.

LEV

HPLC

UV λ0220nm

Pharm

aceutical

form

ulations

No

HypersilBDS

C18(5

μm)

Isocratic

30°C

Potassium

dihydrogen

orthophosphate

buffer/

acetonitrile/m

ethanol

(650:250:100,v/v/v)

(pH

5.2,

TEA)

1.0

107–22

72007

[130]

ENO

HPLC

UV

(PDA)

λ0280nm

Hum

anplasma

Amnioticfluid

MAR

ZorbaxEclipse

XDB-C

18(5

μm)

Gradient

40°C

15mM

citratebuffer

pH3.2,

9%acetonitrile,

5%methanol,5mM

tetram

ethylammonium

brom

ide

1.5

190.035–30.0

(LEV,

MOX)

0.035(LEV,

MOX)

2007

[63]

LEV

PEF

CIP

LOM

MOX

OXO

GAT

HPLC

FL λex/em0295/

480nm

Rat

plasma

NOR

Shimadzu

Shim-

PackCLC-O

DS

(5μm

)

Isocratic

28°C

2.5mM

phosphoricacid/

methanol/acetonitrile/TEA

(pH2.8)

(64.8:15:20:0.2,

v/v/v)

NR

NR

0.02–0.60

0.02

2007

[94]

MOX

HPLC

FL λex/em0464/

537nm

Hum

anplasma

NOR

Phenomenex

C18(5

μm)

Isocratic

Acetonitrile/10mM

orthophosphoricacid

(pH

2.5)

(80:20,v/v)

1.2

8.0

0.015–2.70

0.015

2007

[95]

ULI

HPLC

FL λex/em0280/

425nm

Hum

anplasma

LOM

Diamonsil

C18(5

μm)

Isocratic

30°C

Acetonitrile/0.5%

TEAbuffer

(pH3,10%

phosphoric

acid)(21:79,v/v)

1.0

6.5

0.01–1.00

0.01

2007

[96]

LEV

Stereospecific

HPLC

UV λ0293nm

Pharm

aceutical

form

ulations

No

RP-C

18(5

μm)

Isocratic

r.t.

Methanol/w

ater,10

mM

L-leucine,5

mM

copper

sulphate

(88:12,v/v)

1.0

200.5–400

NR

2007

[134]

LEV

HPLC

FL λex/em0295/

440nm

Hum

anplasma

Terazosin

KromasilC18

(5μm

)Isocratic

25°C

Phosphate

buffer

pH3.0,

0.01%

TEA/acetonitrile

(76:24,v/v)

1.0

NR

0.0521–5.213

0.0521

2007

[97]

GAT

HPLC

PDA

λ0293nm

Hum

anplasma

CIP

XTerraMSC18

(5μm

)Isocratic

r.t.

0.025M

disodium

hydrogen

phosphate

pH3.0/acetonitrile

(80:20,v/v)

1.0

7.5

0.10–6.0

0.1

2006

[61]

PAZ

HPLC

FL λex/em0335/

450nm

Hum

anplasma

–Lichrospher

C18

Isocratic

0.5%

trifluoroacetic/

methanol(60:40,v/v)

––

0.024–19.58

–2006

[135]

ULI

LC

ESI-MS/M

SHum

anplasma

OFL

Diamonsil

C18(5

μm)

Isocratic

20°C

Methanol/w

ater/formic

acid

(70:30:0.2,v/v/v)

0.5

5.0

0.025–5.0

0.025

2006

[62]

m/z350→

248

(ULI)

m/z362→

261(IS)

LEV

HILIC

MS/M

SHum

anplasma

CIP

AtlantisHILIC

silica

column(5

μm)

Isocratic

30°C

Acetonitrile/ammonium

form

ate(100

mM,

pH6.5)

(82:18,v/v)

0.5

5.0

0.01–5

0.01

2006

[98]

m/z362.7→

261.2

(LEV)

m/z332.3→

231.1

(IS)

MOX

HPLC

FL λ e

x/em0290/500nm

Hum

anplasma

OFL

Supelco

LC-H

isep

(5μm

)Isocratic

Acetonitrile/0.25M

sodium

phosphate

(pH

3)(5:95,

v/v)

1.0

8.0

3–1,300

3.0

2006

[99]

GAT

HPLC

PDA

λ0293nm

Sem

enNo

X-Terra

RP-18

(5μm

)Isocratic

r.t.

10mM

TBA

phosphate,

25mM

citric

acid/

acetonitrile

(90:10,v/v)

0.4

5.0

2–20

(UV)

2.3(U

V)

2006

[100]

FL λex/em0292/

480nm

0.02–2(FL)

0.03

(FL)

112 J. Sousa et al.

Tab

le5

(con

tinued)

FQs

Technique

Detectio

n(m

/z;nm

)Sam

ple

ISColum

nElutio

nMobile

phase

Flow

rate

(mL/m

in)

Run

time

(min)

Calibratio

nrange

(μg/mL,μg

/g)

LLOQ

(μg/mL,μg

/g)

Year

Ref.

LEV

HPLC

UV

Pharm

aceutical

form

ulations

No

LiChrospher

RP-18

(5μm

)Isocratic

r.t.

Water/acetonitrile/TEA

(pH

3.3,

phosphoric

acid)(80:20:0.3,v/v/v)

1.0

4.0

4.0–24.0

0.46

(LEV)

2006

[129]

PEF

λ0295nm

(LEV)

0.25

(PEF)

LOM

λ0279nm

(PEF)

0.52

(LOM)

GAT

λ0288nm

(LOM)

0.39

(GAT)

λ0293nm

(GAT)

LEV

HPLC

FL λex/em0295/

440nm

Hum

anplasma

MOX

Nucleosil100-5C18

Nautilus

(5μm

)Gradient

Acetonitrile/0.01M

sodium

dihydrogen

phosphatepH

2.7

1.5

180.1–15

(LEV)

0.05

(LEV)

2006

[101]

MOX

LEV

0.2–3.5(M

OX)

0.2(M

OX)

LEV

HPLC

FL λ e

x/em0296/504nm

Hum

anplasma

MOX

YMCPro

C18(5

μm)

Gradient

20°C

NR

0.25

NR

0.1–6(plasm

a)0.1

2006

[102]

Dialysates

0.1–5(dialysate)

GAT

HPLC

UV λ0286nm

Pharm

aceutical

form

ulations

Hum

anplasma

–Mediterranea

C18

Isocratic

Acetonitrile/m

ethanol/

water(40:40:20,v/v/v)

(pH2.7,phosphoricacid)

1.0

–0.1–25

0.00577

(plasm

a)2006

[136]

LEV

HPLC

UV λ0293nm

Hum

anplasma

Neutrophils

No

LiChropherRP-18

(5μm

)Isocratic

Acetonitrile/phosphate-

buffered

salin

e,5mM

TBA

brom

ide,5%

TEA

(pH

2.2)

(85:15,v/v)

NR

NR

0.05–5(plasm

a)0.025

2005

[32]

MOX

FL λ e

x/em0278/515nm

2.5–200

(neutrophils)

LEV

HPLC

UV

(PDA)

Hum

anurine

No

LiChrospher

100

(5μm

)Gradient

Acetonitrile/0.1M

phosphoricacid,sodium

hydroxidebuffer

(pH3.0)/0.01M

n-octylamine

(pH3.0,phosphoricacid)

1.0

1550–150(LEV)

7.3(LEV)

2005

[103]

MOX

λ0292nm

(LEV)

(8:46:46,v/v/v)

8.8(M

OX)

GAR

λ0294nm

(MOX)

(40:30:30,

v/v/v)

20–60

(MOX,G

AR)

6.0(G

AR)

λ0282nm

(GAR)

LEV

HPLC

UV λ0299nm

Hum

anplasma

MOX

SupelcosilABZ+

(5μm

)Isocratic

Water,0.4%

TEA

(pH

3,orthophosphoricacid)/

acetonitrile

(83:17,v/v)

1.2

120.25–25

(plasm

a)0.20

(plasm

a)2004

[104]

BAL

1–6(BAL)

0.40

(BAL)

Bonetissues

0.5–10

(bone)

0.5(bone)

LEV

HPLC

column

switching

FL λ e

x/em0296/504nm

Hum

anserum

No

SupelcosilABZ+

(5μm

)Isocratic

r.t.

