Available online at www.sciencedirect.com

Talanta 74 (2008) 14631475

Development and optimisation of a singtwtre

uvris Anance

er 202007

Abstract

A unique ceutiby chemom the dand -blockers classes. Performing the SPE on Oasis MCX (mixed-mode cation exchange), the sequential elution of each pharmaceutical classis achievable, allowing a high purity level of extracts as well as high recovery rates. Performing a unique sample preparation results in animportant save of time. The extracts were then analysed by LC/MS/MS, using a Hibar Purospher Star column for -blockers and an X-Bridgecolumn for corticosteroids with formate buffer (pH 3.8)/AcN and water/AcN mobile phases, respectively. This work also includes a study of thechromatograapplied to enup to 318 ngeffluents. Asmethod can 2007 Else

Keywords: Ph

1. Introdu

Quantificern since trisk [14].order to peunchangedorganisms

Humantreatment pefficiency iing the pasenvironmental occurre

CorresponE-mail ad

0039-9140/$doi:10.1016/jphic and mass spectrometric parameters in order to increase the analyte signal. The optimised SPE-LC/MS/MS method was thenvironmental samples from sewage treatment plant (STP). -Blockers and corticosteroids were detected, respectively, in concentrationsL1 (sotalol) and 174 ng L1 (cortisone), in STP influents. Moreover, both pharmaceutical classes have also been detected in STPfar as we know, this is the first paper reporting the detection of corticosteroids in environmental waters. The developed analytical

be used in further studies to investigate the environmental contamination by these drugs.vier B.V. All rights reserved.

armaceuticals; Water analysis; SPE; -Blockers; Steroids; Liquid chromatography; Mass spectrometry

ction

cation of drugs in the environment is a matter of con-hey potentially represent an important environmentalIndeed, these substances have been developed in

rform a biological effect and are partially excretedby the body. Thus, they can affect non-targeted

and be harmful for the ecosystem.medications reach the environment through sewagelant (STP). Effectively it was recognised that STPs very low for this kind of micropollutants, allow-sage of these compounds and their release into thet. Several authors have reported the environmen-nce of drugs in surface-, ground- and tap-water,

ding author. Tel.: +33 4 72 43 11 52; fax: +33 4 72 44 62 02.dress: arnaud.salvador@univ-lyon1.fr (A. Salvador).

as well as in STP influents and effluents [512]. Many stud-ies report the quantification of large amounts of non-steroidalanti-inflammatory drugs (aspirin, paracetamol) [1315], antide-pressant (fluoxetine, paroxetine) [15], antibiotics (amoxicillin,sulfamethoxazole) [1517], steroid sex hormones (estrone,estriol) [1820] or cardiovascular drugs (atenolol, dilitiazem)[11,15,16,21]. -Blockers (cardiovascular drugs) have beenreported to contaminate both American and European watersin a large number of studies [13,2125], with concentrationsup to 2000 ng L1. The occurrence of steroids in environmentalwaters has been confined to the study of the estrogens subclass[1820]. Few papers report the contamination by other steroidclass molecules, such as cholestanes (cholesterol), progesta-gens (progesterone, levonorgestrel) or androgens (testosterone)[2628]. However, no environmental measurements were doneconcerning corticosteroids although they are consumed in largequantities as anti-inflammatory or antiallergenic drugs. More-over, this pharmaceutical class has been underlined as a potential

see front matter 2007 Elsevier B.V. All rights reserved..talanta.2007.09.038for the LC/MS/MS analysis ofclasses residues in sewage

Anne Piram, Arnaud Salvador , Jean-Yves GaUniversite de Lyon, Lyon 1, UMR 5180, CNRS, Laboratoire des Science

69622 Villeurbanne, FraReceived 27 April 2007; received in revised form 5 Septemb

Available online 9 October

extraction procedure leading to the separation of 2 different pharmaetric tools. From only one sampling, this analytical method allowsle extraction procedureo pharmaceuticalatment plantt, Pierre Lanteri, Rene Faure

lytiques, 43 Boulevard du 11 novembre 1918,

07; accepted 28 September 2007

cal classes molecules has been developed and optimisedetermination of 21 pharmaceuticals from corticosteroids

1464 A. Piram et al. / Talanta 74 (2008) 14631475

environmental risk by a study of prioritisation based on a mod-elisation [29].

The aimpharmaceustructurespharmaceusumption (and the infin the envicosteroid cnecessary sconcerning

The diffilinked withthey contaipersonal cafactor in thetal concentEuropean aconcentratethis sample(SPE) [52is importancontinuouscedures abenvironmeto improveThen, it is uallows theone samplielution of e

The extrmeans of standem mamental anathe mass sThis paperoptimisatio

2. Experim

2.1. Chem

Betametisol (98%)(D), fluniso(Fc), predncinolone (9acebutololride (99%)(100%) (M(99%) (Pr)(S), and tiSigmaAldfumarate (1bourne, Uwas provid

LC/MS grade (AcN), formic acid (85%) (FOA) and ammo-nium formate (99%) were obtained from Fisher Scientific (Val

il, Fed bas g

Wych cog L

ide (r sto50 m

cortird wepark.2 autionosterAcNolutiation

ampl

ironSTP

urbis, 2ngst an

raneat 4fter t

ampl

plesom W

extrasyrinrs lere.

