2.1 INTRODUCTION

The analysis of impurities in drug substances, beginning with the initial

screening and ending with the use of validated methods in routine quality control

and quality assurance, is becoming an increasingly challenging task along the

pharmaceutical value delivery chain. This compendium offers guidance on how

Rapid Resolution Liquid Chromatography (RRLC), in combination with mass

spectrometry, can improve overall workflow for analyzing and identifying

impurities in drug substances.

The pharmaceutical industry routinely uses high performance liquid

chromatography (HPLC) to quantitative levels of impurities at the mandated level

of 0.1% [l-31. However, pharmaceutical drug substances and their impurities are

often structurally similar and therefore may co-elute. The evaluation of peak

purity is a key component of method development and validation (i.e. specificity).

Analytical techniques typically used to increase the analyst's confidence in the

purity of chromatographic peaks include orthogonal separations, selectivity testing

using potential impurities and fraction collections followed by alternate testing.

These off-line techniques can be time consuming and ineffective, especially at the

early stages of drug development when impurities are unknown. Diode array

detection (DAD) has also been widely used to provide chromatographic peak

purity determinations [4-81. Although it was demonstrated that DAD can provide

rapid on-line determinations, disadvantages such as inability to consistently detect

an impurity below 0.5 per cent co-eluting with a high level main analyte and a

requirement for some separation of analytes were acknowledged. Analytes must

also have dissimilar UV spectra to achieve low detection levels. This shortcoming

is important in the pharmaceutical industry as impurities typically have UV

spectra similar to the main analyte. Coupling HPLC with mass spectrometry (LC-

MS) offers an alternative mode of detection which might be exploited for rapid

on-line HPLC peak purity assessment. Unlike DAD, LC-MS has the potential,

with the exception of isobaric impurities, to provide detection capabilities for all

impurities. Mass spectrometric detection offers the added ability to provide mass

and structural information on the co-eluting impurity. LC-MS can provide high

sensitivity for a wide range of compound classes relevant to the pharmaceutical

industry while simultaneously providing chromatographic selectivity and mass

selectivity. Indeed, LC-MS has been used to demonstrate method specificity and

to screen drug purity [9,10]. MS has also been implemented as a tool to determine

HPLC peak purity during the analysis of a few particular compounds [ll-141.

Bylund applied mathematical modelling to LC-MS peak purity determinations

using both real and simulated data [IS]. Bryant [16] compared electrospray

ionization (ESI) LC-MS and LC-MS-MS with DAD for examination of co-eluting

impurities in famciclovir and ropinirole. Tlus study demonstrated that for an

impurity set with similar W spectra, MS could be optimized in such a way to

consistently detect semi-coeluting known impurities at 0.1% whereas DAD could

not. All examined compounds contained m i n e groups, which typically provide

good sensitivity by ESI [17]. Salau [18] determined HPLC peak purity of

pesticide mixtures by thermospray MS. This study demonstrated the suppression

effects of exactly coeluting compounds in MS and that mathematical modelling

can detect coeluting compounds at the 5% level. This high detection limit is

attributed to the fact that thermospray exhibits greater noise and lower sensitivity

relative to other ionization techniques such as atmospheric pressure ionization

(API).

Fisher [19] examined the use of API LC-MS and LC-MS-MS techniques

to be used as chromatographic peak purity tools using one drug coeluted with four

impurities at levels ranging from 0.05% to 5.0%. Data indicated that, despite

spectral comparison of the pure compound with that of the spiked samples,

unambiguous detection of coeluting impurities was possible only at the 0.4%

level, falling short of the mandated level of 0.1%. The application of MS as a peak

purity tool for additional separation techniques was also discussed by Fanali [20].

A preliminary study examined ESI- MS as a tool for peak identification, peak

purity testing and selective monitoring of overlapping peaks with capillary

electrophoresis (CE). Other studies have interfaced MS with gas chromatography

(GC) to determine peak purity [21, 221. These GC and CE applications illustrate

the potential flexibility of MS as a general tool for peak purity. These studies

referenced above provide some evidence for the use of MS as a chromatographic

peak purity tool in specific instances. However, these studies do not provide an

evaluation of the general applicability of API LC-MS to detect low levels of

unknown impurities coeluting with a high level main analyte.

The impact of ionization technique (i.e. thermally assisted ESI and API)

was not sufficiently explored. As it is widely known that response factors vary

using mass spectrometric detection, it is important to test a diverse array of

compounds and impurities to provide a robust evaluation. This paper presents the

evaluation of API MS as a general tool for detection of coeluting unknown

impurities in HPLC. The investigation employed a single stage MS to facilitate

broad applicability. Data was obtained on an m y of drugs and impurities

coeluting at levels ranging from 10% down to 0.1%. The compounds tested

included acids, bases and zwitteriohs possessing a wide range of polarities,

spanning a mass range typical of pharmaceuticals and with varying ionizabilities.

Discussions of additional procedures and factors that can affect the application of

API MS to HPLC peak purity investigations are also discussed.

2.1.1 Impurities in drug substances

In general, impurities in drug substances are addressed from two perspectives:

The chemical aspects, which encompass classification and identification of

impurities, are in effect how to generate reports, set appropriate

specifications and describe analytical procedures.

Even more important are the safety aspects for the patients, who will be

using the final product when a new drug is brought to the market.

Comparative studies and genotoxicity testing are of increasing importance

in this context.

From the point of view of regulatory bodies, such as the US FDA, EMEA,

etc., impurities in drug substances are classified in these categories:

Organic impurities (process and drug related)

Inorganic impurities

Residual 'solvents

2.1.2 Case study

The five Application Notes in this compendium show a typical workflow

in an analytical method development and QAtQC laboratory during drug

development and commercialization, that is the analysis and determination of

production- related impurities in an active pharmaceutical mgredient. Analytical

procedures are essentially improved using Rapid Resolution LC by speeding up

the analysis and, combined with TOF, providing accurate mass information in the

initial phase of the investigation. There are multiple sources for organic impurities

at various concentration levels. They may arise most likely during the synthesis,

and storage of the active drug substance, but also during the manufacturing

process andlor storage of the final drug product and can come from:

Starting materials

By-products

r Intermediates

Degradation products

Reagents and ligands, but also from packing materials.

According to the FDA Guidelines "Impurities in Drug Substances",

identification of impurities below apparent levels of 0.1 % is generally not

considered necessary. However, identification should be attempted for those

potential impurities that are expected to be unusually potent, producing toxic or

pharmacologic effects at a level lower than 0.1 %. In all cases, impurities should

be qualified.

2.1.3 Analysis of by-products

The analytical procedures described in this document focus on the

discovery of starting materials, by-products and intermediates, and how they can

most effectively be determined early in the identification process. Further

information about the analysis of packaging material and organic volatile

impurities (OVI) can be found in the appendix. Impurity analysis follows the

typical method development workflow, method optimization / transfer and

subsequent routine use under cGMP conditions (Fig. 2. I).