10mM

KH2PO4buffer,

2mM

TBA

brom

ide

(pH

2.5)/acetonitrile

(88:12,v/v)

1.2

130.125–4.00

(LEV,

MOX)

0.125(LEV,

MOX)

2004

[55]

GAT

0.1625–0.5000

(GAT)

0.1625

(GAT)

MOX

GRE

HPLC

UV

(PDA)

λ0278nm

Hum

anurine

No

LiChrospher

100

RP-18(5

μm)

Gradient

Acetonitrile/0.1

Mphosphoric

acid,sodium

hydroxidebuffer

(pH3)/

0.01

Mn-octylamine

(pH

3,phosphoric

acid)

1.3

104–80

3.5

2004

[67]

(8:46:46,v/v/v)

(36:32:32,

v/v/v)

PAZ

Stereospecific

HPLC

UV λ0332nm

Pharm

aceutical

form

ulations

No

Shimadzu

CLC-O

DS

(10μm

)Isocratic

r.t.

8mM

L-phenylalanine,

6.3mM

copper

sulphate

(pH

3.5,NaO

H)/

methanol(70:30,v/v)

1.0

352.48–248

(S-enantiomer)

NR

2004

[137]

2.52–252

(R-enantiomer)

GAT

HPLC

UV λ0293nm

Hum

anserum

CIP

Adsorbosphere

HS

C18(5

μm)

Isocratic

23°C

10mM

SDS,1

0mM

TBA

acetate,25

mM

citric

acid/acetonitrile

(50:50,v/v)

1.0

7.5

0.100–100.000

(serum

)0.100(serum

)2003

[71]

Urine

1–150(urine)

1(urine)


Tab

le5

(con

tinued)

FQs

Technique

Detectio

n(m

/z;nm

)Sam

ple

ISColum

nElutio

nMobile

phase

Flow

rate

(mL/m

in)

Run

time

(min)

Calibratio

nrange

(μg/mL,μg

/g)

LLOQ

(μg/mL,μg

/g)

Year

Ref.

LEV

HPLC

UV λ0280nm

Hum

anplasma

Tinidazole

RP-C

18(5

μm)

Isocratic

r.t.

Potassium

phosphate

buffer/acetonitrile

(pH

2.6)

(82:18,v/v)

NR

NR

NR

0.1

2003

[105]

Urine

LEV

HPLC

LEV,GAT,

MOX,TRO:

Hum

anplasma

CIP

Adsorbosphere

HS

C18(5

μm)

Isocratic

r.t.

10mM

SDS,10

mM

TBA

acetate,25

mM

citricacid/

acetonitrile

(pH3.4)

(57:43,v/v)

1.0

NR

UV:

NR

2002

[70]

CIP

UV

(PDA)

λ0293nm

LEV

0.05–10.00(LEV)

GAT

FL λ e

x/em0280/450nm

CIP

0.10–10.00

MOX

CIP:

CIP

(GAT,MOX)

TRO

UV

(PDA)

λ0280nm

GAT

0.20–10.00

FL λ e

x/em0293/500nm

(CIP,TRO)

FL:0.020–10.00

LEV

HPLC

FL λex/em0310/467

nm

Microdialysates

Plasm

aCIP

HypersilODS

(5μm

)Isocratic

45°C

0.11%

phosphoricacid,

1MTBA(pH3.0)/

acetonitrile

(88.5:1.5,v/v)

0.4

120.0156–5.0

(microdialysates)

0.0156

(microdialysates)

2002

[107]

0.02–12.5

(plasm

a)0.02

(plasm

a)

LEV

(and

metabolites)

HPLC

FL λ e

x/em0295/490nm

Softtissue

CIP

WatersSym

metry

C18(5

μm)

Gradient

Water/m

ethanol/T

EA

(pH

3)1.5

140.1–40

NR

2001

[108]

Bone

(750:250:4,v

/v/v)

Bile

(600:400:4,v

/v/v)

Serum

SPA

HPLC

FL λ e

x/em0275/464nm

Hum

anurine

Salicylic

acid

RPcolumn

Isocratic

30°C

0.5M

tetraethylam

monium

brom

ide/0.02

Mphosphate/methanol

(8:40:52,v/v)

1.0

8.0

0.05–4.00

NR

2001

[109]

CLI

HPLC

UV λ0340nm

Hum

anplasma

NOR

RP-C

18

Isocratic

0.05

Mcitricacid,1.15mM

TBAhydroxide,0.1%

ammonium

perchlorate/

acetonitrile

(80:20,v/v)

NR

NR

0.025–10

(plasm

a)0.025(plasm

a)2001

[11]

2.5–200(urine)

2.5(urine)

GAT

HPLC

ESI-MS/M

SHum

anplasma

CIP

Astec

Cyclobond

I2000

(5μm

)Isocratic

0.1%

form

icacid

0.4

3.0

0.01–1.00

NR

2001

[110]

m/z376→

358

(GAT)

m/z332→

314(IS)

GAT

HPLC

FL λ e

x/em0295/480nm

Hum

anserum

TRO

Nucleosil-100-5

C18(5

μm)

Isocratic

r.t.

12mM

TBA

phosphate

(pH

3.48,phosphoric

acid)/acetonitrile

(83:17,v/v)

1.0

15(serum

)0.06–4(serum

)0.06

(serum

)2000

[111]

Urine

8.0(urine)

3.5–600(urine)

3.5(urine)

GEM

HPLC

MS/M

SHum

anplasma

[13C2H3]GEM

PLRP-S

(5μm

)Isocratic

40°C

0.01

Mam

monium

acetate

buffer

(pH

2.5,

trifluoroacetic

acid)/

acetonitrile

(70:30,v/v)

1.0

1.5

0.01–5.00

0.01

2000

[112]

GRE

HPLC

UV λ0

296nm

(plasm

a)Hum

anplasma

Urine

AT-4117

TSK

gelODS-

80TM

(5μm

)Isocratic

35°C

5%acetic

acid,

1%triethylam

ine/

acetonitrile/m

ethanol

(70:15:15,

v/v/v)

0.8

NR

0.05–2.5(plasm

a)0.05

(plasm

a)2000

[54]

λ0340nm

(urine)

0.5–500(urine)

0.5(urine)

MOX

HPLC

UV λ0296nm

Plasm

aENR

SupelcosilABZ+

(5μm

)Isocratic

Acetonitrile/10mM

potassium

dihydrogen

phosphatebuffer

(pH

4.0,

orthophosphoric

acid)(18:82,v/v)

1.25

130.025–3.2(plasm

a)0.03

(plasm

a)2000

[113]

Lungtissue

0.25–16.0

(lung)

0.4(lung)

114 J. Sousa et al.

Tab

le5

(con

tinued)

FQs

Technique

Detectio

n(m

/z;nm

)Sam

ple

ISColum

nElutio

nMobile

phase

Flow

rate

(mL/m

in)

Run

time

(min)

Calibratio

nrange

(μg/mL,μg

/g)

LLOQ

(μg/mL,μg

/g)

Year

Ref.

GRE

HPLC

FL λ e

x/em0338/425nm

Rabbitplasma

ENR

NovapackC18

(5μm

)Isocratic

r.t.

0.020M

potassium

dihydrogen

phosphate,

6mM

phosphoric

acid,12

mM

tetraethylam

monium

brom

ide/acetonitrile

(22:78,v/v)

(pH

3,NaO

H)

1.0

NR

0.005–4.000

0.010

2000

[114]

TRO

HPLC

FL

Serum

No

Nucleosil100-5

Isocratic

r.t.

Acetonitrile/10mM

TBA

phosphate

0.9

200.07

–7.5

0.07

1999

[115]

λ ex/em0275/405nm

C18(5

μm)

(pH

3.68)(25:75,v/v)

IEX-H

PLC

Urine

Nucleosil100-5

Acetonitrile/sodium

hydroxide,phosphoric

acid

(pH

3.6)

(62.2:37.8,v

/v)

1.0

100.5–

500.50

SA

(5μm

)

GRE

HPLC

UV

Hum

anplasma

AT-4117

C18YMCpack

A-312

(5μm

)Gradient

50°C

(A)5%

acetic

acid

1.0

250.05–5(plasm

a)0.5(urine)

1999

[116]

CIP

λ0280nm

Urine

(B)Acetonitrile/m

ethanol

(50:50,v/v)

0.5–

500(urine)

0.05

(plasm

a)

SPA

HPLC

UV

Plasm

a,urine

GRE

C18YMCpack

A-312

(5μm

)Isocratic

50°C

5%acetic

acid,1%

TEA/

acetonitrile/m

ethanol

(76:12:12,

v/v/v)

1.2

150.025–1.5(plasm

a)0.025(plasm

a)1999

[48]

λ0290nm

(plasm

a)0.5–

50(urine)

0.5(urine)

λ0364nm

(urine)

SPA

HPLC

UV

Pharm

aceutical

form

ulations

No

Shim-packCLC-

ODS(5

μm)

Isocratic

r.t.