h 4of

L)d. Tminge ped w.5 moster:0.1,.1, v

eOs weids ationof this study was to analyse simultaneously 2tical classes: -blockers and corticosteroids whoseare given in Tables 1 and 2 , respectively. Thesetical classes were selected because of their high con-e.g. 35 t year1 in France for propranolol -blocker)ormation deficiency for the corticosteroids impactronment. Analytical method development for corti-ompounds analysis in environmental samples wasince so far no methods were proposed by the literaturethis matrix (water).culty of tracking trace pollution of drugs in STP isthe complexity of environmental matrices. Indeed,

n lots of other organic pollutants such as pesticides orre products. Moreover, there is an important dilutionenvironment which results in expected environmen-

ration in the ng L1 range, as it has been found in bothnd American waters [528]. Thus, it is necessary toand purify samples before analysis. In the literature,preparation is mainly done by solid phase extraction8,3033]. The development of multiresidue methodst in environmental analysis of pharmaceuticals andefforts have been made to design new analytical pro-

le to quantify traces amounts of pharmaceuticals inntal waters. These recently developed methods aimthe total analysis time and reduce the analysis cost.seful to develop a unique extraction procedure, whichextraction of both pharmaceutical classes from onlyng, on the same SPE sorbent, with a sequential finalach class.acted sample analysis should be then carried out byensitive and selective detection tools, such as thess spectrometers, which are widely used in environ-lysis. A liquid chromatographic device is coupled topectrometer as reported in literature [528,3033].also presents a study for the LC/MS/MS parametersn in order to improve the analyte response.

ental

icals

thasone (97.8%) (Be), budesonide (99.2%) (Bu), cor-(Hy), cortisone (98.3%) (C), dexamethasone (98%)lide (98.6%) (Fs), fluocinolone acetonide (99.6%)isolone (99%) (Pl), prednisone (99%) (P), triam-8.2%) (T), triamcinolone acetonide (99.3%) (TA),hydrochloride (100%) (Ac), alprenolol hydrochlo-(Al), atenolol (99%) (At), metoprolol tartrate salt), nadolol (100%) (N), propranolol hydrochloride, pindolol (99%) (Pi), sotalol hydrochloride (99%)molol maleate salt (99.9%) (Ti) were provided byrich (Saint Quentin Fallavier, France). Bisoprolol00%) (Bi) was provided by Sequoia research (Pang-

nited Kingdom). Methanol HPLC grade (MeOH)ed by SDS-Carlo-Erba (Peypin, France). Acetonitrile

de Rueprovidwater w(High

Eac1000 macetonblockeA firstthe 11standawas prthe dar

4,by dilCorticwater/these sprepar

2.2. S

EnvBenitemainlyanalyssampliinfluenmembstoreddays a

2.3. S

Sam3 cc) frphasea 404(Villiesoftwa

Eac400L(

A.Piram

etal./T

alanta74(2008)14631475

1465

Table 1Structures and mass spectrometric parameters used for the analysis of corticosteroids

Molecule [CAS] Structure [M + H]+(m/z)

Product ion(m/z)

Orifice voltage(V)

Ring voltage(V)

Collision energy(eV)

Retention time(min)

Betamethasone (Be) [378-44-9] 393.0 373.1 41 240 13 6.3

Budesonide (Bu) [51333-22-3] 431.0 413.3 6 140 15 11.0

Hydrocortisone (Hy) [50-23-7] 362.9 327.2 31 200 29 3.8

Cortisone (C) [53-06-5] 361.0 163.1 26 160 31 4.1

Dexamethasone (D) [50-02-2] 393.0 373.1 41 240 13 6.5

1466A.Pira

met

al./Talanta

74(2008)14631475

Flunisolide (Fs) [3385-03-3] 435.0 321.1 6 150 19 7.9

Fluocinolone acetonide (Fc) [67-73-2] 453.1 413.2 11 160 17 8.7

Prednisolone (Pl) [50-24-8] 360.9 343.1 11 140 15 3.6

Prednisone (P) [50-03-2] 358.9 313.0 21 180 17 3.8

Triamcinolone (T) [124-94-7] 391.2 375.0 46 270 15 2.4

Triamcinolone acetonide (TA) [76-25-5] 435.0 415.1 6 150 15 7.6

A. Piram et al. / Talanta 74 (2008) 14631475 1467

Table 2Structures and mass spectrometric parameters used for the analysis of -blockersMolecule [CAS] Structure [M + H]+

(m/z)Production (m/z)

Orificevoltage (V)

Ringvoltage (V)

Collisionenergy (eV)

Retentiontime (min)