Fig.2.1 Typical workflow in an analytical method development and

QAIQC laboratory

Regulatory authorities worldwide like ICH, USFDA, Canadian Drug and

Health Agency are emphasizing on the purity requirements and the identification

of impurities in Active Pharmaceutical Ingredients (APls). The process called

qualification of the impurities is acquiring and evaluating data that establishes

biological safety of an individual impurity; thus establishes biological safety of an

individual impurity; thus, emphasizing the need and scope of impurity profiling of

dmgs in pharmaceutical research. Impurities in drug substances and drug products

are classified as shown in Table 2.1.

Table 2.1 Classification and source of impurities in drug substance and drug products

Type of impurity Source of impurity

Process related: Dmg substance Starting material Intermediate By product Impurity in starting material Residual organic solvents Reagents, catalysts

Process related: Drug product Organic or inorganic Reagents Catalysts

Degradation: Drug substance or drug Organic degradation products- Product related substances

Degradation: Drug products Excipient interaction

Different official regulatory authorities have different definitions for

impurities: ICE defines irnpurities as by-products,' degradation products,

interaction products, intermediates, penultimate intermediates, related products

and transformation products. USP defines impurities as impurities in official

articles, ordinary impurities, and organic volatile impurities.

2.1.4 HPLC with 10 pm particle columns

. Although GC capillary columns were available in the early to mid 1970s,

these glass columns were difficult to work with and reactive. For these reasons in

the 1970s packed GC columns (as was used in HPLC) were commonly employed

for the analysis of seized drugs. For HPLC a major breakthrough occurred in 1972

with the popularization of silica based microparticulate packings [23] containing

totally porous 5 and 10 pn particles. In 1973 it became commercially feasible to

modify the silica surface via silanization and this led to the first 10 p reversed-

phase columns [24]. These columns contained a bonded non-polar moiety, which

in combination with a polar eluent separated solutes in order of their

hydrophobicities. It is interesting to note that HPLC using the 10 pm particle

packings and GC using these same packings (packed columns) gave comparable

separations for seized drugs [25, 261. A comparison of packed column GC and

HPLC for heroin analysis is shown in Fig.2.2 and 2.3.

Fig.2.2 Packed Column GC-FID chromatogram of heroin and related compounds. (Conditions: injection size 1.4 pL; column 6 ft x 114 in. (2 mm, id.) packed with 3% OV-1 on Chromosorb W-HP 801100 mesh. Temperature 235 OC)

Fig.2,3 HPLC chromatogram of heroin exhibit at sample concentration of 10 mglmL (Conditions: injection size 5 pL; column, 300 mm x 3.9mm 10 pm Porasil; 25% ammonia, methanol, chloroform (water washed) ( I t 200 +BOO); flow rate 2.0 mLlmin with W detection at 254 nm )

Ion pair chromatography and reversed-phase chromatography /27, 281

were important developments in the analysis of seized drugs, Using reversed-

phase chromatography it is preferable to analyze basic drugs in the un-ionized

state. This however, requires a mobile phase pH above 8. Unfortunately at such

high pH values modified silica particles become unstable and silica becomes

soluble. At lower pH value of 3.5, most basic drugs are ionized and thus have little

or no affinity for the non-polar bonded phase and instead interact with residual

silanol groups through ion exchange or adsorption mechanisms, Such interactions

can result in severe tailing. A possible solution to this problem is to form a

lipophillic ion pair complex with the salt of the basic drug using a counter ion

such as an alkyl sulfonate. This complex is far more retentive when used with a

non-polar bonded phase. The procedure also permits the concomitant detection of

acidic drugs for they will be neutral at such pH and also retained, To demonstrate

this, a 300 mm x 4 rnm 10 pm Bondapak C18 was applied to the analysis of a

wide variety of drugs of forensic interest including ergot alkaloids,

phenethylamines, opium alkaloids (heroin excluded), local anaesthetics,

barbiturates and other drugs of forensic interest [29]. The use of reversed. phase

ion pair chromatography allowed these compounds to be separated wing a single

mobile phase (40% methanol, 59% water, 1% acetic acid, 0.005 M

heptanesulfonic acid (pH 3.5). To further exploit the advantages of liquid phase

over gas phase separations the role of the stationary and mobile phases for

reversed- phase ion pair chromatographic spinition was investigated by the

author. Using Bondapak C18, Bondapak alkyl phenyl and Bondapak CN columns

and a mobile phase containing 1% acetic acid, a study was made of the effect of

counter ion size, counter ion concentmtion, water-methanol ratio and basicity on

the retention factor (k) and selectivity a values [30, 311. For a given stationary

phase and water-methanol ratio, the k values of the weakly acidic barbiturates and

an un-ionized base were fairly independent of counter-ion type and concentration.

For the ionized bases at a given mobile phase, the k values increased with

increasing size of counter-ion with both the C18 and alklyphenyl columns. For the

ionized bases on the C18 and alkylphenyl stationary phases and a given mobile

phase, the ratio of k values for any given set of counter-ions was fairly constant.

In general, higher variations in k with size of counter-ions were observed on the

C18 stationary phase than the alkyl phenyl stationary phase. These k ratios were

independent the size of the solute, but varied with the charge of the base. The

variation of k values with counter ion size on the C18 and alkylphenyl columns

was fairly independent of the methanovwater ratio employed. For the C18 and

alkylphenyl columns, the k of ionized bases increased with an increase in counter-

ion concentration especially for the more hydrophobic counter-ion ions. For the

CN column, no significant variation of k with counter-ion size or concentration

was observed for any of these basic drugs.

For similar solutes which differed in aliphatic character, increasing the

amount of water at a given counter ion (0.005 M concentration) increased values.

For the C18 and alkylphenyl stationary phases, increasing the size of the

alkylsulfonate counter-ion in mixtures of acids and ionized bases selectively

increased the retention of the bases. Although the values of ionized basic

compounds invariably changed with counter-ion ion size, there was no

generalized trend other that that the highest values were usually obtained using the

least hydrophobic counter ion. In most instances, the values of bases were

invariant to changes in counter ion concentration. Although for most solutes the

retention order was C18 > alkylphenyl > cyano at a given mobile phase

conditions, the C18 phase was preferred. The C18 phase was found to exhibit

greater overall selectivity than the other phases.

Based on these studies a wide variety of drugs of forensic interest could be

analyzed with superior resolution and speed over previously reported

methodology using a C18 stationary phase by replacing the heptanesulfonate

counter ion with methanesulfonate and using two isocratic mobile phases at

different methanol concentrations. [32]. None of these systems was satisfactory

for the separation of the major constituents in heroin. Therefore a third mobile

phase containing phosphate buffer, methanesulfonate and acetonitrile (pH 2.2)

was developed for the analysis of heroin samples [33]. Phosphate buffer was used

in place of acetate buffer because of its lower UV cut-off. For all three mobile

phases the use of a relatively high methanesulfonate concentration (0.02 M)

diminished the variation of retention time with concentrdtion of bases in the

working sample concentration range of 0 to 1.0 mg/mL.