5%acetic

acid/m

ethanol/

acetonitrile

(70:15:5,

v/v/v)

1.0

1010–40

NR

1999

[128]

λ0292nm

Raw

material

CLI

Stereospecific

UV

Hum

anplasma

PD

138312

CrownpackCR(+)

column(5

μm)

Isocratic

35°C

Water,0.1mM

decylamine/methanol

(pH

2,perchloric

acid)

(88:12,v/v)

1.0

750.04

–2.5

0.04

1998

[117]

HPLC

λ0340nm

HPLC

PD

118012

BDS-H

yoersil

C18(5

μm)

Isocratic

25°C

Acetonitrile/0.05M

citric

acid,1.15

mM

TBA

hydroxide,0.1%

ammonium

perchlorate

(pH

4)(80:20,v/v)

1.0

120.025–10.0

0.025

SPA

HPTLC

FL

Hum

anplasma

No

Silica

gel60

F254

plates

NR

Chloroform/m

ethanol/

form

icacid/water

(35:4:2:0.25,v/v/v/v)

NA

NA

0.1–

0.8

0.05

1998

[118]

λ em0365nm

CIP

HPLC

UV

Growth

media

SPA

Alltech

Adsorbosphere

HSC18(7

μm)

Isocratic

25°C

Phosphate

buffer,0.2%

TEA,0

.2%

SDS(pH

3)/

acetonitrile

(65:35,v/v)

1.75

NR

0.1–

20.0

0.1

1998

[69]

OFL

λ0280nm

Mueller–Hinton

broth

SPA

SPA

CIP

MOX

HPLC

FL

Hum

anplasma

OFL(saliva)

Nucleosil100C18

Gradient50°C

0.01

MTBA

sulphate,

0.05

Msodium

dihydrogen

phosphate

(pH

3)/acetonitrile

1.0–

1.3

100.005–1.50

(plasm

a)0.0025

1997

[119]

λ ex/em0296/504nm

Urine

On-columnfocusing

(5μm

)0.0025–1.00

(urine)

Saliva

0.010–1.00

(saliva)

LEV

Stereospecific

HPLC

UV

Hum

anplasma

CIP

InertsilODS-2

(5μm

)Isocratic

35°C

5mM

copper(II)sulphate

pentahydrate,10

mM

L-isoleucine/methanol

(87.5:12.5,v

/v)

1.0

150.08

–5.18

(plasm

a)NR

1997

[120]

λ0330nm

Urine

23–1,464(urine)

TEM

Stereospecific

UV

Hum

anserum

No

ZorbaxSil(5

μm)

Isocratic

r.t.

Hexane/methylacetate/

methanol/ammonia

water

(150:100:10:1)

0.8

NR

0.0–

5.00

(serum

)NR

1996

[121]

HPLC

λ0280nm

Urine

0.0–

0.50

(urine)

Ovomucoid-

aminopropyl

silicagel

0.02

Mphosphatebuffer

pH7.0/acetonitrile

(92:8,

v/v)

1.0


Tab

le5

(con

tinued)

FQs

Technique

Detectio

n(m

/z;nm

)Sam

ple

ISColum

nElutio

nMobile

phase

Flow

rate

(mL/m

in)

Run

time

(min)

Calibratio

nrange

(μg/mL,μg

/g)

LLOQ

(μg/mL,μg

/g)

Year

Ref.

TRO

HPLC

UV

Hum

anserum

Methyl-TRO

Nova-Pack

C18(4

μm)

Isocratic

r.t.

0.04

Mphosphoricacid/

acetonitrile/TBA

hydroxide/0.005M

dibutylaminephosphate

(pH3)

(83:16.85:0.05:0.1,

v/v/v/v)

0.6(serum

)10

0.1–

200.1

1996

[60]

λ0275nm

Urine

0.7(urine)

SIT

HPLC

Photolysis-FL

Hum

anserum

DX-9484

InertsilODS-2

(5μm

)Isocratic

25°C

Tetrahydrofuran/50mM

sodium

phosphatebuffer

pH2.0/1M

ammonium

acetate(19:81:1,v/v/v)

1.0

140.004–1.880(serum

)NR

1994

[122]

λ ex/em0280/430nm

Urine

0.073–9.680(urine)

FLE

HPLC

UV

Serum

No

Radial-Pack(4μm

)Isocratic

Methanol/acetonitrile/0.4

Mcitric

acid

(3:1:10)

1.0

300.3–

10NR

1994

[138]

LEV

λ0340nm

(FLE)

SPA

λ0275nm

(LEV,

SPA

,CLI,TOS)

CLI

TOS

SPA

IEX-H

PLC

FL

Hum

anserum

OFL(plasm

a)Nucleosil100SA

(5μm

)Isocratic

r.t.

Acetonitrile/0.1M

phosphoricacid,23mM

NaO

H(pH3.82)

(75:25,v/v)

1.5

NR

0.1–

2.0(serum

)NR

1992

[59]

λ ex/em0295/525nm

Urine

1.56

–50

(urine)

Stool

0.5–

20.0

(stool)

TEM

(and

metabolites)

HPLC

FL

Plasm

aAT-57084

Adsorbosphere

HS

C18(7

μm)

Isocratic

r.t.

Acetonitrile/0.04M

H3PO4,

0.01

MNaH

2PO4,0.2%

SDS,

0.005M

N-

acetylhydroxam

icacid

(53:47,v/v)

1.5

100.05

–10.0

(plasm

a)0.01

(plasm

a)1991

[123]

λ ex/em0280/389nm

Urine

FLE

HPLC

FL(FLE)

Serum

UrineBile

No

UltrasphereC18

(5μm

)Isocratic

r.t.

5mM

TBA

brom

ide,10

mM

sodium

dihydrogen

phosphatemonohydrate/

acetonitrile

(pH

2.0)

2.0

4.0

0.0025–20.000

(serum

)0.0025

(serum

)1989

[66]

λ ex/em0277/445nm

0.050–20.000

(urine)

0.050(urine)

0.025–20.000

(bile)

0.025(bile)

(90:10,v/v)

TEM

FL(TEM)

(79:19,v/v)

0.010–20.000

(serum

)0.01

(serum

)

λ ex/em0275/450nm

0.200–20.000

(urine)

0.20

(urine)

0.100–20.000

(bile)

0.10

(bile)

TOS

FL(TOS)

(78:18,v/v)

0.020–20.000

(serum

)0.02

(serum

)

λ ex/em0265/433nm

0.500–20.000

(urine)

0.50

(urine)

0.250–20.000

(bile)

0.25

(bile)

CIP

ciprofloxacin,

CLI,clinafloxacin,

ENO

enox

acin,ENRenroflox

acin,FLEflerox

acin,FQsfluo

roqu

inolon

es,GARgareno

xacin,

GATgatifloxacin,

GEM

gemifloxacin,

GREgrepafloxacin,

ISinternal

standard,LEVlevo

flox

acin,LOM

lomefloxacin,

MARmarbo

flox

acin,MOXmox

ifloxacin,

OFLofloxacin,

OXO

oxolinic

acid,PA

Zpazuflox

acin,PEFpeflox

acin,SITsitaflox

acin,SA

Rsaraflox

acin,SPAsparflox

acin,TEM

temafloxacin,

TOStosuflox

acin,TRO

trov

afloxacin,

ULIulifloxacin,

HPLChigh

-perform

ance

liquidchromatog

raph

y,HPTLChigh

-perform

ance

thin-layer

chromatography,

IEX-H

PLC

ion-exchange

high-perform

ance

liquidchromatography,

LLOQ

lower

limitof

quantification,

Ref.references,FLfluorescence,PDAphotodiode

array,

UVultraviolet,λ

wavelength,λ

ex/emexcitatio

nandem

ission

wavelengths,B

ALbronchoalveolarlavage,S

DSsodium

dodecylsulphate,TBAtetrabutylam

monium,T

EAtriethylam

ine

116 J. Sousa et al.

SDS and its concentration were studied; the retention andresolution of the five fluoroquinolones increased with in-creasing SDS concentration from 5 to 20 mM, but this wasnot observed for the retention of cinoxacin. In addition, thesame authors found that the resolution did not improvesignificantly above SDS concentrations of 10 mM, whileretention varied from 7 to 9 min.

