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BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr. 18: 759784 (2004) Published online in Wiley InterScience (www.interscience.wiley.com).
Simultaneous chiral analyses of multiple analytes DOI: 10.1002/bmc.447
REVIEW REVIEW 759
Simultaneous chiral analyses of multiple analytes: case studies, implications and method development considerations
Nuggehally R. Srinivas*
Drug Development, Discovery Research, Dr Reddys Laboratories, Bollaram Road, Miyapur, Hyderabad 500 049, India Received 9 August 2004; accepted 2 September 2004
ABSTRACT: The eld of chiral separations had a modest beginning some two decades ago. However, due to rapid technological advancement coupled with simultaneous availability of innovative chiral stationary phases and novel chiral derivatization agents, the eld of chiral separations has now totally outpaced many other separation elds. Keeping pace with rapid changes in the eld of chiral separations, investigators continue to add stereoselective pharmacokinetic, pharmacodynamic, pharmacologic and toxicological data of new and/or marketed racemic compounds to the literature. Examination of the evolution of chiral separations suggests that in the beginning many investigators attempted to separate and quantify a single pair of enantiomers, adopting either direct (separation made on a chiral stationary phase) or indirect (separation made following precolumn conversion of enantiomers to corresponding diastereomers) approaches. However, more recent trends in chiral separations suggest that investigators are attempting to separate and quantify multiple pairs of enantiomers with available technologies. Added to this, some interesting trends have been observed in many of the recently reported chiral applications, including preferences regarding internal standard selection, mobile phase contents and composition, sorting out issues with mass spectrometric detection, determination of elution order, analytical manipulations of metabolite(s) without reference standards and addressing some specicity-related issues. This review mainly focuses on chiral separations involving multiple chiral analytes and attempts to justify the need for such chiral separations involving multiple analytes. In this context, several cases studies are described on the utility and applicability of such chiral separations under discrete headings to provide an account to the readership on the implications of such tasks. The topics of case studies covered in this review include: (a) therapy markersdifferentiation from drug abuse and/or applicability in forensics; (b) role in pharmacogenetic/polymorphic evaluation; (c) monitoring and understaning the role of parent and active metabolite(s) in clinical and preclinical investigations; (d) exploration on the pharmacokinetic utility of an active chiral metabolite vis--vis the racemic parent moiety; (e) understanding the chirality play in delineating peculiar toxic effects; (f) exploration of chiral inversion phenomenon, and understanding the role of stereoselective metabolism. For the further benet of readership, some select examples (n = 19) of the separation of multiple chiral analytes with appropriate information on chromatography, detection system, validation parameters and applicable conclusion are also provided. Finally, the review covers some useful considerations for method development involving multiple chiral analytes. Copyright 2004 John Wiley & Sons, Ltd. KEYWORDS: chiral separations; multiple chiral analytes; chiral stationary phases; chiral derivatization agents; review
*Correspondence to: N. R. Srinivas, Drug Development, Discovery Research, Dr Reddys Laboratories, Bollaram Road, Miyapur, Hyderabad 500 049, India. E-mail: nrsrinivas@drreddys.com DRL publication no. 442. Abbreviations used: 5HMP, 5-hydroxymethoxyphenamine; AGP, -acid glycoprotein; AUC, area under the plasma concentration vs time curve; CBH, cellobiohydrolase; CD, circular dichroism; Cmax, peak plasma concentration; COOH, carboxyl group; CV, coefcient of variation; CYP, cytochrome; DHD, carbamazepine-10,11-transdihydrodiol; DMOA, dimethyloctylalmine; EDDP, 2-ethylidene-1,5dimethyl-3,3-diphenyl-pyrrolidine; ESI, electrospray ionization; FR, ow rate; GITC, 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate; HME, N-ethyl-4-hydroxy methyoxyamphetamine; LOD, lower limit of detection; LLOQ, lower limit of quantitation; MDA, methylenedioxyamphetamine; MDE, N-ethyl-3,4methylenedioxyamphetamine; MHD, 10-hydroxycarbazepine; NCE, novel chemical entity; NDMP, N-desmethylmethoxyphenamine; NSAID, nonstereoidal anti-inammatory drug; 5-OHPPF, 5-hydroxy propafenone; ODMP, O-desmethylmethoxyphenamine; PPF, propafenone; RT, room temperature; SIM, single ion monitoring; SPE, solid phase extraction; SRM, selective reaction monitoring. Copyright 2004 John Wiley & Sons, Ltd.
INTRODUCTION
The presence of chirality in xenobiotics has offered a signicant potential for growth in the development of stereoselective analytical methods, which are needed for the separation and quantication of individual chiral components. The applicability of both direct and indirect methods for chiral analysis of some important racemic medicinal compounds has been reviewed (Srinivas, 2004a,b; Ali and Aboul-Enein, 2003; Haginaka, 2002; Bojarski, 2002; Toyoka, 2002; Sun et al., 2001; Srinivas et al., 1995; Ward, 2002). Both these approaches have withstood the test of time in terms of continued applicability in the eld of chiral chromatographic separations, as evidenced by the introduction of novel chiral derivatization reagents and/or chiral stationary phases. Such recent developments have paved the way for a
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better understanding of the pharmacokinetic, pharmacodynamic and/or toxicological implications of the individual components of the racemate (Srinivas, 2001; Baumann et al., 2002; Burke and Henderson, 2002; Rentsch, 2002; Wainer, 2001; Williams and Wainer, 2002). Additionally, such information has further provided insights for the selection of the appropriate chiral candidate (racemate vs enantiomer) for developmental consideration (Hutt and OGrady, 1996; Srinivas et al., 2001; Baumann et al., 2002; Benedetti et al., 2002). The signicant progress achieved in scientic and technical elds pertaining to chirality is commensurate with the stringent regulatory guidelines on the development of stereoisomeric drugs by regulatory agencies (Birkett, 1989; De Camp, 1989; Srinivas et al., 2001). An examination of the literature on chromatographybased chiral analytical methods revealed that methods were generally developed in the past to facilitate the separation and quantitation of one set of enantiomers. However, in more recent years, the eld has evolved and attention is being paid to the separation and quantication of multiple chiral analytes. This new strategic approach ts well with the need for thorough understanding of the issue or question being considered. For example, to claim that the parent drug is undergoing stereoselective oxidative metabolism it may not be adequate to measure the parent entity alone to show differences in the levels of the parent enantiomers (before and after the reaction or incubation for comparison purposes); it may be necessary to support the claim by measuring the purported metabolite(s) and showing that there is a correlation between the disappearance of the parent enantiomers and the formation of the new metabolite(s). This is illustrated by the work of Zhang et al. (1999), wherein it was unequivocally demonstrated under in vitro conditions that the amount of chiral -hydroxysalmeterol formed was equal to the amount of the loss in the parent salmeterol substratethis also indicated that hydroxyl metabolite was the only product produced upon incubation with human liver microsomes. Since recent reviews in the eld of chiral separations and applications cover exhaustively the analytical aspects of both direct and indirect methods, the focus of this review is to emphasize the need to generate a chiral method that could be used for more than just separating and analyzing a given pair of enantiomers. Therefore, the scope of this review extends to identication of several scenarios where the need to analyze multiple chiral analytes is of paramount importance. Appropriate case studies with relevant clinical/preclinical implications alongside method developmental considerations will also be covered in this review.