The analysis of PCP on parsley or mint leaves was not possible using the

methanesulfonate counter ion, because of interference from compounds present in

the plant material. By using the lipophillic counter-ion ion dodecyl sulfate at a

concentration of 0.02 M, it was possible to manipulate retention so that the acidic

and neutral compounds related to the plant material eluted first, followed by the

basic compound PCP [34]. A major advance in mobile phase optimization

occurred in the 1980 when Glajch [35] outlined an approach for rational method

development for reversed- phase chromatography, based on Snyder's solvent

selectivity triangle [36] and a response surface as described by Snee [37]. This

computerized approach for optimizing selectivity, named by the authors

"overlapping resolution mapping", included keeping the solvent strength constant,

while changing the ty-pe(s) and amount(s) of organic modifier. Different solvents

such as methanol, acetonitrile and tetrahydrofuran have different abilities to

undergo hydrogen bonding, dipole interactions, dispersion, dielectric interactions

and electrostatic interactions. Overlapping resolution mapping was used for the

sepamtion of 26 fentanyl homologs and analogs using a 250 mm x 4.6 mm

Inertisil ODS3V column [38]. This approach predicted an optimum mobile phase

consisting of 81% phosphate buffer (pH 2.1), 4% methanol, lo0? acetonitrile and

5% tetmhydrofuraa

In 1975 a topological index 'tnolecular connectivity (X)" was introduced to

estimate solute cavity surface area [30]. Karger 1401 showed that hydrophobic

selectivity in HPLC can be accounted for by using x values. Using connectivity

indexes for the 26 fentanyl homologs and analogs, hydrophobic selectivity was

found to depend on the position of methylene substitution on the parent fentanyl

molecule and type of substituent [41]. It was M e r found that hydrophobic

selectivity was approximately independent of mobile phase composition for the

solvent mixture used in the overlapping resolution mapping scheme. Similarly,

hydrophobic selectivity was also found to be almost identical on both a 10 pm

particle silica-based 250 mm x 4.6 mm lnertisil ODs-3V column and a 10 pn

particle polymer based 150 mm x 4.6 mm RPI column (under normalized time

conditions). The silica based column exhibited significantly greater efficiency per

column length than the latter column.

2.1.5 HPLC with 3 and 5 pm particle columns

For GC a major breakthrough occurred in 1979 with the introduction of

fused silica capillaries [42]. These columns have much thinner walls than glass

capillary columns and the polyimide coating imparts mechanical strength. As such

they are flexible and have low reactivity. It is not surprising given the relative ease

of use and the significantly greater efficiencies and peak capacities that capillary

GC became popular in the 1980s. The use of capillary GC for heroin analysis is

shown in Fig.2.4. The peak emciencies of HPLC also improved in the late 1970s

and early 1980s with the advent of the smaller 3 and the increased use of 5 pm

particle size columns. Although these columns significantly increased

performance over the 10 pm particle columns, the GC capillary columns still

offered significantly greater peak capacity.

Fig.2.4 Capillary GC-FID chromatogram of major impurities, adulterants and diluents in heroin(Conditi0ns: column 25 m x 0.32 mm, fused silica (0.2 pm) SE-54. Temperature programmed from 150 OC a t 9 OC min -1 to 280°C (held for 0.5 min). Peaks: 1 = nicotinamide-TMS; 2 = acetaminophen- TMS; 3 = meconin; 4 = caffeine; 5 = glucose-TMS; 6 = phenobarbital-TMS; 7 = methaqualone; 8 = N-phenylnapthylamine; 9 = tetracosane (internal standard); 10 = acetylcodeine; 11 = acetylthebaol; 12 = morphine-TMS; 13 = 06-monoacetylmorphine-TMS; 14 = heroin; 15 = papaverine; 16 = phenolphthalein; 17 = noscapine 1431 )

Lurie and Carr conducted a comparison of 3 and 5 and 10 l m particle size

reversed phase columns for the separation of drugs of forensic interest [44]. Due

to the similar selectivity of the stationary phases and the higher efficiencies

obtained using 5 pm particles when compared to 10 pm particles, separations on a

125 mm x 4.6 mm 5 pn particle column were performed in at least half the time

compared to using the 250 mm x 4.6 mm 10 pm C18 particle columns. Rapid

analysis could be obtained using even shorter 3 p particle columns. However,

low plate counts and asymmetrical peaks were observed for basic drugs using the

100 mm x 4.6 mm HS/3 C18 3 pn particle column due to a large number of

unbonded silanol sites present on these particles. For the HS15 C 18 5 p particle

column M e r reductions in retention times and different selectivities were

obtained when the m i n e modifier hexylamine was added to the mobile phase

containing phosphate buffer pH 2.2. An approximately 9 fold decrease in retention

time could be obtained for the separation of heroin and its major by-products over

an earlier separation (10 p particles and no modifier) by using a combination of

smaller particle sized columns and an m i n e additive. Even after the addition of

hexylamine to the mobile phase to block unbonded silanol groups, the separation

of basic drugs on the 3 pm column was still inadequate. Using the HSl5 CIS 5 pm

particle column a separation of heroin and its major by-products, plus additional

by-products as well as major adulterants, was developed by studyiig the effect of

mobile phase parameters including amine concentration, organic modifier type

and eluent pH [45] (Fig.2.5). Since a lower solvent strength was necessary for

early eluting compounds and a higher solvent strength was needed for the more

hydrophobic adulterants, gradient elution was used. For the separation of acidic

and neutral acetylated rearrangement products of opium alkaloids, Lurie and Allen

used two 30 mm x 4.6 rnm 3 pm HSl3 C18 columns in series [46]. A separation

was developed based on the solvent optimization triangle.

Lurie used a 125 mm x 4.6 mm HS15 C18 5 p particle column for

analysis of manufacturing by-products in seized cocaine exhibits. Similar as in the

previous separation for PCP on plant material, 0.02 M dodecylsulfate was used in

order to control retention so that the retention order was acids, mono-protic

mines and di-protic mines. The chromatogram consisted first of carboxylic

acids such as benzoic acid, cinnamic acid (cis and trans) and several isomers of

truxillic and truxinic acids; next the mono-protic amines benzoylecgonine, cocaine

and cinnamoylcocaine (cis and trans) and finally isomers of the di-protic amine

truxilline. Lurie wed a 110 mm x 4.7 mm Inertisil ODs-3, 5 pm particle column

with isocratic conditions for the separation of benzodiazepines [47]. Anabolic

steroids were separated using a 250 mm x 4.6 mm Inertisil 5 p particle column

and a methanol water gradient [48].