Moreover, the analysis of fluoroquinolones by RP-HPLCis frequently associated with band broadening and peaktailing that result in lower plate numbers and poor peakresolution, thus compromising column efficiency [139].These phenomena are probably caused by secondary inter-actions between free silanol groups of the stationary phasesupport and amine basic groups of fluoroquinolones [55,88]. In fact, although silica is a good support material for thereversed-phase stationary phase because it is able to modifythe surface silanol groups, some of these remain unreactedowing to the steric hindrance of the silane reagent [139].Because of their acidic character these free and exposedsilanol groups are likely to interact with basic analytes,namely with the amine group of fluoroquinolones. Thesesilanophilic interactions may occur through ion exchange(electrostatic interaction) or hydrogen bonding and are at theorigin of the peak tailing effect [140]. This problem may beovercome either by choosing the appropriate pH of mobilephase or by using amine additives. In the first case an acidicmobile phase decreases silanophilic interactions because inthis condition both basic groups of fluoroquinolones andfree silanol are protonated and less able to interact with eachother; as a result silanol sites are shielded from fluoroqui-nolones [139]. The role of the amine modifiers added to themobile phase is to interact with silanol sites, thus competingwith fluoroquinolones and reducing the probability of theirinteraction [55, 139]. Kim et al. [139] selected five differentfluoroquinolones with three to five nitrogen atoms, one ofthem being GEM, and investigated how to improve peakshapes by optimizing the pH of the mobile phase and byadding N,N-dimethyloctylamine (DMOA). Using a silica-based monomeric C18 stationary phase, they found that asthe DMOA concentration increased (0 to 40 mM) the peakshapes were sharpened, the peak asymmetry decreased andthe plate number increased. Additionally, it was observedthat DMOA reduced the retention time of the compounds,but at concentrations equal to or higher than 30 mM thevalues remained constant because at that concentration theamine additive had probably blocked all the silanol sites. Inorder to evaluate the tailing suppression effect of the pH ofthe mobile phase in the separation of fluoroquinolones, Kimet al. [139] used a mobile phase at pH 2 and compared theresults with those obtained for a mobile phase at pH 4, bothwithout additive; the chromatograms showed better peakshapes at pH 2. They also tested the chromatographic sep-aration of the compounds by combining the addition of

DMOA (30 mM) with the mobile phase at pH 2; as a resultthe tailing was eliminated and the best peak shapes wereachieved. Those authors concluded that an acidic mobilephase, addition of amine additives or both were good strat-egies to reduce peak tailing and improve the peak resolution.

For the determination of the new fluoroquinolones, theHPLC methods discussed in this review reveal that the useof amine additives (often referred to as ion-pair reagents orcompeting-base reagents) in the mobile phase is a commonprocedure (Table 5). In general, the mobile phases containone of two types of amines, a tertiary amine, triethylamine(TEA), or a quaternary ammonium salt, such as the tetrabu-tylammonium salt which is most frequently used. The use ofa primary amine, n-octylamine, as tailing suppressor wasonly used in two techniques [67, 103]. Liang et al. [70]studied three competing-base reagents, specifically TEA,tetrabutylammonium acetate (TBAA) and acetohydroxamicacid (AHAA); the best peak shape and resolution of LEV,ciprofloxacin, GAT, MOX, TRO and cinoxacin wereobtained with 10 mM TBAA, thus it was selected for themobile phase. For the simultaneous separation of sevenquinolones, Nemutlu et al. [63] also tested different ion-pair reagents to remove the peak tailing. They comparedTEA, tetramethylammonium bromide (TMAB) and tetrabu-tylammonium hydroxide (TBAOH) and observed that thebest chromatographic peak shapes and shorter analysis timewere attained with TMAB. Nguyen et al. [55] investigated theinfluence of tetrabutylammonium bromide (TBAB), TMABand hexadecyl-trimethylammonium chloride (HDTACl); thelater was immediately rejected because of the unreliable re-tention times and TBAB was chosen as the best amine addi-tive, providing the best peak shapes and resolution for thechromatographic separation of LEV, GATand MOX. Further-more, the concentration of TBAB in mobile phase was alsoassessed by increasing it from 2 to 10 mM; the results revealthat peak resolution did not improve for higher concentrationsand thus, 2 mM TBAB was selected for the final mobilephase. Indeed, the amount of amine added to the mobile phaseis also an important feature to be considered in the optimiza-tion step and it is related to the quality of the silica base of thestationary phase [140]. De Smet et al. [88] investigated theeffect of three concentrations of the amine additive TBACl(0.010, 0.025 and 0.05 M), and observed reduced retentiontimes and improved peak shapes of ofloxacin, ciprofloxacinandMOXwith increasing TBACl concentration; however, theresolution of the fluoroquinolones did not improve; therefore,the best retention and peak shapes were obtained with0.025 M TBACl.

Nevertheless, some disadvantages of the addition of amineadditives have been pointed out by a few authors, particularlythe preparation of mobile phase is more complex, the columnequilibration is slower, the separation of analytes is highlysensitive to temperature, the baseline drifts and there are


problems in column maintenance and incompatibility withmass spectrometry (MS) detectors [61, 87]. It should, howev-er, be kept in mind that the reduction of peak tailing isimportant not only to improve the quality of the chromato-gram but also to achieve a reproducible and reliable quantifi-cation of the components of a mixture. As mentioned abovethe peak tailing effect is inherent to silica-based columns; thesubsequent troublesome chromatographic separation of basiccompounds has stimulated the development of speciallydesigned, deactivated silica-based stationary phases, in whichsilanophilic interaction is decreased, or of alternative polymer-based stationary phases. End-capping is a technique used tocover residual silanols that remain unreacted in a bondedreversed stationary phase; it is usually carried out with smallcompounds that are able to penetrate into the silica surface andgenerate trimethylsilyl groups, blocking silanophilic interac-tion [140]. This type of deactivated silica-based stationaryphase improves the performance of the column in separatingbasic compounds; however, end-capping agents cannot blockthe free silanols perfectly and anti-tailing amines are stillneeded [141]. Liang et al. [70] used an end-capped Adsorbo-sphere HS C18 column for the separation of LEV, ciproflox-acin, GAT, MOX, TRO and cinoxacin, but peak tailing stillhad to be reduced probably because of the presence of 1% offree silanols in this commercialized column. Another factorthat could influence the peak resolution is the bonded phase inthe silica support. The most common bonded phases areoctadecyl (C18 orODS) and octyl (C8) carbon chains (Table 5).Nemutlu et al. [63] studied the performance of two end-capped columns, Zorbax Eclipse XDB-C18 and ZorbaxEclipse XDB-C8, and selected the C18 column that offersbetter chromatographic peak shapes and resolution. In fact,C8 columns are less sterically hindered at the silica surfacethan C18 columns and consequently the basic compoundsinteract more easily with silanol groups, which might explainthe difference found between the two columns in the peaktailing effect [87]. Polymer-modified silicas have also beendesigned to deactivate silanol sites and, therefore, producesymmetrical peaks of basic compounds. Kim et al. [139]evaluated the chromatographic separation of five fluoroquino-lones in a polymer-coated silica-based stationary phase, theCapcell-pack C18 column. The packing material of the columnconsists of silica particles covered with a monolayer of siliconepolymer that protects the silanol groups against secondaryinteractions, thereby eliminating peak tailing effects. Someauthors defend the use of Supelcosil ABZ+columns as theyprovide a high level of silanol deactivation and selectivity overthe conventional or other deactivated C18 reversed-phase col-umns [104, 113]. This column has amide groups within thebonded phase (C18) that repel charged compounds (like pro-tonated fluoroquinolones), thereby protecting them from sila-nophilic interaction; additional shielding was also attributed tothe hydration layer of the silica surface. The chromatograms

obtained by Lemoine et al. [113] and Djabarouti et al. [104]showed better peak shapes with the Supelcosil ABZ+column.Nguyen et al. [55] tested two silica-based deactivated columns(Kromasil C18 and Supelcosil ABZ+) and one polymer-basedcolumn (Asahipack ODP50) to separate three fluoroquino-lones. The latter column has a conventional C18 chain bondedonto a porous polymer of polyvinyl alcohol, offering a verysimilar selectivity to a silica-based C18 column with no silanolsites. Although not using this alternative packing material, theSupelcosil ABZ+column gave the best peak shapes andefficiencies.