Acebutolol (Ac) [37517-30-9] 337.0 116.2 21 150 29 7.9

Alprenolol (Al) [13655-52-2] 249.9 116.2 16 140 23 9.2

Atenolol (At) [29122-68-7] 267.0 145.1 21 150 33 3.8

Bisoprolol (Bi) [66722-44-9] 326.1 116.2 21 140 25 8.7

Metoprolol (M) [51384-51-1] 268.0 116.3 21 150 23 8.1

Nadolol (N) [42200-33-9] 310.1 254.1 21 140 23 6.6

Pindolol (Pi) [13523-86-9] 248.9 116.2 16 130 23 7.5

Propranolol (Pr) [525-66-6] 259.9 116.3 16 140 23 9.1

Sotalol (S) [3930-20-9] 272.9 255.1 6 120 17 3.9

Timolol (Ti) [26-839-75-8] 317.0 261.1 16 140 23 7.8

1468 A. Piram et al. / Talanta 74 (2008) 14631475

Both eluates were evaporated to dryness in a Speed Vac fromSavant (Ramsey, Minnesota, USA). The dry residues were dis-solved in 400L of a mixture AcN/water (25:75, v/v). Analiquot of 100L was then injected in the LC/MS/MS device.

2.4. Liquid chromatographytandem mass spectrometry

The HPLC device consisted of Agilent 1100 series pump andautosampler (Massy, France). As corticosteroids and -blockerspossess very different physicochemical properties (regardingtheir hydrophobicity or chemical functions), they could notbe analysed on a same chromatographic column. -Blockerswere chromatographied on a 5m Hibar Purospher Star column(250 mm 4.6 mm I.D.) from Merck (Lyon, France), operat-ing at 1 mtwo solvenmate acidi(Table 3a).C18 X-Bridoperating ausing twowas AcN (performed

The HPquadrupoleCanada) eqing in posiprocessingsoftware. Tpolypropylcity, USA)in the rang

The nebwere set at400 C forauxiliary gwas set at 5sitions diffedetermined20 points. Tteroids andparametersTable 2 forthe autotun

ing each molecule separately. Product ions used for monitoringwere selected based on their significance in the MS/MS spectra.For this study only one MRM (Multiple Reaction Monitoring)transition was used although a second transition is generallyrequired to confirm the presence of an analyte by using the ratioMRM1/MRM2. As the mass spectrometer used in this study didnot permit to work with large number of MRM transitions, itwas not possible to analyse a large number of compounds withtwo MRM transitions. So we decided to use only one transi-tion. During method development we noticed that when a peakwas observed on STP sample extracts, at the required retentiontime, with the first MRM transition, the presence of the ana-lyte was always confirmed by the second MRM transition. Thismethodology was considered as acceptable for method devel-opment. However, consecutive routine analysis will require an

satioo incnsitiEC.

ults

iquidis

demntal aallymet

race

quireant apmee stitranroidssor/pimilmpouth ditalkm/z

rolol(pr

Table 3HPLC solven

(a)Total time (m ime (

025

10a A1 = formb B = can.c A2 = ultraL min1. A gradient elution was performed usingts: A1 was formate buffer (10 mM ammonium for-fied to pH 3.8 with FOA) and solvent B was AcNCorticosteroids were chromatographied on a 3.5mge column (150 mm 2.1 mm I.D.) from Waters,t 0.3 mL min1. A gradient elution was performedsolvents: A2 was ultra-pure water and solvent BTable 3b). Both chromatographic separations wereat room temperature.LC device was coupled to a Sciex API 300 triple

mass spectrometer from MDS Sciex (Toronto,uipped with a TurboIonspray Source (TIS) operat-

tive ion mode. Instrument control, data analysis andwere performed using the associated Analyst 1.4.1he mass spectrometer was initially calibrated usingene glycol as standard (Applied Biosystems, Foster, setting the resolution, as peak width at half height,e 0.7 0.1 amu.ulizer (zero air) and the curtain gas flow (nitrogen)10 arbitrary units. The TIS source was operating at

corticosteroids and at 500 C for -blockers, with theas flow (zero air) set at 8 L min1. The TIS voltage000 V. As both peak width and number of MRM tran-red in these two analytical methods, dwell times werein order to define chromatographic peaks with abouthen, the dwell time was 100 and 50 ms for corticos--blockers, respectively. Other mass spectrometricare summarised in Table 1 for corticosteroids and-blockers. These parameters were optimised usinge feature of the Analyst 1.4.1 software, by infus-

organiorder ttwo tra96/23/

3. Res

3.1. Lanalys

Tanronme

especispectroraphynot reimportdevelomay bMRMticosteprecurduce s(3) cobut wia cross

with ametopteroids

t gradient for the separation of (a) -blockers and (b) corticosteroids(b)

in) Flow rate (mL min1) %A1a %Bb Total t1 85 15 01 85 15 61 55 45 91 55 45 20

ate buffer.