Fig.2.5 HPLC chromatogram of major impurities and adulterants in heroin (Conditions: injection size 50 pL; column, 110 mm x 4.7mm 5 pm Inertisil ODs-3. Initial conditions: 5% methanol, 95% phosphate buffer (0.023M hexylamine, pH 2.2). Final conditions: 30% methanol, 70% phosphate buffer (0.023M hexylamine, pH 2.2), 20 minute linear gradient. Hold final conditions for 8 minutes; flow rate 1.5 mLImin)

2.1.6 HPLC with multiple detectors

The use of smaller size columns and mobile phase optimization has been

discussed in this chapter as means of increasing HPLC performance. Another way

of increasing this technique's performance is to increase the specificity of

detection. This is important in both qualitative and quantitative analysis. For

HPLC the relatively low peak capacity means that the reliability of compound

identification by retention time only (even for screening purposes) is poor. Baker

[49] showed that for 78 drugs separated by reversed- phase HPLC using a 300 mm

x 4 mrn 10 pm Bondapak C18 column, only 9% could be uniquely identified by

relative retention time alone. However, using relative retention time and 254:280

nm absorbance ratios (peak area or peak height ratios) and 95% of the drugs could

be uniquely identified.

Absorbance ratios will also provide information on peak purity which is

immensely important for quantitative analysis. Multi-wavelength detection also

allows more accurate and selective detection by using wavelengths close to the

absorbance maximum of a given solute. Other means of increasing specificity of

detection include fluorescence (FL), electrochemical (EC) and mass spectrometric

(MS) detection. The key to using these detection schemes with HPLC was the

commercial development of the thin-layer amperometric detector in the 1970 [SO]

the diode array (PDA) W detector in 1976 [51] and the thermospray (TSP)

interface in 1984. [52] Lurie used 220:254 nm absorbance ratios, as well as

relative retention times, for the screening of by-products and adulterants in heroin

samples. These ratios were also used to determine the peak purity of heroin.

Absorbance ratios of 215:230 mn were used, along with relative retention times,

to distinguish between 27 homologs and analogs of fentanyl. Lurie and Allen

employed dual UV detection, a programmable fluorescence detection and

electrochemical detection for the sensitive and selective detection of acidic and

neutral acetylated rearrangement products of opium alkaloids. For these solutes

higher UV wavelengths gave greater specificity, but not necessarily greater

sensitivity of detection. Although many of these compounds fluoresce,

fluorescence detection is more specific at trace levels because of its significantly

greater sensitivity for a smaller proportion of solutes, In contrast, for

electrochemical detection in the oxidation mode (0.85 V), only a relatively small

percentage of compounds will be electrochemically active with a sensitivity

approaching UV detection. Diode array (PDA) UV detection with the ability to

generate UV spectra "on the fly" offers significantly greater specificity for

qualitative analysis that the use of absorbance ratios. In addition this detection

mode allows W spectra to be generated across the peak to ascertain peak purity.

Lurie and McGuinness used relative retention times and PDA detection in

series with dual electrochemical detection to screen for the presence of by-

products and adulterants in heroin exhibits. For dual electrochemical detection in

the parallel mode, relatively high oxidation potentials of 1.0 and 1 . I V were used.

Response ratios were reported as one of three broad categories in order to

minimize changes in ratios which result from drifts in the reference redox

potential, sample over potential effects, or changes in temperature. Peak area

responses normalized to an internal standard nalorphine were used in order to

minimize effects due to electrode pacification. Library search sofhhrm was

developed for searching UV spectrum g e n d by the PDA detector and applied

for various applications including the identification of by-products and adulterants

in heroin samples 1531. Apex spectra at a measured retention time were searched

versus a library containing standard compounds. For a peak containing an impure

spectrum the upslope spectrum was successfully used to identify the peak.

Cocaine exhibits, Dual UV detection at 2 15 and 277 nm was employed. The lower

wavelength was used to measure all solutes except compounds that have a

cinnamoyl moiety. The higher wavelength allows for the sensitive and more

selktive detection of the latter compounds. The acquisition of UV spectra greatly

facilitated the determination of peak identity and purity. For the separation of

twenty benzodiazepines there was extensive overlap in retention times using UV

detection at 230 nm. However, all compounds gave unique UV spectra via PDA

detection. Three of the benzodiazepines (not well separated) were

electrochemically active in the oxidation mode and could be fkther identified by

employing PDA detection in series with dual electrochemical detection in the

parallel mode at 1.0 and 1.1 V. The benzodiazepines were also analyzed

separately using TSP-MS detection. Significantly enhanced specificity of analysis

over retention time alone was obtained since all solutes except clorazepate gave

[M+H]+ ions as the base peak. For the latter peak the base peak is [M + H -C02 - H20)+ ion. For most benzodiazepines, additional specificity is imparted to the

TSP mass spectra due to the presence of either a single C1 or Br atom or two C1

atoms, which give rise to ions due to natural isotopic abundances. Single ion

monitoring can be used to deconvolute the overlapping peaks. Lurie employed

PDA detection to enhance the specificity and sensitivity of analysis of anabolic

steroids. Although some of these solutes have identical UV spectra, for the few

unresolved peaks the UV spectra of the pair of anabolic steroids are unique. For

most steroid preparations only one of such a pair of compounds would be present.

The sensitivity k d specificity of quantitative analysis was enhanced by using

multiple wavelength detection at 210,240 and 280 nm.

In order to enhance the specificity of electrochemical, PDA or TSP-MS

detection for certain compounds, postelution photo irradiation was investigated

[54, 551. Continuous on-line post-elution photo irradiation wing a W lamp is a

means of converting eluting compounds into one or more photoproduct(s) prior to

detection. Continuous post-column photolytic derivatization using dual

electrochemical detection at 0.75 and 1.1 V facilitated the identification of cocaine

and selected adulterants, several of which had no response with the lamp off.

Cocaine under lamp-on conditions exhibited significantly greater specificity using

electrochemical versus UV detection.

Although the use of PDA detectors considerably increases selectivity by

providing UV spectra, UV spectra are not generally considered tools for absolute

peak identification due to the fact that compounds with similar structure may

produce identical spectra. As an alternative, TSP-MS can also significantly

increase selectivity by providing molecular weight information. However, usually

little or no additional information beyond molecular weight is obtained.

Additionally, certain compounds are not detected using TSP-MS. These

limitations can be overcome using post-elution irradiation. For example,

structurally related pairs morphine and 06- monoactetyl morphine and MDA and

MDMA each give similar UV spectra under lamp off conditions, but are easily

distinguished when the lamp is turned on. When using TSP-MS under lamp off

conditions, the structurally related pairs 03-monoacetylmorphine and 06-

monoacetylmorphine, and cannabidiol and A9-THC each give only identical rnlz

328 and m/z 3 15 (M + H)+ ions, respectively. However, under lamp on conditions

the thermospray mass spectra consisting of multiple ions, each are vastly different.

Barbiturates give no detectable TSP mass spectra under lamp-off conditions, but

multiple-ion mass spectra after post-elution irradiation.