In line with the continuous improvement of column tech-nology, a recent study on the chromatographic behaviour ofLEV, ciprofloxacin and MOX on three new types ofreversed-phase stationary phases was reported by Chamseddinand Jira [142]. The separation of these three fluoroquinoloneswas first performed on a sub-3-μm column (Thermo BDSHypersil® C18); this shorter column packed with 2.4-μmparticles proved to be as efficient as longer columns packedwith 5-μm particles, enabling faster analysis and solventsaving. Then, a comparative study of three other differentcalixarene-bonded stationary phases was also carried out.Calixarenes are macrocyclic molecules composed of phenolicunits, chemically bonded to the silica surface, which providespecial retention characteristics and selectivity. Besidesbeing used as reversed phases, they support additional inter-actions (e.g. π–π interactions) compared with conventionalalkyl silica-bonded phases. Chamseddin and Jira [142]also investigated the effect of the ring size of the threeused calixarene-bonded phases and found that the sepa-ration of LEV, ciprofloxacin and MOX was achieved ona calix[4]arene and calix[6]arene phases, but no separa-tion between LEV and ciprofloxacin was possible on acalix[8]arene column. This could be due to strongerπ–π interactions in smaller calixarenes. Finally, thesame authors evaluated the chromatographic behaviourof the aforementioned fluoroquinolones on a monolithicstationary phase. This type of column consists of highlyporous rods of silica forming a dense network of macro- andmesopores, a structure that allows high flow rates with lowpressures and without loss of resolution. The three compoundswere separated within 4 min at flow rate 5 mL/min [142].

The scope of conventional HPLC techniques was recentlyextended with the development of ultra-performance liquidchromatography (UPLC) which uses smaller particles (sub-2 μm) as column packing material and ultra-high pressures(maximum of 15,000 psi). This technique allows a reduction ofanalysis time and improvement of resolution and sensitivity[73]. The small-sized particles of the UPLC technology providenarrower peaks, increasing the theoretical plate number andsubsequently resolution; because the height of narrower peaksis higher, an increase in sensitivity is also obtained. For thequantitative analysis of SPA in bulk drug and pharmaceutical

118 J. Sousa et al.

formulations, Gupta et al. [73] developed a stability-indicatingUPLC method and compared it with a conventional HPLCmethod. The performance parameters determined for bothchromatographic methods show that elution time of SPA wasreduced tenfold in UPLC and theoretical plates were threefoldhigher than in HPLC technology. Moreover, a reduced tailingfactor and better separation efficiency were achieved in UPLCanalysis as compared with HPLC. In the work by Gupta et al.[73] and Jain et al. [85] the suitability of the proposed UPLCmethod was analysed in terms of retention time, tailing factorand/or theoretical plate counts and the results were within theaccepted limits. From their results it is easy to understand thatUPLC is a technology that provides faster analysis withoutsacrificing resolution and sensitivity, thus gaining considerableinterest in the pharmaceutical and biomedical fields. Indeed, thedevelopment of liquid chromatography methods is commonlyguided by the requirements of selectivity, linearity, sensitivityand reproducibility; however, a method permitting a simple andrapid procedure is also a valuable factor for clinical routineanalyses and pharmacokinetic studies, in view of the largenumber of samples that usually need to be tested. In thiscontext, the development of new methods employing UPLCtechnology has emerged with success in the bioanalytical field.

On the other hand, direct injection of biological sampleswould eliminate extensive and time-consuming pre-treatment steps usually required prior to HPLC analysis,but it causes clogging and deterioration of conventionalRP-HPLC columns [99]. Thus, to avoid these problemsnew strategies have been described in the literature. Micellarliquid chromatography (MLC) was developed by Rambla-Alegre et al. [89] for the determination of five fluoroquino-lones in urine samples. This technique is an RP-HPLCprocedure with a micellar mobile phase that enables thedirect injection of diluted urine samples into the chromato-graphic system without any further pre-treatment steps. Themicellar media of the mobile phase provided by the SDSreagent solubilizes the proteins of the matrix which arewashed away and elute with the solvent front. Anotherinteresting strategy is the fully automated HPLC methodproposed by Nguyen et al. [55] for the determination ofthree fluoroquinolones by direct injection of human serumwithout any prior sample clean-up. In this work clean-upand chromatographic separation were performed by acolumn-switching procedure which allowed the on-line de-termination of the drugs in serum samples, improving theefficiency of analysis for a large number of samples. Thecolumn-switching technique involves two different columnsoperating in two sequential steps. In the first step serumsamples are directly injected onto the pre-column; the ana-lytes are firmly retained in this column while the biologicalmatrix is drained away by the eluent to the waste. In thesecond step, the analytes are transferred to the analyticalcolumn to be separated and quantified. Another strategy

consists in the use of a RAM column as reported byLaban-Djurdjević et al. [99] for the determination of MOXby direct injection of human plasma samples. The RAMcolumn used in this method is a shielded hydrophobic phase(SHP) column. The stationary phase is a hydrophobic bond-ed phase of phenyl groups coated with a hydrophilic net-work of bonded polyethylene oxide. The hydrophobic phaseacts as a conventional phase partitioning, while the hydro-philic polymer, due to its pore size, excludes large mole-cules such as proteins [143]. When plasma samples areinjected, the proteins are size-excluded by the outer hydro-philic polymer and flushed off with the solvent front; theanalytes are smaller and can penetrate through to the innerhydrophobic phase where they are retained and separated.The advantages of direct injection HPLC methods are evi-dent and include simple sample preparation, shorter analysistime, low cost of analysis and good recovery of analytes [99,143]. However, MLC offers poor resolution; this is thereason why Rambla-Alegre et al. [89] had to use two dif-ferent sets of analytical conditions to analyse the fluoroqui-nolones of two different groups because the simultaneousseparation of all five fluoroquinolones was not possible. Thecolumn-switching technique requires special and additionalequipment for the analysis so is not feasible in all laborato-ries. Although the RAM column technique seems to be thesimplest one, SHP column procedures have shown a lack ofsensitivity for analysing low blood levels of drugs as shownby the high value of the limit of quantification of MOX(3.0 μg/mL) reported by Laban-Djurdjević et al. [99].

The chemical structure of most fluoroquinolones has oneor two chiral centres (Fig. 2). As a consequence, thesecompounds may exist as racemates, enantiomers or diaster-eoisomers [144]. Enantiomers of chiral drugs often exhibitdifferent pharmacokinetic and pharmacodynamic propertiesthat may induce different clinical responses. This has increasedthe concern about drug stereochemistry; as a consequence,detailed studies not only on the racemate but also on eachenantiomer are nowadays required by drug regulatory author-ities for approval of any new chiral drug [145]. Therefore, theneed has arisen to develop analytical techniques for the enan-tiomeric separation and quantification, to support pharmacoki-netic and pharmacodynamic studies and quality controlprocedures for determination of chiral purity of drugs. It is wellknown that enantiomers typically only differ in the spatialarrangement of the same atoms around the chiral centre; hence,most of their physicochemical properties are identical. Conse-quently, their differentiation and, in particular, chromatographicseparation are not possible in achiral environments. SomeHPLC methods have been developed to introduce chirality inthe process either directly using a chiral stationary phase (CSP)or indirectly by derivatization of analytes with a chiral reagentor addition of chiral additives to the mobile phase [121]. In thederivatization strategy the enantiomers are derivatized before