-pure water.n of MRM transitions by retention time windows, inrease the number of followed MRM transitions up toons per analyte, as lauded by the European directive

and discussion

chromatographytandem mass spectrometry

mass spectrometry is the best detection tool for envi-nalysis because of its high sensitivity and selectivity,

in the MRM mode. Moreover, operating the masser in the MRM mode allows a shorter chromatog-since a perfect separation of all the compounds isd using this acquisition mode. Nevertheless, somespects have to be considered in multiresidue methodnt and in some cases, chromatographic separationll required: (1) compounds measured at the samesition (e.g. betamethasone and dexamethasone cor-

which are diastereroisomers and have the sameroduct ion transition); (2) compounds which pro-ar (overlapping) product ions upon fragmentation;nds that are measured using the same product ion

fferent precursor ions, in order to avoid the risk ofphenomenon, which is the case of most -blockers

116 product ion (acebutolol, alprenolol, bisoprolol,, pindolol and propranolol) and some corticos-ednisolone/cortisone and budesonide/fluocinolone

min) Flow rate (mL min1) %A2c %Bb

0.3 70 300.3 60 400.3 0 1000.3 0 100

A. Piram et al. / Talanta 74 (2008) 14631475 1469

Fig. 1. Mass e) andand sotalol (S

acetoniderespectivel

The coumass spectchromatog

Thus, itration capaenrichmentpreparationcation limicompositiospectrometphases abletrometer iosalts are suthe sourcemethod senby studyinthe analytedetect enviwas reporte

Chromato facilitatespectrometAnalysis morganics maqueous phof FOA, fowith FOAaddition uptative comcorticostero-blockers

For cortmixtures ofwith AcN.was perform

omaeparaxam

reso

res ainte

moleatogduehe cavehromimp

n tesgh ubec

c sepatog

usespectrometric responses of 2 representatives corticosteroids: betamethasone (B).

have a similar product ion at m/z 343 and 413,y).ples of molecules quoted above have to reach the

rometer separately, and to be previously separated byraphy.

is important to develop a chromatographic sepa-ble of separating these critical pairs. Although anfactor of 1000 can be expected from the sample

, each parameter susceptible to lower the quantifi-ts is important to consider, such as mobile phasen. Then, it is also interesting to improve the massric sensitivity by using chromatographic mobileto increase the analyte ionization in the mass spec-

n source. Indeed, the organic solvent or the addedsceptible to interfere with the ionization process inof the mass spectrometer and affect the analyticalsitivity. So the quantification limits were lowered

g the influence of the mobile phase composition ons mass spectrometric response and it is possible to

1

ing chrwere s

and defactorystructusimilarThesechrompeaks,

In tvent gwith ceratedcolumAlthouchosengraphichromwas toronmental concentration in the ng L range, as itd in literature [528,3033].tographic mobile phases were then chosen in order

the analyte ionization in the source of the masser. Each compound was analysed in Flow Injectionode (FIA) by testing different combinations of hydro-ixtures. MeOH and AcN were tested with differentases: ultra-pure water, acidified water with 0.1%rmate buffer (10 mM ammonium formate acidifiedto pH 3.8) and basified water (ammonia solutionto pH 11). The analyte responses of 4 represen-

pounds of both families are presented in Fig. 1. (2ids: betamethasone and cortisone, in white; and 2

: propranolol and sotalol, in grey.)icosteroids the best responses were observed withunmodified water with MeOH or unmodified water

Therefore, the chromatographic gradient separationed with water/AcN mixtures (Table 3b). The result-

water (0.1%(Table 3a) wa satisfacto

3.2. Solid

Solid phment factoas flow ratin each sterately resultime consuence of seveand optimisallow the evof experimabout the reinformationcortisone (C), and 2 representatives -blockers: propranolol (Pr)

togram is shown in Fig. 2. The critical analyte couplested within less than 12 min, excepted betamethasone

ethasone. Indeed it was not possible to achieve a satis-lution for these two compounds since their chemicalre very narrow (diastereoisomers), which results inractions with the chromatographic stationary phase.cules will subsequently be quantified together. The

raphy of budesonide resulted in two chromatographicto the presence of two epimers.ase of -blockers, the use of MeOH as organic sol-better responses. However, this solvent did not fitatographic separation criteria. Indeed, MeOH gen-ortant peak tailing whatever the chromatographicted. Thus, AcN should be used as organic constituent.nmodified water gave the best results, it has not beenause of the risk of poor robustness of the chromato-aration. Finally, the best compromise between a goodraphic separation and a satisfactory analyte responsea mixture of formate buffer and AcN or acidifiedFOA) and AcN. A formate buffer/AcN gradientas selected. This mobile phase composition allowed

ry separation in less than 10 min as shown in Fig. 3.

phase extraction

ase extraction efficiency (purification and enrich-r) is linked to a large number of parameters suches, composition, and elution solvent volumes usedp of the procedure. Studying each parameter sepa-ts in a high number of experiments, which should beming. Therefore, evaluating simultaneously the influ-ral parameters is interesting for method developmentation. Here is the advantage of chemometrics, whichaluation of several parameters by a restricted number

ents. This approach first requires a small knowledgetention of target molecules on the SPE support. Thiscan be obtained from a preliminary study.