2.1.7 Supercritical Fluid Chromatography

In 1982 Novotny and Lee 1561 reported on a workable instrument for

capillary supercritical fluid chromatography (SFC), which led to the first

commercial instrument in 1985. Because the diffusion coefficient of solutes in

supercritical fluids is about ten times greater than that in liquids (approximately

10000 times less than in gases) [57], capillary SFC could provide better resolution

and faster analysis than HPLC. In addition capillary SFC, which operates at lower

temperatures than GC, would be more favourable for the analysis of t h d l y

labile compounds. Next compared to GC capillary SFC would be more favourable

for the analysis of relatively non-volatile solutes. Finally, capillary SFC using

pure carbon dioxide as the mobile phase is compatible with the universal flame

ionization detector. Therefore it was not surprising that capillary SFC generated

much interest and excitement as a possible alternative technique to HPLC.

Lurie in 1988 was the first to demonstrate a multidimensional approach

coupling HPLC with capillary SFC [58], a schematic of which is shown in Fig.

2.6. The relatively non-volatile high molecular weight isomeric millines (eleven

possible isomers of molecular weight 658), eluted with methylene chloride, were

he* cut from an HPLC size separation column onto a capillary SFC system.

Unfortunately, the partial separation obtained for these solutes using SFC was not

better than could be obtained using HPLC.

Fig.2.6 Schematic diagram of coupled HPLC- capillary SFC system [From

reference 581

2.2 SEPARATION AND QUANTIFICATION OF IMPURITIES:

CONCEPTS OF UHPLC

Among impurity profiling tools, the separation technique such as HPLC,

TLC and GC are the most preferred. Even though thin layer chromatography has

been used extensively in the past, cunently pharmaceutical analysis adores HPLC

as its core separation tool. It is not only the inherent quality that comes with

HPLC but it is its accuracy, fine tuned results and hyphenation with other

detectors that made it the most versatile technique among separation methods. Gas

chromatography, even though an ~ ~ ~ u r a t e method owing to its destructive nature

it has been always considered as an alternative technique. It is applicable for

special impurities estimations like residual solvents in activate pharmaceutical

ingredients and formulations. Capillary electrophoresis (CE) and supercritical

fluid chromatography (SFC) are the other promising separation technologies that

gave an edge to separation science.

Since its discovery, HPLC has undergone sea changes with respect to its

technical capabilities. In this direction the latest development has given rise to an

improved version called Ultra High Performance Liquid Chromatography

(UHPLC). The whole idea of introducing the UHPLC systems is to reduce

analysis time considerably without compromising on performance. UHPLC

increases throughput as well as decreased flow rates. Some of the worth

mentioning features of the individual modules of an UHPLC and their

characteristics are as follow.

2.2.1 The,auto samplers

The present day auto samplers can deliver with high precision submicrolitre

volumes (0.1 pl to 1500p1) to the flow at any mobile phase flow rate and pressure.

They possess the zero cany over property, which is highly desirable for impurity

profiling studies.

2.2.2 Photodiode array detector (PDA)

The PDA detector plays a crucial role in the identification of compounds and

determination of peak purity. The diode array detector can acquire data from 190-

950 nm with a speed and frequency of SoHz for both spectra and signals. Up to

eight signals at different wavelengths can be acquired apart from recording high-

resolution UV spectra with a wavelength accuracy of i 1 nm.

2.23 The Pump

The pumps deliver flow rates of 0.05 mVmin to 5 ml/min. The high-

pressure tolerance limit ranges fieom 600 ban to 1500 bar in some systems.

Column thermostat will have a temperature range of 0°C to 100aC with

tempaature stability of < M.OS°C. Apart from the above mentioned advakced

characteristics in various modules of an UHPLC, an another aspect that needs to

be mentioned here is its hyphenation and compatibility with various other

detectors. The detectors that can be conveniently connected to monitor the eluent

of an HPLC are:

Mass Spectrometers (LC-MS) (LC-MS-MSn)

Nuclear Magnetic resonance spectrometer (LC-NMR)

Others detectors include LC-ELSD, LC-CLND, LC-PDA etc.

2.2.4 Column the heart of HPLC

The aim of HPLC analysis is to separate efficiently the closely related

individual components of a mixture to a best possible level and within a shortest

possible time with a high degree of accuracy and repeatability. Even though

various functions of an HPLC are controlled and dictated by individual modules

of an HPLC, column is responsible for the separation. It is the stationary phase

(Packing material) in combination with the mobile phase which is responsible for

the separation. There are varieties of column stationary phases that are available

as commercial products. To name some are C I R , ~ ~ , C4, Cyano (CN), Phenyl etc.

In general in most of the cases it is the silica that is used as a stationary support for

the above mentioned functionalities which are bonded covalently to the oxygen of

Si-0. The most widely used column technology for the analysis of majority of

small molecule pharmaceuticals is the Cls phase. Keeping the other things

constant the most important parameter that has a direct bearing on separation is

the particle size of the stationary phase. The relation between the resolution, plate

number, retention and resolution are given by the formula,

Thus, resolution (Rs) increases with increase in column length (L) or decrease in

particle size (dp). Even though increase in column length increases resolution, it

increases retention proportionately, thus increasing the analysis time multiple

folds. Hence the best choice is to decrease the particle size to increase resolution.

This idea has constantly evolved giving b i to the sub 2 pm particle columns.

The history of development of column stationary phase particle technology is

mentioned in Table 2.2. However, the decrease in particle size of the stationary

phase exerts high resistance to the flow, leadiig to increase in column pressure.

To tackle this problem the HPLC system has been redesigned to operate and

withstand high pressures. This concept has given birth to the ultra high

performance liquid chromatographic system (UHPLC).

The advanced technical features in these systems are:

(A)The upper pressure limits range from 600 to 1500 bar. As against 400 bar of

the traditional HPLC system

(B) High frequency data acquisition detector system (typically 80Hz)

Thus by increasing the pressure tolerance, columns with particles as small as

1.8 pm can be used. Various particle sizes and their nominal pressures are

summarized in the following table with column and mobile phase conditions:

100~4.6 rnm, 1 .OmVmin, 5050 (vlv) water: Acetonitrile (Table 2.3).

Table 2.2

History of commercial HPLC column particle development and tbeir efficiency

Year of Nominal size Approximate

Acceptance plates11 5cm

1950s 100pm Irregular shaped 200

1967 50pm Glass beed - pellicular 1000

1972 1 Opm 6000

1985 5 ~ m 12000

1992 3 - 3.5 pm 22000

1998* 1.5 pm* non - porous 30000

1999 . 5.0 pm pellicular 8000**

2007/2008 2.7 pm pellicular 32000***

+non porous silica or resins

**300 A' pore for protein

***90-120 A' Pore

Table 2.3

Various particle size columns and their back pressure for a given mobii phase and flow rate condition

Particle size Pressure Theoretical

Psi bar Plates (N)

As the chromatographic run time decreases with increase in performance of the

column, the peak widths also will decrease considerably. The decreased peak

widths may lead to loss of sensitivity. Hence, the data acquisition rate has to be

increased to keep in phase with decreased peak widths. To address this issue

detector with high efficiency data acquisition (8OHz) was introduced.