chromatography using a pure chiral reagent to form a mixtureof diastereoisomers with distinct physicochemical propertiesallowing separation in a conventional achiral HPLC column.Taking advantage of the carboxyl group of the fluoroquino-lones, some authors have used chiral amines to form diaster-eoisomers, after activation of that carboxylic function [144].Lehr and Damn [146] describe a chromatographic method forthe determination of (R)-ofloxacin and (S)-ofloxacin (usuallynamed as LEV, and 8 to 128 times more potent in vitro than theR-enantiomer) by coupling L-leucinamide to the enantiomersvia diphenylphosphinyl chloride activation. A similar tech-nique was developed by Machida et al. [147] to obtain the L-valinamide diastereoisomeric derivative of GAT. As an alter-native to the derivatization of the carboxyl group, the aminefunction was shown to be more effective in some fluoroquino-lones because the distance to the chiral centre of the enan-tiomers is shorter. Matsuoka et al. [121] investigated theseparation of TEM enantiomers by coupling the chiral reagent(S)-(−)-N-1-(2-naphthylsulphonyl)-2-pyrrolidine carbonyl-chloride (L-NSCP) to their piperazine group. The other indirectstrategy to quantify separately each enantiomer involves theuse of a chiral ligand and a suitable transition metal ion in themobile phase [144]. These chiral mobile phase additives inter-act with enantiomers during their passage through the columnby an exchange mechanism, forming diastereomeric com-plexes that can be easily separated on conventional reversed-phase columns [120, 134, 144]. Chiral ligand-exchange chro-matography is applied to compounds which possess electron-donating heteroatoms or π-electron-donating double bonds,like fluoroquinolones, to be incorporated into the opticallyactive metal ion coordination sphere. In general, the metal ionsmost commonly used are bivalent copper cations because of therapid formation and excellent stability of their diastereoisomerscomplexes [120, 144]. Wong et al. [120] reported a stereospe-cific method for the determination of LEV in human plasmaand urine using copper ions and L-isoleucine. These additiveswere also selected by Liu et al. [148] for the separation of ULIenantiomers in quality control tests. Yan et al. [134] developedand discussed a ligand-exchange chromatography method forchiral separation and quantification of LEV and its R-enantio-mer to control the chiral purity of the drug. They added thechiral ligand L-leucine and the metal ion, copper (II), to themobile phase. In this phase they form a coordination complexthat combines with the enantiomers through their carboxylicand keto groups, yielding two ternary diastereomeric com-plexes, which have different configurations. These complexesbind to the achiral stationary phase to generate a chemicalequilibrium. The difference in the stability of those diastereo-meric complexes affects the equilibrium leading to distinctivechromatographic behaviours. In general, the trans-complex ismore stable than the cis-complex and thus the latter is elutedearlier [134, 137]. As a result, the enantiomers can be separatedon a conventional HPLC column. Yan et al. [134] also studied

the effects of ligands, organic modifier, pH of mobile phase andtemperature on the chiral separation. They found that at a pH ofmobile phase lower than 3.5 enantioselectivity would hardly bedetected, and when the pH value exceeded 5.0 the copper ionwould precipitate and block the chromatographic system. Inaddition, at high temperatures, the adsorption of the ternarycomplexes to the stationary phase decreases as they migratefaster along the column. Therefore, low temperatures willcontribute to a better enantiomeric separation. Similar studieswere conducted by Zhang et al. [137] for the chiral separationof PAZ enantiomers, the S-enantiomer being much more activethan the R-enantiomer. Amongst other factors, the effects of L-phenylalanine, L-valine and L-alanine as chiral ligand agentswere investigated and L-phenylalanine showed the best enan-tiomeric resolution. This was tentatively attributed to the pres-ence of a large group (phenylmethyl group) on the α-carbon ofthe ligand, leading to an enhanced space-excluding steric func-tion and to an increased lipophilicity of the diastereomericcomplexes.

The direct approach corresponds to the chromatographicresolution of enantiomers on a CSP. There are a large varietyof distinct CSPs, but fluoroquinolone enantiomers have onlybeen separated on cellulose, protein or crown ether-basedcolumns [144]. HPLC methods using a crown ether-basedCSP were successfully developed for fluoroquinolones con-taining a primary amino functional group [149]. For exam-ple, Brodfuehrer et al. [117] and Lee et al. [150] used acommercially available crown ether-coated Crownpack CR(+) column for the separation of CLI and GEM mesylateenantiomers, respectively. However, the resolved enan-tiomers have long retention times in both methods, makingthem unsuitable in terms of analytical run times. Moreover,because Crownpack CR (+) column is prepared by dynamiccoating of a chiral crown ether on silica gel surface, it isrecommended not to use a mobile phase with more than15% of methanol to avoid deterioration of the CSP; thus,reducing retention times by increasing the proportion ofmethanol is a useless endeavour [149–151]. To overcomethis problem, another crown ether CSP was developed in-house by covalently bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to a silica gel surface [151, 152]. Thistype of chiral column was more effective in the enantiomer-ic separation of GEM mesylate and its analogues, enablingthe use of a higher percentage (80%) of methanol. Themethod proposed by Hyun et al. [151] has revealed anexcellent resolution of GEM mesylate enantiomers within15 min, whereas on a Crownpack CR (+) column more than1 h was required in the method reported by Lee et al. [150].The mechanism of chiral recognition on crown ether CSPsis not completely understood. It has been reported that theinteraction between the primary ammonium ion of the ana-lyte and the oxygen atoms of the crown ether of the CSPleads to the formation of a transient diastereomic complex

120 J. Sousa et al.

which is essential for the enantioselectivity of this type ofchiral chromatography. Consequently, acidic modifiersadded to the mobile phase protonate the primary aminogroup of fluoroquinolones, enhancing their complexationinside the chiral cavity of the crown ether ring of the CSPand therefore contributing to a more significant discrimina-tion between the enantiomers [144, 150–152]. Grellet et al.[144] concluded that this mechanism of chiral recognitionmight explain the better separation of enantiomers whichhave the primary amino group adjacent to the chiral centre.Some protein-based CSPs have also been used for the enan-tioseparation of fluoroquinolones. This type of CSP in com-parison to the crown ether CSP has the advantage ofallowing the resolution of a wider variety of analytes andthe use of an aqueous mobile phase as in RP-HPLC [144].Lehr and Damn [146] developed a method to quantify (R)-ofloxacin and its S-enantiomer (LEV) in biologic fluids byusing a CSP with bovine serum albumin immobilized onsilica gel. Another protein-based column consisted of anovomucoid conjugated to aminopropyl silica gel and itwas used for the separation of racemic TEM in serum andurine, reported by Matsuoka et al. [121]. In both methodsthe chromatography was performed with a neutral or basicmobile phase. For the first time, Sun et al. [153] havedetermined ofloxacin enantiomers on a cellulose-basedCSP. In conclusion, several techniques for the enantiomericseparation of fluoroquinolones are still needed, according tothe differences in their chemical structure and location ofchiral centres. The chiral discrimination by the biologicalsystems is important for antibacterial activity when thechiral centre is close to the quinolone core, as in the caseof racemic ofloxacin (LEVand (R)-ofloxacin). However, formolecules with the chiral centre further away from thequinolone core the enantiomeric differences in antibacterialactivity and pharmacokinetic properties were less significantor almost non-existent; this is the case with most of thefluoroquinolones of the third and fourth generation (e.g.GAT, TEM, CLIN) [111, 144]. Despite these facts, enantio-meric studies are still needed to establish the existence ofenantiomeric differences in pharmacodynamics and pharma-cokinetics of new fluoroquinolones.

Another type of liquid chromatography found in theliterature for the determination of the new fluoroquinolonesis ion-exchange HPLC (IEX-HPLC). This method is a well-established analytical technique for the separation ofcharged molecules and has been widely used for the purifi-cation of proteins. Fluoroquinolones, like proteins, have atleast two ionizable groups, thus ion-exchange chromatogra-phy can be applied for their quantitative determination.Separation in ion-exchange chromatography is based onthe charge distribution of the analytes and their differentdegrees of interaction with the stationary phase. Chargedanalytes bind by ionic interaction with oppositely charged

ligands immobilized on the stationary phase which are inequilibrium with counter ions of the mobile phase. Theseionic interactions can be controlled by the ionic strength andpH of the mobile phase. For example, to optimize retentionof charged analytes in the stationary phase, the mobile phasemust have a low ionic strength (salt concentration). However,the elution of the analytes is more easily achieved at a higherionic strength which is important to obtain an adequatechromatographic run time. In addition, pH of mobilephase influences the ionizable state of the analyte andthus the overall charge of the molecule. To the best ofour knowledge, only two ion-exchange chromatographymethods have been described by Borner et al. [59, 115]for the determination of SPA and TRO, respectively, inhuman matrices (Table 5). In both methods, separationwas performed on a cation-exchange column with abenzenesulphonic acid on the silica surface. This func-tional group is negatively charged and is responsible forthe retention and separation of positively charged molecules.The mobile phase used in the aforementioned methods has alow pH value (3.80 and 3.60, respectively); in this acidicmedium fluoroquinolones become protonated and positivelycharged. Furthermore, sodium hydroxide was added to themobile phase to provide the counter ions necessary for theexchange between the fluoroquinolones.