1470 A. Piram et al. / Talanta 74 (2008) 14631475

Corticosare basic mproperties,meric mixeselected: Osorbent isteroids) andas -block

The evocompositioelution solvmixtures, cof the moltion of theorder to rinless retaineused to eluto have a scorticosteroin acidifieda second wIndeed, acitheir acidicFig. 2. LC/MS/MS chromatogram of a 5 ng injected corticosteroids so

teroids are non-polar molecules whereas -blockersolecules with pKas around 9. According to these

in order to have a single sample preparation, a poly-d-mode cation exchange reverse phase sorbent wasasis MCX. Indeed, with apolar interactions MCXable to retain apolar molecules (such as corticos-with ionic interactions they can retain cations (such

ers when protonated).lution of the extraction yield of target analyte with then of the eluant mixture was evaluated. The volume ofent used was fixed to 1 mL. In acidified MeOH/water

orticosteroids extraction yield varied with the polarityecules. It has been concluded that (1) the composi-eluant mixture should be less than 20% MeOH inse the cartridge without eluting triamcinolone, thed corticosteroid and (2) the percentage of MeOH

te corticosteroids has to be higher than 60% in orderatisfactory elution of budesonide, the most retainedid. Moreover, no elution of-blockers was observedmixtures. These results were interesting as it allowedash of the cartridge with 100% of acidified MeOH.dic pH condition maintains these molecules understructures, thus, they keep fixed on ion exchange

sites, whereonly subjecfrom the cpindolol w

These pand -blocples withsame sampis firstly puwas washe

Previoutake care ovolume loaefficiency,An improvthen requir

To imprformed on Pscenario siniest matrixproblemati

The ninrate used tolution (see abbreviations in Table 1).

as in basic media,-blockers become neutral and areted to apolar interactions. To remove all -blockersartridge, 95% of basified MeOH was necessary asas not eluted with lower MeOH percentages.reliminary experiments showed that corticosteroidskers could be extracted from environmental sam-

a unique solid phase procedure. Indeed, from thele loading on the cartridge, corticosteroids fractionrified then eluted. And secondly, -blockers fractiond then eluted on this same cartridge.s results can be strongly improved. Indeed, it does notf important parameters such as the influence of theded into the cartridge, nor evaluate the purificationwhich could be evaluated by measuring matrix effect.ement of the method on environmental samples wased.ove the SPE protocol, all the experiments were per-ierre Benite STP influents in order to cover the worstce untreated STP waters are expected to be the dirt-

found in the environment and consequently the mostc.e following parameters were firstly studied: the flowpercolate the sample on the cartridge, the sample vol-

A. Piram et al. / Talanta 74 (2008) 14631475 1471

ume loadedcorticosterocentage forvolume useume used tThis study,to fix 5 crit15 mL minanalytes noorder to shume percoaffect the eto 400 mLuntil 4 chrof the volu(15 mL) dwas maintaThis washfrom the cafor-blockwere obtain28% (95:5,(98:295:5purificationFig. 3. LC/MS/MS chromatogram of a 100 pg -blocker solution

, the eluant volume and methanol percentage for theids wash step, the eluant volume and methanol per-the corticosteroids elution step, the acidified MeOHd to wash the -blockers fraction, the eluant vol-

o eluate -blockers and its percentage of ammonia.which would not be detailed in this paper, allowed

ical parameters. The flow rate for loading (from 5 to1) did not influence neither the retention of targetr interferences and was then fixed to 15 mL min1 inorter the sample preparation time. The sample vol-lated was studied from 100 to 1000 mL and did notxtraction yield of target molecules. It was then fixed(leading to a 400L extract) in order to performomatographic injections per sample. The increaseme percolated to clean up the -blockers fractionid not increase the purification, and subsequently itined to 1 mL of MeOH acidified with 0.1% FOA.permits to eluate the remaining apolar interferentsrtridge, allowing to achieve a high purification statusers extracts. The best extraction yields for-blockersed by eluting with 1.5 mL MeOH/ammonia solutionv/v), when 0.51.5 mL of MeOH/ammonia solutions, v/v) were tested. These conditions gave satisfactorys.

The 4 rthanks to aant volumebetween 0.tion (called+1). The e(called X3)+1); its com70% MeOH

Table 422 factorial de

Exp.

12345678(see abbreviations in Table 2).

emaining parameters were studied more precisely,screening design (as shown in Table 4). The elu-for the corticosteroids wash step (called X1) varied

5 mL (level 1) and 1.5 mL (level +1); its composi-X2) was 10% MeOH (level 1) or 15% MeOH (levelluant mixture volume used to elute corticosteroidsvaried between 0.5 mL (level 1) and 1.5 mL (levelposition (called X4) was 65% MeOH (level 1) or(level +1).

sign of the experiment

Step

1st wash 1st elution

V (mL) %MeOH V (mL) %MeOH0.5 10 0.5 651.5 10 1.5 650.5 15 1.5 651.5 15 0.5 650.5 10 1.5 701.5 10 0.5 700.5 15 0.5 701.5 15 1.5 70

1472 A. Piram et al. / Talanta 74 (2008) 14631475

Table 5Corticosteroids extraction yields measured for the design of experiments (%)Exp. Pl T Fc Be-D C P Hy Bu Fs TA

1 6 5 5 3 13 7 8 2 8 62 82 70 70 66 84 74 82 38 52 593 16 13 8 15 23 16 19 10 10 144 1 1 1 1 7 1 1 6 3 45 79 75 91 81 79 86 92 129 81 926 74 69 86 73 84 77 73 52 90 907 7 5 12 6 10 6 8 18 7 58 61 44 72 75 65 61 104 19 48 57

For each analyte 2 responses were considered. Firstly theextraction yield was calculated following equation:

Yield (%) = AreaAAreaB

100 (1)

where AreaA is the analyte chromatographic area measuredwhen a spiked matrix was extracted and AreaB is the analytechromatographic area measured when an unspiked matrix wasextracted then its eluate was spiked.