2.2.5 Preparative HPLC

Preparative HPLC is the most efficient and convenient tool for the

purification of wide varieties of pharmaceutical, chemical, biological and plant

extraction products. Preparative HPLC derives its high efficiency from its highly

sophisticated instrumentation as well as efficient column technology. In the

pharmaceutical industry, scientists o h come across compounds closely related

to each other such as related substances, degradation products, chiral impurities

(enantiomen) etc., which need to be isolated and characterized in their pure form.

Most of these separations cannot be achieved by traditional column

chromatography or crystallization techniques. Thus, the Preparative HPLC in

combination with highly efficient preparative HPLC columns is the solution for

the difficult separations of related substances.

2 3 SPECTROSCOPIC TOOLS FOR THE STRUCTURE ELUCIDATION

OF UNKNOWN IMPURITIES1 DEGRADANTS: HRMS AND NMR

2.3.1 High Resolution Mass Spectrometry (HRMS)

Mass spectrometry is a powerful analytical tool for the analysis of molecule,

both small and large. It can be used to identify unknown compounds, quantify

known compounds, and obtain information about chemical structure of both small

and large biological molecules. The ability of mass spectrometry to do all of this

from minute amounts of substances, often less than a billionth of a gram makes it

a highly useful technique in both established and emerging branches of science.

The versatility of this technology lies in its sensitivity and applicability to large

bio-molecules such as proteins, peptides etc. to small organic molecules.

The world's first commercial mass spectrometer was launched in 1948

which made use of electron impact ionization (El) with a mass range of 300 amu

and limited resolution. During 1950s time-of flight and quadruple analysis were

conceived. The next major development was of gas chromatography and its

coupling with mass spectrometry. This is not only allowed the first time direct

analysis of mixture of analytes but was also a trigger for the development of

present day mass spectrometry. For the past 30 years new ionization techniques

like fast particle desorption, electrospray ionization and matrix-assisted laser

desorptionlionization were discovered, The ionization techniques were invented

and developed in order to analyse complex molecules in their intact native form

and to aid hyphenation with other separation techniques like HPLC.

Combination of two analytical instruments yields great amount of

analytical information simultaneously and rapidly. Most of the current technical

advancements in analytical instrumentation are aimed at hyphenation. Direct

coupling of Gas spectrometer with MS was initial step towards hyphenation. The

gaseous molecules need to be ionized before they are fed in to the mass

spectrometer. Ionization is a very crucial process in mass spectrometry.

Generation of ions instantaneously h m neutral molecules is what happens inside

an ionization source. Electron impact ionization utilizes fast moving electrons to

knockout electrons from neutral molecules forming ions which are sucked in to

the mass spectrometer by means of electrometer by means of electrostatic

attractions and vacuum. This process is harsh and mainly gives fragments of the

molecular ion peak. This is the technique still vastly used in GC- MS systems

apM from chemical ionization. These techniques cannot be applied to the LC-MS

hyphenation where the eluent is a liquid. For this purpose electro-spray ionization

was invented and put in to use in early 1980s. In this technique highly charged

liquid droplets dispersed fiom a capillary in a high electric field are evaporated

spontaneously by hot nebuliser gas such as nitrogen. The free charged particles

formed in this process are attracted towards the MS inlet by electrostatic fields.

There are varieties of mass analysers available today with varied

analytical capabilities such as mass range and resolution. The most basic analysers

will be the quadruple mass spectrometers. Followed by TOF and other mass

analysers such as ion traps, ion cyclotron resonance MS etc. Single quadruple

mass spectrometers are the most vastly used machines in today's world for both

qualitative and quantitative analysis. Single quadruple mass spectrometric

experiments yield mass to charge ration of an ion rounded to its nearest whole

number. High resolution mass spectrometers such as time-of-flight and double

focusing FT-ICR mass spectrometers are capable of measuring the exact mass of

an ion. This is useful for interpretation because each element has a slightly

different mass defect. This "mass defect" is the difference between the mass of the

isotope and the nominal mass (which is equivalent to the number of protons and

neutrons). The atomic mass scale is defined by carbon-12 with a mass of exactly

12.0000 u (Table 2.4). The exact mass of a specific isotope is determined relative

to 12c by high resolution mass spectrometry. High resolution mass spectrometry

can distinguish compounds with the same nominal mass but different exact mass

caused by different elemental composition.

Molecular formula of molecular ion can be determined directly by

comparison of ion mass at high resolution with possible compositions using

accurate masses of individual isotopes. Thus the exact mass calculated fiom the

54

following table gives a value of 28.03 130 for ethane. When the mass is measured

by HRMS and if it yields a mass value 28.03130 it not only conforms ethane

molecular formula but also distinguishes from other two compounds yielding

similar molecular weights such as N2 and CO (Table 2.5).

Table 2.4

Exact masses of different elements

Table 2.5

Identification of compounds by HRMS

Molecule Exact Mass

Atom

Exact Mass

Table 2.6

I2c

12.000000

'H

1.007825

Identification of compounds by HRMS

Molecule Exact Mass

CH20 30.01056

The nominal mass of CH20, C2Ha and NO is 30 amu. The ordinary mass

spectrometer cannot distinguish them as all three yields the same mass of 30 m u

6~

15.99491

1

14.00307

whereas, the HRMS can distinguish them according to their exact masses as

mentioned in the table 2.6. Thus applying the above. masses to calculate exact

mass of any unknown molecular formula can be predicated. Calibration is crucial

for HRMS instrumentation.

2.3.2 Nuclear Magnetic Resonance P M R )

Nuclear magnetic resonance (NMR) spectroscopy is a powerfil and

versatile spectroscopic technique for the investigation and understanding of

unknown molecular structures and dynamics. Apart from structure elucidation

NMR is finding applicability in other pharmaceutical analysis also. This technique

is applicable to both liquid and solid samples. It is an extremely useful technique

as it is anon destructive analytical tool. NMR was developed in the year 1946 and

over the next 50 years NMR evolved in to premier organic spectroscopic

technique.

NMR spectroscopy makes use of the magnetic properties of nuclei inside the

atoms to yield chemical information of each atom in a molecule. According to

quantum mechanics, magnetic momentum of the nuclei arises from a net spin of

the sub atomic particle (electrons, protons and neutrons). In some atoms, the spins I2 16 32 are paired up ( C, 0, S) and some nuclei posses over all spin

( 1 ~ , 1 3 ~ , 1 9 ~ , 1 5 ~ , 3 1 ~ ) . If both the number of neutrons and the number of protons are

even, the nucleus has no spin. If the number of neutrons plus the number of

protons is odd, then the nucleus has a half-integer spin (i.e. 112, 312, and 512). If

the number of neutrons and the number of protons are both odd, then the nucleus

has an integer spin (i.e. l , 2 and 3).