Hydrophilic interaction liquid chromatography (HILIC) isa useful technique for highly polar analytes; these are poorlyretained in a reversed-phase column and require a highlyaqueous mobile phase, which causes dewetting of the station-ary phase and decreased sensitivity in electrospray ionization(ESI) mass spectrometry. HILIC uses a polar column and alow aqueous/high organic mobile phase. Ji et al. [98] reportedthe advantages of this type of HPLC coupled to MS detectionwhich will be further detailed below.

High-performance thin-layer chromatography (HPTLC)is a different chromatographic technique that can also besuccessfully applied to separate analytes in a mixture.HPTLC is a simple and economic method with a goodselectivity and sensitivity for the determination of SPA andGEM mesylate in human plasma samples as reported byMody et al. [118] and Rote and Pingle [90], respectively. Inthese methods, HPTLC was performed on a plate coatedwith a thin layer of silica gel used as the stationary phase.The mobile phase moved through the silica gel by capillaryaction under high pressure, carrying the analytes which wereseparated according to their different migration velocities.Because the diffusion time is determined by the solventfront and is the same for all the analytes, the differences inthe migration distance are used to identify and evaluate theperformance of the chromatography by calculating the re-tention factor, Rf. The mobile phase of HPTLC methodsdeveloped by Mody et al. [118] and Rote and Pingle [90]consisted of a mixture of organic and aqueous solvents,


globally apolar. In the case of the Mody et al. [118] themobile phase was chloroform/methanol/formic acid/water(35:4:2:0.25, v/v/v/v). Rote and Pingle [90] added ammoniato the mobile phase in order to obtain well-defined spotswithout a tailing effect; the final mobile phase consisted ofethyl acetate/methanol/ammonia (8:4:3, v/v/v). In both tech-niques the determination of SPA and GEM mesylate wasachieved by densitometric scanning of the spots at 365 and254 nm, respectively.

In summary, all the topics referred to in this section arerelevant contributions to the development and optimizationof chromatographic techniques for the determination offluoroquinolones. The choice of the column type and mobilephase composition depends primarily on the chemistry andproperties of the fluoroquinolones and whether they arepresent in biological samples or in pharmaceutical formula-tions and bulk drug. The difference between these two typesof matrices lies in other components that may coexist (evenafter sample preparation) in the sample with different andsometimes unexpected chromatographic behaviour, hinder-ing chromatographic separation. In biological samples suchcomponents are mainly endogenous (e.g. hormones), where-as in pharmaceutical formulations synthesis intermediates,excipients, degradation products and impurities are likely tooccur. Another difference is the amount of fluoroquinolonepresent in the sample, which is typically much lower inbiological matrices than in pharmaceutical formulations.Therefore, in the last case the carry-over effect is an impor-tant issue that must be carefully evaluated.

Detection

The selection of a methodology for the detection of compo-nents of a mixture eluted from the chromatographic columnis an important step in chromatography. Three types ofdetection systems are commonly cited in the literature forthe quantitative determination of new fluoroquinolones: UV,fluorescence and MS detectors (Table 5). Amongst thesedetection systems, the first two have been the mostfrequently used, taking advantage of the optical proper-ties of the fluoroquinolones, namely their molar UVabsorption coefficient and fluorescence efficiency [66];however, in the last 2 years MS/MS detectors have beenfrequently used (Table 5). In practical terms the selection ofthe optical detection method is based on the sensitivity(expressed by the detection and quantification limits) andselectivity required in each study.

In UV detection, the wavelength of the incident lightmust be selected in accordance with the absorption charac-teristics of the analyte; in principle, it should correspond toits maximum absorbance to enhance sensitivity. However,care should be taken to minimize the absorption of theinterfering endogenous substances, providing a more

specific detection of the analytes. For determination ofSPA, Kamberi et al. [48] observed higher signals in plasmaat a wavelength of 290 nm; in urine a wavelength of 364 nmwas chosen because the main concern was not the degree ofsensitivity but the need to reduce the interference of endog-enous substances (Table 5). The same approach was used inthe determination of GRE in plasma and urine [54]. UVdetectors can be used with gradient elution if the solventsare mostly transparent in the wavelength range of interest;this is the case with water, methanol, acetonitrile and tetra-hydrofuran, commonly used as mobile phase solvents. Be-cause the mobile phase is never totally transparent, possiblefluctuations in the mobile phase absorption may occur ow-ing to composition changes during the gradient elution. Tominimize their influence, a fixed wavelength was used as areference for the determination of four antibiotics in urinesamples by Ocaña González et al. [103]. In this work, themaximum absorbance wavelength of each compound wasselected and the absorbances were measured with respect tothe reference value.

Fluoroquinolones usually emit strong fluorescence due tothe aromatic quinolone core when irradiated with electro-magnetic radiation of specific wavelength. Fluorescencedetection is an extremely useful method because it is morespecific and sensitive than UV detection [59, 96, 115]. Theincreased specificity of fluorescence as compared with UVmethods results in sufficiently clean chromatograms andtherefore allows simple and fast sample preparation [115].Here again, the selection of the wavelength of maximumexcitation and emission for each analyte is an importantpoint in the optimization of the technique, as it gives evenhigher sensitivity for the determination of the analytes. Theexcitation and emission wavelengths selected for each chro-matographic method are indicated in Table 5. Some authorsused a time-programmed wavelength fluorescence detectorto change the wavelengths according to the eluted analyte[88]. Moreover, in a small number of articles it was reportedthat the intensity of the fluorescence emission of the analy-tes is influenced by the pH of the solution [96, 99, 109]. Itwas consistently observed that at lower pH values, thefluorescence intensity and consequently the response signalof the analyte were higher. During the development of themethod proposed by Wen et al. [96], the fluorescence spec-tra of ULI in mobile phases at different pH (2.3, 3.0, 3.8 and4.5) showed that the fluorescence intensity decreased withthe increasing of pH value at a determined excitation wave-length. For detection of SPA derivative and MOX, the maxi-mum fluorescence intensity was reached with a solution of pHof 3.0–3.4 and 3.0–5.0, respectively [99, 109]. Furthermore,Laban-Djurdjević et al. [99] used SDS as a displacing reagentand reported that its addition greatly improved the fluores-cence intensity of MOX owing to the creation of a hydropho-bic environment in which intramolecular quenching of

122 J. Sousa et al.

fluorescence is hindered. Although having the typical struc-ture of fluoroquinolones, SIT has weak fluorescence proper-ties and conventional HPLCwith fluorescence detection is notsensitive enough to determine its usual concentrations inbiological samples [122]. Aoki et al. [122] developed the firstchromatographic method for the determination of SIT in bio-logical samples (serum and urine) by means of HPLC withphotolysis-fluorescence detection. In this work, SIT elutedfrom the chromatographic column was irradiated with UVlight and the resulting photoproducts were detected by theirfluorescence intensity. It was found that photoproducts of SITemit much stronger fluorescence than the intrinsic fluores-cence of SIT. Hence, post-column photolysis improved thesensitivity of the determination of SIT by detecting its photo-products. On the other hand, the IS used in this method had nopositive influence in the fluorescence intensity by UV irradi-ation. SIT has three halogen atoms in 1-fluorocyclopropyl, 6-fluoro and 8-chloro moieties of the molecule [122], whereasthe IS has just one fluorine atom at the 6-position (character-istic of the fluoroquinolones). Therefore, it has been suggestedthat the introduction of halogens into the molecule will de-crease the fluorescence intensity. Aoki et al. [122] pointed outthat post-column derivatization of SIT by photochemical re-action enhanced the fluorescence intensity due to the elimina-tion of a chlorine atom at the 8-position or 1-fluorocyclopropylmoiety in the 1-position of the molecule. Conversely, the ISwas shown to be stable after UV irradiation; in fact the mole-cule has no halogen atom at the 1- or 8-position. Tanaka et al.[154] described the same HPLC method but with a differentpost-column photochemical reactor that consisted of two quartzflow cells. The performance of this new reactor was comparedwith that of a conventional tubular polytetrafluoroethylene(PTFE) coil reactor. It was reported that fluoride is liberatedfrom PTFE tubing when irradiated with UV, becoming morebrittle and causing, eventually, its rupture. In addition, tetrahy-drofuran in the mobile phase also reduces the durability of thePTFE reaction coil. Therefore, quartz flow cells have obviousadvantages over the PTFE reaction coil as photochemicalreactors as they ensure not only a better transparency to theincident UV radiation, but also inertness and resistance to lightand heat produced by the UV lamp and to organic solvents inthe mobile phase. Owing to the smaller volume of quartz flowcells, the application of this new reactor to the fluorescencedetection of SIT resulted in improved peak shape and shorterchromatographic run times [154]. Like SIT, SPA has a halogenatom at the 8-position of the molecule and emits weak fluores-cence. To enhance fluorescence intensity of SPA, Du et al.[109] developed a pre-column derivatization HPLC methodwith fluorescence detection for the determination of that ana-lyte in urine. In this study, SPA was oxidized by nitrous acidbefore reacting with potassium iodide to form a stronger fluo-rescent SPA derivative. After these chemical reactions thesample was injected into the chromatographic system and

SPAwas determined through its derivative. Another fluorimet-ric HPLC method based on pre-column derivatization wasdeveloped for the quantification of MOX in plasma by TatarUlu [95]. In this method, MOX is derivatized with 4-chloro-7-nitrobenzodioxazole (NBD-CI) reagent and then determinedby HPLC. Although MOX does not have poor intrinsic fluo-rescence, the purpose of this modified fluorimetric HPLCmethod was to increase the sensitivity by derivatization, allow-ing the precise and accurate quantification of the lower con-centrations of MOX in plasma.