Secondly, the matrix effect was measured following equation:

Matrix effe(

AreaB)

where Arepure solven

Certainlwhen compmany errorcomponentof the analshould be r

The extrondarily coof experim

ments has been duplicated. The experiments were reproducible,except those which gave very poor extraction yields (under 5%)but this cannot misshape the interpretation. In Fig. 4, the effectsof the screening model (yield = b1X1 + b2X2 + b3X3 + b4X4) arepresented. The higher the absolute value of the effect (b1, b2,b3, b4), the higher the influence of the corresponding parame-ter on the response. As showed in Fig. 4, each tested parameterhas a strong influence on the extraction yield, since the out-put effects values are meaningful. The signal of the measuredresponse ( or +) is directly linked with the sign ( or +) of theexperimental design parameter tested. b1, b3 and b4 are positivefor most analyte, thus the levels +1 of the parameters X , X

incshouse. Ied 101) o

easeordeeme

ddehow

ticosH/w

Fig. 4. Influeelution; b4: %ct (%) =AreaC

1 100 (2)

aC represents the analyte chromatographic area ints.y the presence of matrix components in the extract,ared to standard solution in pure solvent, can cause

s, which can lead to inaccurate results. These matrixs can promote either ion suppression or improvementyte ionization in the electrospray interface [34], andemoved whenever possible.action yield was headlined and matrix effect was sec-nsidered. The extraction yields obtained for each runental design are shown in Table 5. Part of the experi-

and X4ter X2responacidifi(X3 = +in incr

Inimprovof Mostudy sall corof MeOnce of the tested parameter on the extraction yield. b1: volume used for the first wash;MeOH used for the first elution.1 3rease the response. b2 is negative, thus the parame-ld be fixed to the level 1 in order to increase then other words, washing with 1.5 mL (X1 = +1) with an% MeOH mixture (X2 = 1) and eluting with 1.5 mLf an acidified 70% MeOH mixture (X4 = +1) resultedd extraction yields.r to decrease the eluant mixture volume, a finalnt was performed by using the optimisation modulev.7 software (from Umetrics, Umea, Sweden). Thiss that it is possible to obtain a satisfactory elution ofteroids with X3 = 0, i.e. by eluting with only 1 mLater/FOA (70:30:0.1, v/v/v).b2: % MeOH used for the first wash; b3: volume used for the first

A. Piram et al. / Talanta 74 (2008) 14631475 1473

Table 6Linearity range and IDL for the LC/MS/MS analysis

Molecule Linearity range(g L1)

Correlationcoefficient

IDL(pg)

CorticosteroidsBetamethasone + dexamethasone 1400 0.999 32.0Budesonide 1100 0.999 0.7Cortisol 4400 0.993 7.7Cortisone 1400 0.998 1.3Flunisolide 1100 0.999 2.5Fluocinolone acetonide 1100 0.999 0.6Prednisolone 1400 0.992 9.1Prednisone 1400 1.000 6.7Triamcinolone 1100 0.996 77.3Triamcinolone acetonide 1100 0.998 0.5

-BlockersAcebutolol 0.1400 0.992 0.3AlprenololAtenololBisoprololMetoprololNadololPindololPropranoloSotalolTimolol

The finaMoreov

tant matrixunder 10%have to benot be donAnother ming with dthe isotopithe naturalanalyte/deudent of maand moreomercially aeliminated,the standar

3.3. Method performance evaluation

Calibration plots were obtained by standard solution analy-sis at eight concentrations for each analyte. The linearity of themethod was evaluated between 1 and 400g L1. To determinethe best weighting factor, concentrations were back-calculatedand the residual plot examined. The model with the lowestbias and the most constant variance across the concentrationrange was considered the best suited. A linear regression (1/x2weighted for-blockers and 1/x for corticosteroids) gave the bestfit for the concentration/detector response with R2 values above0.99 and residues less than 15% for each analyte in the concen-tration range tested (Table 6). As no blank matrix was foundout within the tested samples, it was not possible to evaluatethe method sensibility on environmental samples. Instrumen-tal detection limits (IDL) were thus calculated as the minimum

d mass giving a signal to noise ratio 3. The IDLs ranged77.

rumof t

riancch acy otedL1

7.ana

wor

nvirles

nviro

Table 7Extraction yie

Corticosteroid

BetamethasonBudesonideCortisoneCortisolDexamethasoFlunisolideFluocinolonePrednisolonePrednisoneTriamcinolonTriamcinolon0.1100 0.995 1.00.1400 0.993 0.40.1100 0.985 0.10.1400 0.990 0.50.1400 0.991 0.30.1400 0.992 0.3

l 0.1100 0.990 0.20.1400 0.991 2.00.1400 0.989 0.9

l protocol was described in detail in Section 2.er, we noticed that each experiment resulted in impor-

effects, except those that gave extraction yields. To avoid matrix effect higher purification wouldreached; unfortunately we have shown that it coulde without strongly decreasing the extraction yield.ethod consists in compensate matrix effect by work-

injecte0.1 and

Inst(n = 5)day vafor eaaccura

conduc500 ngTable

Theof thisit on emolecu

3.4. Eeuterated compounds for each analyte. In this casec molecule undergoes the same matrix effect than

compounds, thus the chromatographic area ratioterated compound results unchanged and is indepen-trix effect. However, this method is very expensive,ver most studied deuterated molecules are not com-vailable. As matrix effect could not be completelyit was necessary to quantify unknown samples by

d addition method.