Copyright 2004 John Wiley & Sons, Ltd.
THERAPY MARKERSDIFFERENTIATION FROM DRUG ABUSE AND/OR APPLICABILITY IN FORENSICS

Case study of selegiline Selegiline undergoes rapid metabolism with the formation of metabolites such as desmethylselegiline, methamphetamine and amphetamine upon oral administration. These metabolites of selegiline were observed in pure isomeric forms [i.e. ()-enantiomer] in both plasma and urine. Owing to the non-detection of the parent selegiline in either plasma or urine, it is clinically important to monitor the levels of several of the metabolites to monitor the progress of seregiline therapy. Additionally, monitoring of metabolites of selegiline is vital to distinguish methamphetamine abusers. In this regard, one may consider the use of desmethylselegiline to serve as a marker for distinguishing between selegiline therapy and methamphetamine abuse. However, this metabolite appears to disappear rapidly from plasma and the amount excreted in urine is in low and variable quantities (<1% of the administered dose). In this situation, a plasma sample withdrawn after several hours following selegiline therapy may not show levels of desmethylselegiline, therefore making it difcult to distinguish between clinical use of selegiline and methamphetamine abuse. In contrast, the distinction could be easily achieved if one paid attention to the chirality of the multiple analytes. The urine of methamphetamine abusers carries the (+)enantiomer of both amphetamine and methamphetamine. Therefore, development of a senstive chiral method-based gas chromatography to discriminate isomeric forms of multiple analytes was considered the best option to discriminate between clinical therapy and methamphetamine abuse. Additionally, the authors focused on developing a direct method instead of an indirect approach where there may have been an opportunity for the introduction of spurious peaks of the antipode, thereby compromising the distinction between the two groups (Hasegawa et al., 1999). Case study of new ecstasy analog Recently Brunnenberg and Kovar (2001) described a chiral chromatographic method based on -cyclodextrin column for the separation of multiple analytes. In their report, the chiral method was capable of separating the enantiomers of the ecstasy analog, (R,S)-N-ethyl3,4-methylenedioxyamphetamine, and its metabolites, O-glucuronyl-(R,S)-N-ethyl-4-hydroxy-3-methoxyamphetamine and (R,S)-3,4-methylenedioxyamphetamine. On the basis of this evaluation, the authors concluded that the new ecstasy analog undergoes stereoselective disposition in humans with the levels of R-enantiomer of
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N-ethyl-3,4-methylenedioxyamphetamine predominantly found in plasma. Meanwhile, the S-enantiomers of Oglucuronyl-N-ethyl-4-hydroxy-3-methoxyamphetamine and 3,4-methylenedioxyamphetamine were reported to dominate in the plasma (Brunnenberg and Kovar, 2001).
ROLE IN PHARMACOGENETIC/POLYMORPHIC EVALUATION

Case study of stereoselective analysis of citalopram and its metabolites Recently, Holmgren et al. (2004) have developed a sensitive chiral liquid chromatographic assay for the enantiomers of citalopram and its metabolites, desmethylcitalopram and didesmethylcitalopram. The assay was utilized to measure the concentrations of the various analytes in several forensic autopsy cases to enable the genotyping for the polymorphic CYP2C19 and CYP2D6 isozymes. In the samples examined, the stereoselective S/R ratio for citalopram concentrations was approximately 0.67 and a similar S/R ratio of 0.68 was noted for the desmethylcitalopram. The authors found that increasing citalopram concentrations tended to increase S/R ratios and, likewise, led to an increased desmethylcitalopramcitalopram ratio. The authors concluded that, given the importance of citalopram usage, and with the added caveat of two polymorphic CYP isozymes involved in its metabolism, enantioselective analysis would provide vital information for the interpretation of forensic toxicology ndings. Case study of altered pharmacokinetics of mianserin and desmethylmianserin in presence of thioridazine, a CYP2D6 inhibitor (Yasui et al., 1997) A sensitive and selective liquid chromatographic assay was developed for the simultaneous quantication of the enantiomers of mianserin and desmethylmianserin in clinical plasma samples. Since mianserin and its active metabolite, desmethylmianserin, undergo stereoselective metabolism via CYP2D6 isozyme, it was important to understand the kinetic disposition of the multiple analytes when a well-known CYP2D6 inhibitor was introduced in the therapy. Therefore, a study was designed to address the effects of thioridazine, a specic CYP2D6 inhibitor, on the stereoselective disposition of both mianserin and desmethylmianserin at steady state in a cohort of depressed patients. The inhibitory role played by thioridazine was demonstrated by increased plasma concentrations of the S-enantiomer of mianserin (almost 2-fold increase from 78.2 to 150.8 nm), although the steady-state concentrations of the R-enantiomer appeared to be relatively stable before (39.8 nm) and
after (39.5 nm) thioridazine treatment. The effects of thioridazine appeared to be more dramatic for the active metabolite desmethylmianserin, where steady-state plasma concentrations of both enantiomers increased 23-fold when compared with pre-treated values. For example, after thioridazine treatment, the values for Rand S-enantiomers were 115.6 and 24.4 nm, respectively, as compared with the corresponding values of 42.6 and 11.9 nm, respectively, prior to thioridazine treatment. Therefore, availability of an assay to monitor multiple chiral substrates delineated the stereoselective inhibitory role of a CYP2D6 inhibitor. Case studystereoselective analysis of mephenytoin and its metabolites for polymorphic investigations of CYP2C19 and CYP2B6 isozymes Although CYP2B6 is a relatively minor isozyme as compared with other drug-metabolizing CYP isozymes, considerable variability has been reported due to the observations of several mutations in the expression of this isozyme. In the context of this isozyme, Smephenytoins N-demethylation pathway has been used to characterize the in vitro CYP2B6 activity, while Smephenytoins oxidative pathway leading to the formation of the 4-hydroxy mephenytoin has been used to characterize the in vitro CYP2C19 activity. Recently, Jansson et al. (2003) have published a stereoselective liquid chromatographic method utilizing a chiral AGP column for the separation of enantiomers of mephenytoin and the enantiomers of two metabolites, namely nirvanol (N-demethylated product) and 4hydroxymephenytoin. The utility of this method can be extended for monitoring both CYP2C19 and CYP2B6 enzyme activities using mephenytoin as the probe substrate. Additionally, use of the stereoselective assay in generating the plasma proles of the various analytes from extensive and poor metabolizers of CYP2C19 clearly demonstrated the value of using mephenytoin as the probe substrate (Jansson et al., 2003).