More recently MS detection has been used as a highlysensitive and selective detection system for liquid chroma-tography analysis. The basic principle of this detectiontechnique is the measurement of the mass-to-charge (m/z)ratio of ionized molecules. Several approaches are in use toachieve the required ionization which include thermo andelectrospray ionization, particle beam and atmospheric pres-sure ionization. For the determination of pharmaceuticalcompounds for clinical and non-clinical studies, as is thecase of fluoroquinolones, electrospray ionization (ESI) wasfound to be the preferential interface between liquid chro-matography and MS [110]. It involves dispersion of theeluent into a fine aerosol and is particularly useful for largepolar compounds because it avoids their tendency to frag-ment when ionized. Positive ionization is usually the select-ed mode because of the presence of amine and ketonegroups in the chemical structure of fluoroquinolones, whichare easily protonated. The formation of positive ions isenhanced by an acidic mobile phase [110]. Electrospraytandem mass spectrometry (ESI-MS/MS), using multiplerounds of mass spectrometry with molecule fragmentationas an intermediate step, is a more specific and extensivelyused detection method. In the studies reported in the litera-ture (Table 5), fluoroquinolones were protonated by ESI toform the positively charged molecular ion, [M+H]+, namedas the parent or precursor ion. This molecular ion is oftenthe one with m/z corresponding to the most intense peak inthe positive electrospray mass spectrum and is selected forfragmentation, frequently promoted by collision-induceddissociation. Amongst the fragment ions produced, the mostprominent is finally chosen for the sensitive quantificationof the analyte. The precursor-to-product ion transitions aremonitored by multiple or selected reaction monitoring mode(MRM or SRM) [57, 62, 98, 112]. Some authors proposedthe fragmentation mechanism of fluoroquinolones by inter-preting the product ion mass spectrum obtained. In Fang etal. [84], the major fragment ion of LEV that was observedafter collision corresponds to the loss of CO2 at the carbox-ylic group of the molecule. The product ions of GAT areformed by the loss of H2O or CO2 under ESI-MS/MSconditions used by Vishwanathan et al. [110]. In the deter-mination of GEM by Robledo and Smyth [91] and Roy et al.[57], the GEM parent ion loses an H2O molecule to give the


abundant product ion, chosen for the detection monitoringof the ion transition. However, this transition was not theonly one selected for the quantification of GEM by ESI-MS/MS. Doyle et al. [112] chose another product ion; undertheir experimental conditions, an intense signal was detectedand considered to be consistent with the loss of a methoxyradical plus the loss of methanoic acid from the GEMchemical structure. In MS detection, the evaluation of thematrix effect on the ionization of analytes is an importantprocedure that must be taken into account for the validationof the method. The effect of biological matrices is deter-mined by comparing the responses of post-extraction spikedanalytes with the responses of the same analytes in puresolvent [57]. A value of 100% indicates that no matrix effectis observed, whereas a value lower than 100% reveals asuppression effect on analyte ionization by the matrix and avalue higher than 100% points to an ionization enhancement[98]. From representative MRM or SRM chromatograms, itis possible to observe that in most cases there is a narrowdifference between the retention time of the analyte and thatof the IS. This could suggest that the chromatographicmethod is not suitable the for separation and quantificationof analytes; however, MS detection in MRM or SRM modehas the ability to deconvolute signals. For example, in thestudy by Roy et al. [83], ULI and the IS (ciprofloxacin)exhibited the retention times 0.92 and 0.93 min, respective-ly; in the study by Vishwanathan et al. [110], the retentiontime of GAT was 2.15 min and for the same IS (ciproflox-acin) 2.07 min. Ji et al. [98] developed a HILIC-tandemmass spectrometry method to overcome some problemsfound between RP-HPLC and MS detection. In fact, theretention time of fluoroquinolones in RP-HPLC methodswith MS detection was found to be significantly small. Polarcompounds are, indeed, poorly retained on a reversed-phasecolumn which may result in detrimental matrix effects inMS methods with ESI. In addition, the high aqueous contentof RP-HPLC mobile phases does not favour achievement ofideal spray conditions. Therefore, HILIC on a bare-silicacolumn with high organic/low aqueous mobile phase hasbeen used for the determination of the polar LEV in humanplasma [98].

Conclusion

Liquid chromatography is a powerful analytical techniquefor separating closely related chemical species present in amixture. It is, therefore, widely used in many branches ofscience for qualitative identification and quantitative determi-nation of separated species. Several liquid chromatographicmethods for the determination of new fluoroquinolones inbiological matrices and pharmaceutical formulations havebeen discussed in the course of this review, namely RP-

HPLC, IEX-HPLC, HILIC, HPTLC and chiral methods; theirselectivity, sensitivity, accuracy and reproducibility make thema good choice for analysis. The chromatographic conditionswere discussed, in particular for RP-HPLC which is the mostcommonly used approach. It may be concluded that the mobilephases affording the best resolution and analysis time areacidic (pH below the first pKa value) and that amine additivesare essential to avoid band broadening and peak tailing, twocommon problems occurring with fluoroquinolones.

The above liquid chromatographic approaches are oftencoupled with UV, fluorescence and MS detection systems.The last two have proven to be highly sensitive and selec-tive; however, fluorescence detection is particularly relevantin the case of fluoroquinolones, a class of compounds thatemit fluorescence under irradiation; it is also easily availablein analytical laboratories, whereas MS requires more sophis-ticated and expensive instrumentation.

Sample preparation has a high impact on the quality ofdata, especially in the cleanness of chromatograms and inrecovery values. PP, LLE, SPE, ultrafiltration and dilutionare used for the extraction or sample pre-treatment of newfluoroquinolones; in addition, automated techniques havealso been developed as a good alternative, leading to lesstime-consuming procedures and increasing the reliabilityof results. To fully understand how to manipulate thefluoroquinolone samples and the optimization steps ofthe chromatographic methods, the physicochemical prop-erties of these compounds, which are governed by theiramphoteric behaviour, should be considered. The stabil-ity of fluoroquinolones must be evaluated in each case,in acidic, basic, reductive or oxidative conditions as wellas after thermal stress, to avoid degradation processes.One of the major concerns in the use of fluoroquinolonesis their photosensitivity, which is related to their chemi-cal structure.

Overall, it should be noted that a large number of liquidchromatographic methods have been reported for LEV,MOX, GAT and SPA, whereas for the remaining new fluo-roquinolones the available information is sparse.

As the last few years have witnessed a rapid developmentof new sample preparation techniques and improved chro-matographic methodologies with a great impact in bioanal-ysis, it is expected that many of the methods discussed inthis review can be adapated to the emerging trends in bio-analysis. Concerning sample preparation, miniaturizationand automation appear to be the main recent advances. Anexample of miniaturization is the development of MEPSsample treatment leading to reduction of time and solventconsumption. On the other hand, the use of smaller particlesas column packing material and ultra-high pressures(UPLC) as well as monolithic columns are examples thatmay improve the chromatographic resolution and lead tofaster analyses.

124 J. Sousa et al.

Acknowledgments This work was supported by Fundação para aCiência e a Tecnologia through the grant SFRH/BD/69378/2010.

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Analytical methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations by liquid chromatography: a review