Influentand analysmethod. Win 4 aliquotquantity ofconcentratiand alloweties measuhave been

lds and relative standard deviation in this study, on STP influents (n = 5)Yield (%) R.S.D. (%)

e 67 10 A86 12 A71 10 A70 22 B

ne 75 11 M54 19 N

acetonide 84 24 P62 16 P55 13 S

e 63 11 Te acetonide 70 103 pg analyte injected, as shown in Table 6.ental precision was assessed by replicate injectionhe calibration solution in the same day. The intra-e, expressed as relative standard deviation (R.S.D.%)nalyte peak area was usually below 15%. Thef the method was determined by recovery studieson spiked environmental samples (STP influents) at. Extraction yields and their R.S.D. are presented in

lytical protocol was not fully validated since the aimk was to develop an extraction procedure and testonmental samples, in order to investigate if targetwere found in the environment.

nmental analysis

samples from Pierre Benite STP have been extracteded as described above, by the standard additionith this protocol, the environmental sample is divideds of 400 mL. Each aliquot was spiked with a relevantstandard (up to twice the suspected environmental

on). The linearity of standard addition was verifiedd a reliable environmental quantitation. The quanti-red are reported in Table 8. The studied -blockersdetected with concentrations up to 318 ng L1 for

-Blocker Yield (%) R.S.D. (%)cebutolol 68 4lprenolol 61 6tenolol 68 24isoprolol 60 7etoprolol 62 7adolol 60 9indolol 10 24ropranolol 66 7otalol 68 9imolol 62 9

1474 A. Piram et al. / Talanta 74 (2008) 14631475

Table 8Measured concentration in Pierre Benite influents/effluents STP

Compound

CorticosteroidBetamethasBudesonideCortisoneCortisolFlunisolideFluocinoloTriamcinolTriamcinol

-BlockersAcebutololAtenololBisoprololMetoprololNadololPindololPropranoloSotalolTimolol

sotalol. Thdance withthe studiedConcentratcortisol whnous steroinot surpriscentrations

Then, thite STP efflwas to havSTP effluenment was athe target cwhich haddue to thetreatment.STPs and awaters.

To ourcontaminat

4. Conclu

A quanstudy 11 cwaters. Thgle and spethe first prtion of cortof -blockMeOH waanalytes.

This me-blockers

concentrations in Europe or America [528]. It also permittedthe detection of corticosteroids in STP influents and effluents.

emonvir.bothenviures.

wled

autial suthor

nce

CleuCleuCleuFent.Asht.. Beegen

. Cahromat.M. D.

HeberHeber

. ThoZucc.

J. Hil.H. J

CastigInfluents (ng L1) Effluents (ng L1)sone + dexamethasone 15 7

n.d. 3174 22953 637 5

ne acetonide 0.3 11one 31 30one acetonide 40 3

66 0.4170 1.873 1111 110 0.12 0.4

l 92 2318 6532 58

e measured -blockers concentrations are in accor-the amounts reported in literature [13,2125]. All

corticosteroids were detected, excepted budesonide.ions reach 174 ng L1 for cortisone. Cortisone andich are not only prescription drugs but also endoge-ds are continuously discharged in sewages. So it ising these 2 compounds were present to higher con-than other corticosteroids.e developed protocol was applied, to Pierre Ben-uent samples (results shown in Table 8). Our aim

e a first estimation on the quantities discharged byts in environmental waters, i.e. to know if the treat-

ble to totally remove corticosteroids from water. Allompounds have been detected, including budesonidenot been detected in influent samples. This can be

hydrolysis of budesonide conjugates during the STP

This din an estudies

Astestedprocedsample

Ackno

ThefinancThe au

Refere

[1] M.[2] M.[3] M.[4] K.

122[5] D.

167[6] A.C

J. W[7] J.D

Ch[8] J.G

213[9] T.

[10] T.[11] K.V[12] E.