In mechanical terms, the nuclear magnetic moment of a

nucleus will align with an externally applied magnetic field of strength Bo in only

2It1 ways, either with or against the applied field Bo. For a nucleus with spin = %

and a positive gyromagnetic ratio (y) only one transition is possible with a lower

energy level a (spin = + %) (parallel) and a higher energy level

(antiparellel)(spin =-1/2). The rotational axis cannot be exactly parallel or

antiparellel but will presses at an angle with angular velocity

o, is also called Larmor frequency

Each nuclei has a characteristic value of y, related to magnetic moment p

and spin number I

This precession process generates an electric field with frequency o, and if

we irradiate the sample with radio waves (in the MHz frequency range) the proton

will absorb energy and is promoted to the higher energy state. This energy

absorption is called resonance because the frequency of the applied radiation and

the precession coincide or resonate.

With increase in external magnetic field strength (BO), resonance frequency of

a given nuclei also increases (Table 2.7). Thus for different nuclei with different

gyromagnetic ratio (y) different frequencies must be applied to achieve resonates

at 400MHz and carbon at 100MHz.

Table 2.7

Magnetic field strengths and corresponding proton resonance frequencies

BO (Tesla) 'H Frequency (MHz)

1 1.75 500

18.8 800

21.2 900

23.5 lo00

Since the energy changes involved in NMR spectroscopy is very small (radio

fkquency), the signal sensitivity becomes a problem. In Fourier transformation-

NMR all spectral width are irradiated simultaneously with a single radio

hquency pulse. Following imadiation with radio frequency, the nuclei magnetic

moments press away h m applied magnetic field. Once the application of RF is

withdrawn, the magnetization will undergo 'relaxation' process emitting the signal

called 'free induction decay' (FID). FID is a time domain signal, which on Fourier

transformation gives hquency domain signal. This whole process is called a

'scan', which is then repeated several times to improve signal-to-noise ratio.

2.3.2.1 Chemical Shift

Now if all the protons and carbons in a molecule resonate at a single

frequency a single peak would have been obtained, giving no information about

individual protons or carbons. Rut practically the proton and carbon signals from a

molecule are generally spread over a frequency range. This is called the chemical

shift which is measured in terms of ppm scale with reference to TMS

(tetramethylsilane) signal. The chemical shift of individual proton with respect to

TMS indicates the chemical nature of that proton. Chemical shift indicates the

nature of hybridization of carbon, the kind of atom it is bonded to for ex. Oxygen,

halogen, another carbon etc.

Chemically different hydrogen's in an organic molecule do not experience

the same magnetic field. Electrons shield the nucleus thereby reducing the

effective magnetic field and requiring energy of a lower frequency to cause

resonance. On the other hand, when electrons are withdrawn from a nucleus, the

nucleus is de-shielded and feels a stronger magnetic field requiring more energy

(higher frequency) to cause resonance. Thus, NMR can provide information about

an atoms electronic environment.

2.3.2.2 umber of different hydrogens

The area under the NMR resonance is proportional to the number of

hydrogens which that resonance represents. In this way, by measuring or

integrating the different NMR resonances, information regarding the relative

numbers of chemically distinct hydrogens can be found. Experimentally, the

integrals will appear as a line over the NMR spectrum. Integration only gives

information on the relative number of different hydrogen's, not the absolute

number.

23.23 Splitting or coupling

NMR not only gives the total number of protons or carbons but also provides

information on how many hydrogen neighbow exist for a particular hydrogen or

group of equivalent hydrogen's. In general, an NMR resonance will be split into

~ + l . Peaks where N=number of hydrogen's on the adjacent atom or atoms. If

there are no hydrogen's on the adjacent atoms, then the resonance will remain a

single peak, called a singlet. If there is hydrogen on the adjacent carbon atom, the

proton resonance is split into two peaks of equal size, a doublet. Two hydrogen

atoms on the adjacent atoms will split the resonance into three peaks with an area

in the ratio of 1:2:1, a triplet. If there are 3 hydrogen atoms on the adjacent atoms,

the resonance will be split into four peaks with an area in the ratio of 1:3:3:1,

called a quartet. The split distances in terms of hertz are called coupling constant

and is also an indication of the nature and intensity of coupling between protons.

Thus the one dimensional proton and carbon experiments will give

information about the number, their chemical nature and to some extent their

position within the molecule. Additional ID experiments of carbon called DEPT

(Distortion less enhancement by polarization transfer), APT (Attached proton test)

indicate the number of protons directly bond to the carbon atom,

2.3.2.4 Nuclear Overhauser Effect (NOE)

An interesting phenomenon that can be studied in NMR spectroscopy is

through space interaction of protons within a molecule. Saturating the electron

magnetic resonance intensity to be enhanced by a factor of the order of lo3 is

called Nuclear Overhauser Effect (NOE) and was first discovered by Albert

Overhauser in 1953. A proton which is spatially close to another proton by less

than 5 Angstroms will show the transfer of magnetization between each other

when saturated by irradiation. This experiment is carried out by constantly

irradiating one proton and any magnetization picked by other proton is measured.

Both 1D and 2D set of experiments are available named as NOESY experiments.

These experiments will be very useful to confirm s t~~c tures in special cases as

exemplified in this research work.

23.2.5 Other 2D Experiments

The information obtained fiom ID NMR experiments may not be sufficient

to ascertain the structure of the molecule. There are varieties of 2D experiments

that are available to assign positions of different protons, carbons and nitrogen's

in a molecule. These are called 2D correlation experiments. Correlation between

similar atom types (proton-proton) are called homonuclear correlation

experiments (Ex: Cosy). Correlation between different atom types (proton-carbon

or proton-nitrogen) are called hetero-nuclear correlation experiments (Ex: HSQC,

HMBC). All these experiments provide wealth of information about the structure

of the molecule.

2.4 CHIRAL ANALYSIS O F PHARMACEUTICAL COMPOUNDS,

CHlRAL STATIONARY PHASES (CSP)

2.4.1 Chiral analysis of Pharmaceutical Compounds

Any two molecules, which are not super imposable, but are mirror images of

each other, are called chiral molecules. This terminology does not indicate any

aspect of chiral purity or chiral identity but just 1848 by Louis Pasteur, a French

chemist and biologist. For the first time he had separated by hand the two isomers

of sodium ammonium tartrate. Chirality is a hallmark of many molecules existing

in nature and many such molecules are unichiral in nature. For example chiral a-

amino acids, and the peptides and proteins containing them, sugars and their

polysaccharides, steroids, antibiotics and many other compounds from nature are

unichiral. Most of the biologically active molecules obtained from nature are

chiral, for ex. morphine, quinine etc.