MONITORING OF PARENT AND ACTIVE METABOLITE(S) IN CLINICAL AND PRECLINICAL INVESTIGATIONS

Case study of the measurement of esmolol and its acid metabolite in human plasma Tang et al. (2004) described a reversed-phase liquid chromatography coupled with solid phase extraction for the separation and quantitation of esmolol and its metabolite in plasma. The assay followed an indirect method approach by employing 2,3,4,6-tetra-O-acetyl -d-glucopyranosyl isothiocyanate as the chiral derivatization reagent. Since esmolol is an ester drug, the availability of a stereoselective method for the
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determination of the parent and the acid metabolite permitted an in-depth investigation of the hydrolytic potential of esmolol in plasma (Tang et al., 2004). Case study of the measurement of uoxetine and noruoxetine enantiomers in pregnant sheep Kim et al. (2004) performed an elegant study of the stereoselective disposition of uoxetine and noruoxetine in pregnant sheep. In the experimental design, both the ewe and the fetus received an intravenous dose of uoxetine. In addition to maternal and fetal blood, other samples such as fetal amniotic and tracheal uid and maternal urine were collected for the analysis of the parent and noruoxetine enantiomers as well as the corresponding glucuronic acid conjugates. A stereoselective gas chromatographic assay employing mass spectrometric detector was employed to measure the levels of the multiple chiral analytes. While both maternal and fetal blood revealed identical stereoselective disposition of uoxetine enantiomers when administered separately to the ewe and fetus (S/R AUC ratio of approximately 1.65 and 1.73 in ewe and fetus, respectively), there was no formation of noruoxetine in the fetus upon uoxetine administration to fetus and the phase II glucuronic acid conjugation of uoxetine or noruoxetine was absent in the fetal sample. In contrast, the maternal urine following uoxetine revealed the presence of noruoxetine as well as the glucuronides of both uoxetine and noruoxetine. The nding of lack of formation of noruoxetine in the fetus was not surprising because the authors had previous experimental evidence that the fetal liver microsomal incubations revealed lack of conversion of uoxetine to noruoxetine. This particular case study serves as an example of employment of a multiple chiral analyses method having the potential to answer several key questions and bridging the in vitro investigations to in vivo ndings (Kim et al., 2004). Case study of nefopam and desmethylnefopam enantiomersmaking a call on the route of administration Chawla et al. (2003) reported a chiral assay using liquid chromatographic procedure coupled with mass spectrometry for the simultaneous determination of nefopam and its active metabolites, desmethylnefopam enantiomers, in plasma. Since nefopam is used parenterally, little is known about oral administration of this compound, therefore a clinical study was designed to answer questions such as the pharmacokinetic fate of nefopam after oral delivery, formation of the active metabolite in plasma and likely differences in the formation of the active metabolite between oral and intravenous routes. Generally it appeared that the parent
molecule exhibited no stereoselectivity with the comparable plasma concentration vs time curve between the enantiomers after oral or intravenous administration, which translated into similar clearance [53.7 and 57.5 L/h for (+)- and ()-nefopam, respectively] and volume of distribution values [390 and 380 L for (+)- and ()-enantiomers, respectively] after intravenous and bioavailability values [44 and 42% for (+)- and ()-nefopam, respectively] after oral administration. The rst-pass effect was evident after oral administration of nefopam and this perhaps contributed to higher levels of the metabolite desmethylnefopam following oral administration as compared with the intravenous route. Stereoselectivity was apparent in the levels of the desmethylnefopam regardless of the route of administration. One of the important conclusions drawn from this study was that the oral dosing of nefopam was required to achieve higher concentrations of desmethylnefopam and therefore this metabolite can be expected to contribute to nefopams activity only when nefopam is given orally. Case study of oxybutynin and N-desethyloxybutynin enantiomers in oral and transdermal applications A sensitive triplequad liquid chromatographic stereoselective assay was developed for the simultaneous monitoring of the enantiomers of oxybutynin and Ndesethylbutynin in plasma from clinical samples. This investigation examined the stereoselective pharmacokinetics of both oxybutynin and N-desethylbutynin following oral and transdermal delivery of oxybutynin in healthy subjects employing a crossover design format (Zobrist et al., 2001). A pronounced route-dependent stereoselective disposition of oxybutynin was evident in the kinetic parameters. The oral route indicated a pronounced presystemic metabolism of oxybutynin with signicant levels of the N-desethylbutynin enantiomers in the circulation as compared with those observed from the transdermal application. Therefore, application of a stereoselective assay enabled the differentiation of metabolism pattern as well as pharmacokinetic parameters between oral and transdermal routes of delivery of oxybutynin.
EXPLORATION OF THE PHARMACOKINETIC UTILITY OF AN ACTIVE METABOLITE vis-vis THE PARENT MOIETY
Case study of desbutylhalofantrine Using a rat model, the pharmacokinetic disposition of desbutylhalofantrine was evaluated following both intravenous and oral routes of administration (Brocks, 2000). A sensitive and selective stereoselective assay
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was utilized to measure the enantiomers of desbutylhalofantrine and halofantrine in plasma samples (Brocks, 2001). Although elimination half-life values appeared to be comparable between the two enatiomers after either intravenous or oral dosing (range 1423 h), the plasma concentration vs time curve exhibited stereoselectivity. Typically, the levels of the (+)desbutylhalofantrine were greater than those of the ()enantiomer such that (+)/() AUC ratios were approximately 3.7 and 2.8 for oral and intravenous treatments, respectively. A similar 3-fold difference in the ratio of ()/(+)-desbutylhalofantrine was noted for both clearance and steady-state volume of distribution parameters following intravenous dosing. It appeared that more of desbutylhalofantrine was bioavailable (>59%) as compared with the bioavailability of the parent in the same animal species (Brocks, 2000). Additionally, desbuylhalofantrine did not exhibit pronounced stereoselectivity in the rat model, which was evident with halofantrine. Overall, availability of a stereoselective assay revealed that an active metabolite may possess a favorable pharmacokinetic prole that perhaps may be worthy of consideration for development.
administered radioactivity of the parent molecule [4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone] was reported to be localized in the lung. The authors have postulated the likely contribution of the accumulation of the S-enantiomer to the observed selectivity of the metabolite (4-methylnitrosoamino)-1-(3-pyridyl)-1butanone in causing lung carcinoma (Wu et al., 2002).