205[13] M.[14] O.A[15] S.This study demonstrates that corticosteroids reachre continuously discharged by them in environmental

knowledge, this is the first study that demonstratesion of environmental waters by corticosteroids.

sions

titative analytical procedure has been developed toorticosteroids and 10 -blockers in environmentale use of mixed-mode MCX cartridges allows a sin-cific sample preparation. As far as we know, this is

ocedure proposed for the environmental quantifica-icosteroids. Furthermore, it gives a high purificationers, compared to existing methods. Indeed a 100%sh is allowed for these compounds with no loss of

thod was applied on environmental samples and theconcentrations were in accordance with reported

matogr.[16] D. Bend

195.[17] P.E. Stac

D.B. Rei[18] D.W. Ko

Buxton,[19] M.J. Lop[20] A. Salva

102109[21] D.B. Hu

(2003) 1[22] R. Andre[23] T.A. Ter[24] D. Calam

Sci. Tech[25] C. Mie`ge

739.[26] M. Petro

A 974 (2[27] E.P. Kol

6377.[28] K.V. Th

Waldocknstrates that these molecules are a matter of concernonmental context and should be part of monitoring

corticosteroids and -blockers were detected onronmental samples, further work will include a fullvalidation and its application on a large number of

gements

hors acknowledge the Region Rhone-Alpes for itspport and Grand Lyon for assistance on sampling.

s thank also Sanofi-Aventis for its materials help.

s

vers, Ecotoxicol. Environ. Saf. 59 (2004) 309.vers, Toxicol. Lett. 142 (2003) 185.vers, Chemosphere 59 (2005) 199., A.A. Weston, D. Caminada, Aquat. Toxicol. 76 (2006)

on, M. Hilton, K.V. Thomas, Sci. Total Environ. 333 (2004)

rlfroid, A. Van der Host, A.D. Vehtaak, A.J. Schafer, G.B.J. Rijs,er, W.P. Cofino, Sci. Total Environ. 225 (1999) 101.ill, E.T. Furlong, M.R. Burkhardt, D. Kolpin, L.G. Anderson, J.ogr. A 1041 (2004) 171.erksen, G.B.J. Rijs, R.H. Jongbloed, Water Sci. Technol. 49 (2004)

er, J. Hydrol. 166 (2002) 175.er, Toxicol. Lett. 131 (2002) 5.mas, M.J. Hilton, Mar. Pollut. Bull. 49 (2004) 213.

ato, S. Castiglioni, R. Fanelli, J. Hazard. Mater. 122 (2005)

ton, K.V. Thomas, J. Chromatogr. A 1015 (2003) 129.ones, N. Voulvoulis, J.N. Lester, Water Res. 36 (2002) 5013.lioni, R. Bagnati, D. Calamari, R. Fanelli, E. Zuccato, J. Chro-

A 1092 (2005) 206.z, N.A. Paxeus, T.R. Ginn, F.J. Loge, J. Hazard. Mater. 122 (2005)

kelberg, E.T. Furlong, M.T. Meyer, S.D. Zaugg, A.K. Hendersen,ssmen, Sci. Total Environ. 329 (2004) 99.lpin, E.T. Furlong, M.T. Meyer, S.D. Zaugg, L.B. Barber, H.T.Environ. Sci. Technol. 36 (2002) 1202.ez de Alda, D. Barcelo, J. Chromatogr. A 892 (2000) 391.

dor, C. Moretton, A. Piram, R. Faure, J. Chromatogr. A 1145 (2007).ggett, I.A. Khan, C.M. Foran, D. Schlenk, Environ. Pollut. 12199.ozzi, M. Raffaele, P. Nicklas, Chemosphere 50 (2003) 1319.

nes, Water Res. 32 (1998) 3245.ari, E. Zuccato, S. Castiglioni, R. Bagnati, R. Fanelli, Environ.nol. 37 (2003) 1241., M. Favier, C. Brosse, J.P. Canler, M. Coquery, Talanta 70 (2006)

vic, E. Eljarrat, M.J. Lopez de Alda, D. Barcelo, J. Chromatogr.002) 23.odziej, T. Hareter, D.L. Sedlak, Environ. Sci. Technol. 38 (2004)

omas, M.R. Hurst, P. Matthiessen, M. McHugh, A. Smith, M.J., Environ. Toxicol. Chem. 21 (2002) 1456.

A. Piram et al. / Talanta 74 (2008) 14631475 1475

[29] H. Sanderson, D.J. Johnson, T. Reitsma, R.A. Brain, C.J. Wilson, K.R.Solomon, Regul. Toxicol. Pharmacol. 39 (2004) 158.

[30] T.A. Ternes, TrAC 20 (2001) 419.[31] D. Bendz, N.A. Paxeus, T.R. Ginn, F.J. Loge, J. Hazard. Mater. 122 (2005)

195.

[32] H. Budzinski, A. Togola, Spectra Analyse 33 (2004) 1.[33] L. Amendola, F. Molaioni, F. Botre`, J. Pharm. Biomed. Anal. 23 (2000)

211.[34] W.A. Kormacher (Ed.), Using Mass Spectrometry for Drug Metabolism

Studies, CRC press, Boca Raton, FL, 2005, p. 103.

Development and optimisation of a single extraction procedure for the LC/MS/MS analysis of two pharmaceutical classes residues in sewage treatment plantIntroductionExperimentalChemicalsSamplingSample preparationLiquid chromatography-tandem mass spectrometry

Results and discussionLiquid chromatography-tandem mass spectrometry analysisSolid phase extractionMethod performance evaluationEnvironmental analysis

ConclusionsAcknowledgementsReferences