Although they have the same chemical structure, most enantiomers of

racemic drugs exhibit marked differences in biological activities such 8s

pharmacology, toxicology, pharmacokinetics, metabolism etc. There are a number

of examples to state the importance of chirality in h g s and their physiological

effects. Thalidomide is a sedative drug that was prescribed to pregnant women,

from 1957 into the early 60s. It was marketed in at least 46 countries under

different brand names. When administered to pregnant women during the first

trimester of pregnancy, Thalidomide prevented the proper growth of the foetus,

resulting in horrific birth defects in thousands of children around the world. The

drug was found to cause teratogenicity and neuropathy. Because thalidomide

molecule is chiral and the molecule that administered was racemic. One c h i d

form was a sedative, whereas the other one was later found to cause foetal

abnormalities. The tragedy is claimed to have been entirely avoidable had the

physiological properties of the individual thalidomide enantiomers would have

been tested prior to commercialization.

Aspartame is a sweetening agent that is more than a hundred times sweeter

than sucrose. And yet, the other enantiomer molecule is bitter. (S)-carvone

possesses the odour perception of caraway while the other enantiomer (R)-corvine

has a spearmint odour.

Although enantiomers have the same chemical structure, most isomers of

chiral drugs exhibit marked differences in biological activities such as

pharmacology, toxicology, pharmacokinetics, metabolism etc. Some mechanisms

of these properties are also explained. The mechanism of interaction of the chiral

drug and the biological target is now being understood with the help of crystal

structures. Hence, it is very important to understand the role of individual

enantiomers at discovery stage. Thus helping to eliminate the unwanted

enantiomer at the clinical/discovery stage and select and enhance the activity and

reduce the side effects of the selected new chemical entity. This necessitates the

scientific community to enhance and acquire knowledge on enantio selective

synthesis and also equip with strong analytical skills to separate and quantitate the

enantiomers with speed and accuracy.

Among different ways of estimating enantiomers currently, HPLC is one

of the viable options. Different types of chiral stationary phases have been

developed for the separation of enantiomers. There are two types of c h i d liquid

chromatographic separation methods

i) D i t method

By direct diastereomer formation on stationary phase or by addition of

chiral selector in mobile phase

ii) Indirect method

By diastereomer formation by reaction with homochiral reagent

Direct separation method is now the preferred means of achieving c h i d

separation especially using chiral stationary phases whereas the use of chiral

selectors in mobile phases is an expensive process. Indirect method is now a thing

of past as it involves multiple reaction steps and may lead to racemisation during

this process. Thus use of high performance liquid chromatography (HPLC),

capillary electrophoresis (CE), Supercritical fluid chromatography (SFC) and gas

chromatography (GC) instrumentation with chiral stationary phases (CSPs) is the

current trend of chiral analysis both for analytical as well as preparative scale

separations. Different types of detectors have been employed to detect,

quantitative and identify the enantiomers after separation.

2.4.2 Chiral Stationary Phase (CSP)

Chiral Stationary Phase (CSP) is the heart of chiral separation. Various

types of CSPs have been developed and commercialized for the separation of the

racemic mixtures.

2.4.2.1 Macrocyclic glycopeptide antibiotics as CSPs

Macrocyclic glycopeptide antibiotics as CSPs were introduced by

Armstrong in 1994. The antibiotics used for chiral resolution are vancomycin,

vancomycin aglycon, teicoplanin, teicoplanin aglycon, ristocetin A, thistrepton,

rifamycin, fradiomycin, streptomycin, kanarnycin, and avoparcin. However, the

most commonly used antibiotics are vancomycin, teicoplanin, teicoplanin aglycon

and ristocetin A. All of these contain ionisable groups at different pH values in

their structures. Their molecular masses are between 1000 and 2100. The

glycopeptides are amphoteric, containing both ionisable acidic and basic groups.

Thus, they can be positively charged, negatively charged, or neutral depending on

the pH of the mobile phase.

Vancomycin, it is very effective for the enantiorecognition of anionic

compounds particularly those containing carboxylic acid group in their structure.

Its molecular weight of 1449 makes it the smallest of the three macrocyclic

glycopeptides. This selectivity is mainly due to the presence of m i n e groups in

the structure. There are 18 chiral centres in this molecule with 3 cavities. It has a

pendant, freely rotating, disaccharide moiety consisting of D-glucose and

vancosamine and an N-methyl amino acid side chain around three b e d

macrocyclic rings bridged by five aromatic rings linked by ether and peptide

bonds. Vancomycin has nine hydroxyl groups around the "basket shaped" aglycon

and on the attached disaccharide moiety, two mine groups (one primary and one

secondary), and one carboxylic acid group. There are five aromatic ring structures.

Hydrogen donor and acceptor sites are available close to the ring structure.

Several functional groups such as carboxylic, hydroxyl, amino, and amido are

mainly responsible for the ionization of these antibiotics in buffer with various

pHs and compositions and, therefore, it is enantioselective in nature.

Teicoplanin and teicoplanin aglycon: Teicoplanin is a macrocyclic

glycopeptide antibiotic that is structurally related to vancomycin and ristocetin A.

Teicoplanin exhibits a very slight anionic character even at acidic pH. There are

23 chiral centres in this molecule with four cavities. There are three sugar

moieties. Hydrogen donor and acceptor sites are available close to the ring

structures. It also contains a hydrophobic acyl side chain attached to a 2-amino-2-

deoxy-a D-glucopy~anosyl moiety, which activates its surface and enables the

formation of micellar aggregates. This structural feature is characteristic of

teicoplanin. These properties render teicoplanin highly stereo-specific in nature.

Teicoplanin without a sugar part is called teicoplanin aglycon antibiotic contains

almost all of the groups and cavities as in teicoplanin and therefore, has similar

chemical and physical properties. Teicoplanin aglycon does not show the

aggregational behaviour.

Ristocetin, it is the largest of all with a molecular weight of 2066. It has

the greatest number of stereogenic centres, i.e, 37. Ristocetin has structure very

similar to teicoplanin and vancomycin. An aglycon portion with four fused

macrocyclic rings, one tetramxhaxide moiety and six monosaccharides together

with 38 stemgenic centres characterizes it. It bas also seven aromatic rings, 21

63

hydroxyl groups, two primary amine groups, six arnido groups (amide linkages)

and one methyl ester. This adbiotic also contains three cavities.

2.42.2 Polysaccharide based chirnl stationary phases

Polysaccharides are the most abundant optically active natural polymers on

earth. These compounds themselves could not be used as commercial CSPs

because of their poor resolution capacity and problem in handling. The

polysaccharide polymers such as cellulose, amylase and chitin can be readily

derivatized to esters and carbarnates through reaction with acid chlorides and

isocyanates respectively. These polymer chains lie side by side in a linear fashion

in cellulose and in helical fashion in amylase. The CSPs based on polysaccharides

are some of the most popular and show a very high chiral resolving power and are

widely used for both analytical and preparative applications. About 20 derivatives

of amylase and cellulose are commercially available. The structures of some of

these CSPs used in this study are depicted in the following pages.

2.4.2.3 Pirkle-Type chiral stationary phases

The CSPs are broadly classified into two categories called 11-acceptor and

ll-donor phases. Whelk-01 and Whelk-02 are a "hybrid" type of this category,

which is a n-electron acceptor-donor CSP.

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