EXPLORATION OF CHIRAL INVERSION PHENOMENON AND UNDERSTANDING THE ROLE OF STEREOSELECTIVE METABOLISM
Case study of stereoselective disposition and metabolism of obufen (Trejtnar et al., 2003) Flobufen is a novel chiral nonstereoidal inammatory agent. The metabolism of obufen has been reported to be complex with stereoselectivty and formation of a plethora of stereoisomeric metabolites. To understand the disposition of obufen, a stereoselective and sensitive chiral assay procedure was developed for the qunatitation of obufen and four of its isomeric dihydrometabolites. It was apparent that after oral administration the levels of S-enantiomer were much higher than those of the R-enantiomer, leading to the attainment of an S/R plasma concentration ratio of approximately 10. One of the metabolites, namely, 4(2,4-diuorophenyl)-phenylacetic acid, appeared to possess a longer residence time, even exceeding those of the enantiomers of obufen. It has been postulated that this metabolite may contribute to the pharmacological activity of obufen. Case study chiral disposition of methoxyphenaime and its oxidative metabolites in urine The development of a sensitive and enantioselective gas chromatographic assay enabled the measurement of methoxyphenamine and its three primary metabolites in human urine (Srinivas et al., 1989). Methoxyphenamine is metabolized in animals and humans by three distinct primary oxidative pathways, namely Ndemethylation to form N-desmethylmethoxyphenamine (NDMP), O-demethylation to form O-desmethylmethoxyphenaime (ODMP), and 5-hydroxylation to form 5-hydroxymethoxyphenamine (5-HMP). Althoug methoxyphenamine is known to show genetic polymorphism in its metabolism in man, hitherto no work has been carried out to evaluate the stereoslective nature of its disposition. Therefore, the availability of a chiral method prompted the investigation into the chiral aspects of the urinary disposition of methoxyphenamine, NDMP, ODMP and 5HMP in humans. Following a single oral dose of methoxyphenamine, marked enantioselectivity was observed in the urinary levels of
UNDERSTANDING THE ROLE CHIRALITY PLAYS IN DELINEATING A PECULIAR TOXIC EFFECT

Case study of 4-(methylnitrosoamino)-1-(3pyridyl)-1-butanone in rats to understand the mechanism of lung carcinogensis A study was undertaken to evaluate stereoselective disposition and pharmacokinetic characterization, including tissue distribution aspects of 4-(methylnitrosoamino)1-(3-pyridyl)-1-butanone, a chiral molecule and its primary metabolite 4-(methylnitrosoamino)-1-(3-pyridyl)1-butanone, a major carcinogen identied in tobacco smoke (Wu et al., 2002). A combination of chiral assay employing a gas chromatographicthermal energy analyzer and gradient ow liquid chromatography with radioow detection system was employed to carry out the bioanalytical work in assessing the plasma, urine, bile and tissue concentrations of the various chiral analytes (Wu et al., 2002). The results supported pronounced enantioselectivity in the pharmacokinetic disposition of the enantiomers of 4-(methylnitrosoamino)1-(3-pyridyl)-1-butanone. The phase II glucuronic acid conjugatge of the R-enantiomer was identied as the major metabolite. One important observation that came out of this investigation was the stereoselective uptake of S-4-(methylnitrosoamino)-1-(3-pyridyl)1-butanone in the lung tissue (S to R-enantiomer concentration ratio in the lung tissue was reported to be greater than 20). This particular observation gains importance from the fact that almost 75% of the
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methoxyphenamine, NDMP and 5HMP. However, the levels of ODMP appeared to be nonstereoselective in urine. Since methoxyphenamine is known to show genetic polymorphism (Muralidharan et al., 1991), the ndings of enantioselective disposition of methoxyphenamine and two of its three oxidataive metabolites add further complexity and challenges in understanding the metabolic aspects of this drug in extensive and poor phenotypes. Case study of lack of chiral inversion of ragaglitazar in rats During the development of ragaglitazar, an antidiabetic and dyslipidemic agent, a study was undertaken to ascertain the likelihood of chiral inversion of ragaglitazar in toxicology species (i.e. the propensity to generate the opposite enantiomer in vivo was unknown). Mustafa et al. (2004) developed a chiral liquid chromatographic method based on Chiralpak column to quantify both the S-()- and R-()-enantiomers in rat plasma. This newly developed chiral assay was used to unequivocally conrm that ragaglitazar does not undergo chiral inversion to its other antipode in vivo in rat plasma following oral administration (Mustafa et al., 2004). Case study of chiral inversion of pantoprazole enantiomers in rats Pantoprazole is a racemic drug used in the clinic. A study was performed to understand the disposition kinetics including the chiral inversion phenonmenon when the two enantiomers of pantoprazole were separately administered to rats (Masubuchi et al., 1998). A sensitive method was developed to quantify the levels of the enantiomers of pantoprazole, pantaprazole sulfoxide, 4-O-demethyl pantoprazole sulfone and 4O-demethyl pantoprazole sulde using liquid chromatography. The results indicated signicant chiral inversion following the administration of (+)-pantoprazole dosing to rats regardless of the route of administration [approximately 2836% of the total exposure was represented by the ()-enantiomer]. However, no plasma levels of the (+)-pantoprazole were observed following either intravenous or oral administration of ()-pantoprazole to rats. Therefore, the utility of a stereoselective assay delineated the unidirectional inversion of the (+)-pantoprazole to the opposite enantiomer in the rat.
METHOD DEVELOPMENT CONSIDERATIONS

Sample clean-up and sample preparation Simple liquidliquid extraction procedures with acidied or basied plasma are adequate to draw the
enantiomers of interest including the internal standard from the biological matrix. Often, the investigator may also introduce a back-extraction procedure to limit the endogenous interference from the biological matrix. This clean-up procedure may hinder the recovery of the analytes; it also allows greater selectivity and specicity in the analysis. Additionally, it may contribute to a longer shelf-life of the analytical column and, perhaps, prevent frequent build-up of back pressure that may cause deterioration of the performance of the liquid chromatography. Since multiple chiral analytes are being dealt with, it is important to choose buffer systems that are mild in nature and do not cause an artifactual racemization of the enantiomeric analytes and/or stability issues of chemical instability of the analytes. Frequently, multiple extraction steps are needed to eliminate endogenous interferences from the plasmaserum matrix. For example, in the initial stages of method development for ketamine and norketamine enatiomers, it was found that one-step extraction with either 2.5 m sodium hydroxide or 1 m carbonate buffer (pH 10.5) resulted in an interfering plasma peak (Yanagihara et al., 2000); however, washing three times with a combination of the two alkali buffers and cyclohexane solvent minimized this interference (Yanagihara et al., 2000). Additionally, the use of cyclohexane greatly improved the recovery of both ketamine and norketamine enantiomers (>85%) compared with other solvents such as n-hexane (approximately 60% recovery) and methylene chloride (approximately 40% recovery; Yanagihara et al., 2000). When one chooses a solid-phase extraction procedure, there is an abundant choice of simple to specialized SPE cartridges. Appropriate choice and composition of washing and eluting solvents will determine the recovery and cleanliness of the nal product prior to column injection for analysis. It is important to note that the washing solvents should have selectivity towards the endogenous and/or extraneous matters and should ordinarily not elute the analytes of interest. This subject has been extensively reviewed. Column-switching technique enables one to have an efcient on-line sample clean-up operating system. Certain requirements need to be fullled by the mobile phase used for sample clean-up procedures: (a) an inability to denature proteins; (b) buffering capacity that maintains a physiological pH value; (c) organic solvents kept to a minimum (<15%); and (d) weak elution capability to ensure analyte retention on the pre-column. Phosphate-based buffers are commonly employed for on-line clean-up experiments. In some specic examples, due to its lack of compatibility with ESI-MS detection, a different buffering system needs to be employed. A system based on ammonium acetate with an appropriate molarity can be readily employed to overcome this problem. One important aspect of column
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switching relates to the optimization of the time factor for efcient clean-up and subsequent analytical separation (Hasegawa et al., 1999). A premature window of time may not allow for the passage of the so-called extraneous and endogenous materials from the loaded sample and thus may cause problems in the analytical column which employs relatively higher concentrations of the organic solvents. Therefore, the window chosen should be such that it balances between clean-up and short run time for analysis of multiple analytes. Overall, since complexity of sample extraction and clean-up will increase with the analysis of multiple analytes owing to the differences in the respective physicochemical properties, caution needs to be exercised in optimization steps. The work of Lamprecht et al. (2000) describes adoption of efcient column switching to permit the online extraction and separation of atenolol enantiomers following the introduction of diluted urine samples. Thorough optimization was needed to reduce the blank reading which otherwise would have hindered the separation and quantitation of atenolol enantiomers on the teicoplanin column. Careful monitoring of pH, selection of buffer systems and small additions of triethylamine and Tris(hydroxymethyl)aminomethane proved to be useful in reducing the inuence of blank reading to a negligible level (Lamprecht et al., 2000). Xia et al. (2003) have utilized an automated dualcolumn online extraction for the analysis of enantiomers of propranolol, which involved the direct introduction of a small quantity of the biological sample onto one of the extraction columns while the second column was left for equilibration. The approach of using two columns for online extraction helped to avoid delay in the process. Both the columns used in this study showed sufcient ruggedness and sensitivity a total of 300 or more injections were possible without loss of performance (Xia et al., 2003). Internal standard preferences Regardless of the method of extraction adopted, the choice of internal standard can be made based on the recent suggestions of Srinivas (2004a), wherein an opportunity exists for the selection of a closely related analog (single enantiomer or racemate) or a stable label compound of one of the principal analyte(s). If multiple analytes are present, the question that needs to be answered is which analyte to choose in making a stable label compounda solution for this could be to choose an analyte that is commonly seen in all the samples and represents the longest in the systemic circulation. Additionally, the cost and ease of synthesis may also dictate the selection of the stable label internal standard of a given analyte. Another interesting variant has been introduced by Ulrich (2003) in
liquid chromatographic analysis, where two structurally diverse compounds have been used as internal standards (uvoxamine and nisoxetine) for the stereoselective quantitation of uoxetine and noruoxetine. Some 14 years ago, Srinivas et al. (1989) used two diverse compounds, amantadine hydrochloride and dephedrine, as internal standards in chiral analysis using gas chromatography. Although the gas chromatographic column, the composition of carrier gas/make-up gas, ow rates and head pressure on the column were similar, they adopted isothermal conditions for the separation of methoxyphenamine and NDMP, while program temperature-run chromatography was used for the separation of ODMP and 5HMP. Another concept of internal standard selection is to select the opposite antipode as the internal standard for an optically pure single enantiomeric compound after conrming the absence of in vivo conversion of the active enantiomer. An example is provided by the work of Isoherranen et al. (2000), wherein the antipode to levetiracetam, an anticonvulsant agent (chemically S--ethyl-2-oxopyrrolidine acetamide), can be used as an internal standard. In this work, since piracetam (2-oxo-1pyrrolidine acetamide) appeared to possess less than ideal characteristics in terms of its elution and chromatographic properties, the R--ethyl-2-oxo-pyrrolidine acetamide enantiomer would be a better choice as an internal standard. Furthermore, the lack of in vivo conversion of levetiracetam to its antipode supported this selection (Isoherranen et al., 2000). Interestingly, the recent work of Buechler et al. (2003) points to the fact that structurally closely related analogs may not necessarily possess the appropriate chromatographic properties for use as internal standards. In the stereoselective determination of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites, it was necessary to synthesize a new compound, N-ethyl-3,4methylenedioxybenzylamine, to serve as an internal standard to overcome the chromatographic issues associated with structurally related analogs (Buechler et al., 2003). Mobile phase and chromatography Since the literature is lled with information pertaining to both direct and indirect approaches, a literature review of the previous methods may be a quick rst step. For example, Teng et al. (2003) reported a stereoselective procedure with thorough validation only for the separation of ibuprofen enantiomers in rat serum; however, the authors have done enough groundwork to support the use of the method for other NSAIDs in therapy. Several NSAIDs, such as ketolorac, urbiprofen and cicloprofen, were fully resolved under the chromatographic conditions employed for the separation of ibuprofen enantiomers (Teng et al., 2003). For
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example, the use of protein-based chiral columns like chiral-AGP has resulted in some overlaopping peaks between parent and metabolite enantiomers. However, these issues of co-elution have been circumvented by using achiral and chiral chromatography, which imparts a greater degree of selectivity for analytical separation. However, the use of achiralchiral chromatography is typically associated with large baseline disturbances due to the altered composition of mobile phase systems used for the two systems. With this consideration in mind, Kim et al. (2001) have optimized the mobile phase systems for the separation of terbutaline enantiomers and betaxolol, used as an internal standard. Additionally, optimization of the mobile phase in separation of terbutaline enantiomers was required to circumvent the overlapping of an interference peak (Kim et al., 2001). Several case studies are provided to cover the aspects of multiple chiral analytes separation. Separation of mephenytoin, nirvanol and 4-hydroxymephenytoin enantiomers (Jansson et al., 2003). In the separation of enantiomers of mephenytoin, nirvanol and 4-hydroxymephenytoin, a previously used mobile phase comprising 0.3% 1-propanol in sodium phosphate buffer (pH = 7; ionic strength of 0.1) separated the individual pairs of enantiomers when run on an AGP column. However, when the mixture of three racemates (i.e. six enantiomers) was run on the AGP column at the same time, S-mephenytoin co-eluted with the peak corresponding to R-nirvanol (Jansson et al., 2003). Certain organic modiers such as methanol and acetonitile with or without the presence of tetrahydrofuran did not appear to resolve the co-elution issue. Other variants appeared to be detrimental, e.g. decreasing ionic strength of the mobile phase in multiple successive steps from 0.1 to 0.01, they decreased resolution between S- and R-enantiomers of mephenytoin although it appeared to increase resolution between R-nirvanol and S-mephenytoin. When the pH changes were considered, the resolution between S- and Renantiomers was signicantly hampered as the pH dropped from 7 to 6. The authors then focused their attention towards coupling several reversed-phase columns in series to the AGP chiral column. Separation of propafenone and its metabolite (Zhong and Chen, 1999). The strategy applied for the analysis of propafenone and 5-hydroxypropafenone was rst to separate the two analytes on a Nucleosil C18 column. The eluates were separately collected, extracted and injected onto a chiralAGP column to perform chiral analysis. Some interesting mobile phase optimizations were essential for the separate analysis of propafenone and 5-hydroxypropafenone enantiomers. The choice of organic modiers such as 2-propanol or acetonitrile in the mobile phase (10 mm ammonium acetate buffer; pH
5.96) did not completely resolve the enantiomers of propafenone. However, the inclusion of 1-propanol in the same mobile phase dramatically improved the resolution of propafenone enantiomers such that at a concentration of 2.5% of 1-propanol the resolution was about 2.4 while a change in concentration to 8.2% of 1-propanol resulted in a decreased resolution (1.7). In the case of 5-hydroxypropafenone enantiomers, the use of 2-propanol was adequate for the resolution. Using the same mobile phase (i.e. 10 mm ammonium acetate buffer) with 0.9% 2-propanol as organic modier, resolution of 5-hydroxypropafenone enantiomers increased from 2.1 to 2.3 on increasing the pH from 4.1 to 4.5. However, this change in resolution produced a dramatic change in retention times for the R-enantiomer (9.418.7 min) and S-enantiomer (11.7 and 22.3 min). Separation of verapamil and norverapamil enantiomers (Hedeland et al., 2004). Owing to the non-applicability of a previously published of chiral AGP-based separation of verapamil and norverapamil enantiomers, Hedeland et al. (2004) performed some variants in mobile phase optimization to suit the conditions for employing electrospray mass spectrometry. The method originally published by Sandstrom et al. (1999) contained phosphate buffer-based mobile phase with acetonitrile as the organic modier (pH 7). As a result of the poorly volatile nature of the phosphate buffer, this mobile phase was unsuitable for electrospray mass spectrometric detection. Hedeland et al. (2004) adopted a similar procedure of using ammonium acetate, which is a friendlier option for electrospray mass spectrometer. Although the buffering capacity of ammonium acetate was inferior to that of phosphate buffer, it provided chiral separation between the enantiomers of verapamil and norverapamil. It was found that the second eluting peak of verapamil overlapped with the rst eluting peak of norverapamil. However, this did not pose a problem for quantitation since the molecular masses were distinct between the co-eluting pairs. The authors further tweaked the pH of the mobile phase to 7.4 to improve upon the resolution aspects for both verapamil and norverapamil enantiomers. Overall, the newly modied mobile phase system by Hedeland et al. (2004) provided an alternative option for chiral separation of verapamil and norverapamil enantiomers instead of a more cumbersome achiralchiral column switching method and/or chemical derivatization method. Separation of fenuramine and norfenuramine enantiomers following pre-column derivatization (Kaddoumi et al., 2001). In this report, a systematic investigation regarding the effects of different buffers and pH of the mobile phase was carried out on the achiral derivatized enantiomeric analytes on the chosen
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Chiralcel chiral column. The various buffers that were attempted included acetate, citrate, perchlorate and Tris and, regardless of the buffer system employed, the retention time on the column increased as a function of increased concentration and buffering pH. With the exception of perchlorate buffer, where slight resolution was observed between d-enantiomers of fenuramine and norfenuramine, the other buffers failed to resolve the two enantiomeric peaks. The authors then improvised by combining the buffer systems to optimize the resolution between the two enantiomeric peaks. In this process, a mixed buffer system consisting of citrate buffer (0.01 m, pH 2.7) and perchlorate buffer (0.05 m, pH 2.0), produced the best separation between the two enantiomeric peaks (resolution was approximately 0.94). Addressing specicity related issues Example of separation of uoxetine and noruoxetine enantiomers (Gatti et al., 2003). While developing the method for an effective and selective separation of uoxetine and noruoxetine enantiomers, the authors have paid attention to the several concomitant drugs that are typically coprescribed with uoxetine. The list of compounds evaluated for specicity included clomipramine, amitriptyline, nortriptyline, carbamazepine, clozapine, desipramine, dextromethorphan, diazepam, doxepin, imipramine, phenobarbital, phenytoin and valproic acid. The choice of extraction system employed in this study prevented the extraction of acidic compounds. In spite of careful selection and optimization of chromatographic conditions, some compounds, such as desipramine, doxepin and carbamazepine, co-eluted with the peaks of interest. Since this may present an issue in the analysis of samples from patients who are on therapy with the aforementioned compounds, the authors have tackled this potential problem by either reducing the proportion of the organic solvent and/or by adjusting the pH of the mobile phase. Example of separation of methadone enantiomers. Liang et al. (2004) optimized the baseline separation of methadone enantiomers with due attention to the content of organics, amount of modier and pH of the mobile phase in order to avoid chromatographic interference. Also, an extraction procedure was devised such that the chosen solvents and conditions provided the cleanest extractthe solvents tried included hexane, methyl-t-butyl ether and ethyl acetate at different pH ranges, achieved by the choice of various buffers. Since methadone is commonly used in treating cases of drug abuse/addiction, specicity evaluation for likely interference of other commonly used addictive drugs was evaluated. Such drugs and their metabolite(s), including morphine, cocaine, 6-monoecetylmorphine, ecgonine methyl ester and benzylecogonine, were subjected to a
similar extraction scheme and chromatographed under conditions identical to those used for methadone enantiomers. The results revealed that no specicity issues were apparent in stereoselective methodone analysis in the presence of commonly used addictive drugs and their metabolite(s). Example of separation of uvastatin enantiomers in human plasma. Lanchote et al. (2001) have used the combination of solid phase extraction and detection via uorescence to avoid interference from not only endogenous materials but also several other drugs that are co-prescribed with uvostatin. An exhaustive list of commonly prescribed medicines has been proled for interference under the chromatographic conditions for the separation of uvastatin. Some compounds, like quinidine, propranolol, benzyldamine, metoclopramide and clomipramine, were found to be chromatographed on the system without causing any interference. However, several other compounds, like verapamil, sotalol, theophylline, propafenone, trimipramine, lidocaine, nitrazepam, dapsone, digoxin, etidocaine, phenacetin, acetaminophen, aminopyrine, atenolol, amiodarone, carbamazepine, chlorpromazine, captopril, etidocaine and clobazepam, were not detected on the system, even when the run time was extended up to 60 min (Lanchote et al., 2001). Elution order and manipulations for metabolite(s) including conjugates without reference standards It is common practice to have the elution order of the enantiomeric peaks conrmed on the basis of the chosen column and associated chromatographic conditions. This becomes particularly relevant when multiple peaks are being simultaneously eluted. This exercise can be performed by injecting the pure reference standards of the individual enantiomers. In the absence of reference standards, some improvization may become necessary. For example, due to the non-availability of reference standard of 5-hydroxypropafenone enantiomers, Zhong and Chen (1999) used in vivo conversion of propoxyphenone to 5-hydroxy metabolite following oral dosing to generate the pure enantiomeric form of either the R- or S-enantiomer of 5-hydroxy metabolite in plasma. After in vivo conversion, the plasma was extracted and introduced onto the column to check the elution order of either the R- or S-enantiomers. Similarly, Kaddoumi et al. (2001) have used an enzymatic route to prepare enantiomers of norfenuramine for laboratory purposes due to the lack of pure reference standards of norfenuramine enantiomers. In this report, they utilized rat microsomes to generate the norfenuramine after incubations of either pure dfenuramine or dl-fenuramine (Kaddoumi et al., 2001). Another commonly used improvization is to
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inject the pure reference standard of one enantiomer if available and by default; the alternate peak of the racemate was attributed to the other enantiomer. Cerqueira et al. (2003) used the CD spectra of pure metoprolol enantiomers (R- and S-) to assign the elution order for the enantiomers of the metoprolol acidic metabolite. More importantly, following derivatization of the -COOH function, they rechecked the elution order of the metabolite enantiomers to eliminate the possibility of reversal in the elution order due to change in the mobile phase and/or change in polarity of chiral stationary phase (Cerqueira et al., 2003). Additionally, the authors assigned absolute conguration to the enantiomers of metoprolol acidic metabolite in line with those of the parent enantiomers on the basis of a slight change in molecular weight without alteration in the chiral center (Cerqueira et al., 2003). Another example of elution order determination of the metabolite enantiomers using parent enantiomers is that of salmeterol (Zhang et al., 1999). In this study, the elution order of each -hydroxy metabolite of salmeterol was determined following incubation of the pure enantiomer in human liver microsomes followed by chromatography under identical conditions (Zhang et al., 1999). The lack of availability of reference standards of either glucuronidated and/or sulfated conjugates does not present a problem because their concentrations can be accurately measured using the liberated parent molecule as a surrogate following specic enzymatic hydrolysis. Although glucuronidase and/or sulfatase enzyme systems are available, optimization of the amount of enzyme, buffer selection, and time duration for hydrolysis need to be arrived at on a case-by-case basis. Typically an overnight incubation or 24 h incubation with enzyme may be sufcient for the hydrolytic cleavage of the conjugates. Often, the need may arise for quantication of metabolite(s) early in the development program. Reference standards for the metabolite(s) may not be available early in the development program. Identication and characterization of the metabolite(s) in question later in the program would aid in the development of suitable synthetic schemes. Therefore, in such situations, the parent entity may serve as a reference standard for the quantitation of metabolite(s) with the assumption that the UV absorption spectra are similar across the analytes. However, such difculties will not arise if a mass spectrometric detector is employed and, furthermore, employment of different m/z ions for the purpose of quantitation may come in handy, especially if the eluting peaks of multiple analytes overlap. The use of LC-MS, on the other hand, may have problems of signal suppression and/or variable responses, depending on functional group if quantitation is attempted without proper reference standards.
Use of two methods for chiral separation/ quantitation and/or checking for racemization potential of enantiomers Recently, Srinivas (2004a,b) has reviewed the applicability of both direct and indirect approaches in quantitation of several racemates commonly used in clinical practice. In this comprehensive review, 17 case studies of drug racemates, spanning over a decade, are presented, wherein similarity in the pharmacokinetic data between the two methods has been conrmed (Srinivas, 2004a,b). Although the possibility of racemization may not be ruled out in indirect approaches, consideration should be given to employ mild to moderate conditions for chiral derivatization. Therefore, buffer media, ionic strength, temperature and pH for the reaction should be selected with caution to ensure that they do not promote racemization (Srinivas, 2004a,b). In another recent report, two chiral methods using a direct separation approach were employed to verify for the absence of racemization potential (De Gaitani et al., 2003). The authors conrmed that, under varying conditions of ionic strength, pH, and temperature, the parent compound, thioridazine, and its chiral sulfone metabolite did not show any appreciable racemization when tested with two different chiral columns, namely, Chiralpak AD and Chiralcel OD (De Gaitani et al., 2003). The work of Kim et al. (2000) has another twist in the selection of chiral derivatization reagent for the enantiomers of bevantolol. In the published work, three different chiral derivatization reagents were rst employed to derivatize bevantolol enantiomers. These reagents included 2,3,4,6-tetra-O-acetyl-beta-dglucopyranosyl isothiocyanate (GITC), (S)-()--methylbenzyl isothiocyanate and S-()-menthyl chloroformate. Based on the analytical performance and other validation considerations, GITC was preferred to other chiral derivatization reagents for full-scale validation of the stereoselective method for bevantolol enantiomers. In order to conrm the absence of racemization in a denitive manner, the authors employed a Chiralcel OD column-based direct method to validate the results obtained from the indirect GITC method (Kim et al., 2000). In continuation of this theme, Cerqueira et al. (2003) used two methods, both involving direct approaches for obtaining the urinary kinetic data of metoprolol acidic metabolite. One of the methods involved a sample clean-up procedure followed by separation of the metoprolol acidic metabolite under reversed-phase conditions with a Chiralcel-OD-R column, while the other method involved extraction using SPE followed by derivatization (achiral) of the carboxylic acid function of the metoprolol acidic metabolite to permit separation of the enantiomers
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under normal phase conditions with Chiralcel-OD-H column (Cerqueira et al., 2003). The urinary concentrations of the enantiomers of metoprolol acidic metabolite were in agreement between the two methods, such that the correlation coefcient when the two sets of values were regressed was 0.995 or greater. This conrmed the interchangeability of the two methods for the determination of the enantiomers of metoprolol acidic metabolite in urine (Cerqueira et al., 2003). Issues related to mass spectrometric detection Considerations unique to the use of mass spectrometer need to be sorted out prior to quantitation in spite of having xed the biological extraction scheme, mobile phase and chromatographic conditions. For example, in the separation of verapamil and norverapamil enantiomers (Hedeland et al., 2004), SRM mode was used for data collection to obtain the highest possible sensitivity for detecting the peaks of interest. Full-scan daughter ion mass spectra were collected for verapamil, D3-verapamil and norverapamil. This enabled the authors to pick the appropriate fragment ion in terms of its intensity, which in turn happened to be m/z 165 for both analytes and the isotopic internal standard. Since a common fragment ion was being monitored, it meant that chromatography needed to address the resolution aspects to avoid co-elution of analyte peaks (Hedeland et al., 2004). Another important issue may surface during isotopic/stable label internal standard employment relating to the isotopic impurities inherent to the method of making a labeled material which has the potential to interfere in data collection and, therefore, the detection channels need to be monitored to rule out any interference. The issue of ionization suppression is a phenomenon that may arise due to the coelution of multiple analyte peaks that have a common m /z ion for the purpose of monitoring. This aspect should be adequately addressed during method validation and this is easily done by monitoring and plotting the peak area ratio between the internal standard kept at a constant concentration and any one analyte of interest whose concentration is increased. Similar validation experiments can also be performed by switching to other analyte(s) of interest but maintaining the same constant concentration of the internal standard. If signicant suppression in ionization occurs, then it is reected in a decreased ratio as the analyte concentration is increased. Often, a simple experiment to check the response of the individual analyte with and without co-eluting analyte peak (or internal standard) may also provide information on the propensity for ionization suppression. Another commonly observed issue relates to matrix effects. Typically, on reversed-phase columns, the use of mobile phase with high percentage of water content
can lead to matrix effects during LC/MS experiments. Since chiral separations involving a chiralAGP column employ a mobile phase with a high percentage of water in a reversed-phase manner, matrix effects are not uncommon and, therefore, one has to take precautions to circumvent this problem. Liang et al. (2004) have performed a thorough investigation to evaluate matrix effects for the stereoselective determination of methadone. Frequently, optimization and/or maximization of the abundance of the major fragment ion are necessary. For example, to optimize the major fragment ion (m /z 324 or m /z 340) in the quantication of propafenone and 5-hydroxypropafenone enantiomers, certain mass spectrometer parameters such as capillary voltage, tube lens offset, lens voltage, octopole 1 offset and octopole 2 offset were particularly tweaked further (Zhong and Chen, 1999). In the stereoselective determination of the enantiomers methadone and its major metabolite, the following parameters were optimized: mass fragmentation voltage, capillary voltage, nebulizer pressure, drying gas ow and drying gas temperature. Such optimization steps were required to enhance the sensitivity of the signals of both methodone and metabolite enantiomers (Rodriguez-Rosas et al., 2003a). Although normal phase chromatographic conditions were fully optimized for the separation of lercanidipine enantiomers, the mobile phase (hexaneethanol, 95:5 v/v with 0.1% diethylamine) appeared to be incompatible with electrospray ionization such that no peaks corresponding to lercanidipine enantiomers were detected (Jabor et al., 2003). To obtain a compromise between good chromatographic separation and excellent MS sensitivity, the addition of a suitable modier was mandated immediately after post-column elution to facilitate the ionization of the analytes. Therefore, the introduction of a small amount of ammonium acetate (10 mmol/L) to the chromatographic efuent was considered and this resulted in higher intensities of the signals (Jabor et al., 2003). Multiple chiral analyses In a tabular form, several examples of chromatographic separation of multiple chiral (4 to 9 peaks) analytes including validation and applicable conclusion are provided in Table 1.
CONCLUSIONS
Chiral separations of plethora of xenobiotics including those of some therapeutically important drugs have been eloquently practised in the recent decade. The difculties and challenges associated with chiral separations have gradually begun to wane due to the
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signicant progress achieved in the eld of stereoisomeric separations, notably with both direct and indirect tools of separation. Review of the literature in recent years brings several examples of chiral separations where the investigators have taken the challenges of separating multiple chiral analytes with the same analytical method using similar chromatographic conditions. This paradigm shift in the approach to chiral separations, where focus is now also given to additional analyte(s) (metabolite and/or another related analog), will greatly aid in the understanding of complications of isomeric issues which otherwise will plague the development and/or progress of novel chemical entities (NCEs) from discovery phase to the development phase. This review captures the usefulness of establishing chiral assays for multiple chiral analytes with several specic case studies. As the data were being collated, it was obvious that the availability of such analytical methods for multiple analytes would answer several important developmental questions and/or resolve issues to make decisions for NCE development. The key topics under various case studies discussed in this review provide an overview regarding developmental scenarios. Firstly, understanding the role of stereoselective pharmacokinetics/pharmacodynamics both in in vivo and in vitro systems may assist in delineating the developmental path of the racemate and/or the pure enantiomers. Secondly, the availability of chiral method(s) for multiple analytes may nd use in forensic toxicology and additionally may aid in differentiating genuine medical therapy from drug addiction/absuse. Thirdly, the greatest advantage would be in the areas
of pharmacogenetics/polymorphic evaluation, where developmental decisions need to be made from the viewpoint of safety considerations, primarily as a result of altered metabolism and disposition of the drug. Fourthly, the attributes of active metabolite(s) may be readily evaluated for future considerations for drug development and/or substitution for the parent entity in the event of a decision to stop the development of the parent moiety. Fifthly, availability of chiral assays may also provide a platform for delineating peculiar toxic effects and thereby provide other possibilities in designing the next set of chiral NCEs for development. Finally, it would make the investigation into chiral inversion phenonmenon of multiple chiral molecules a routine task in drug development. Although several aspects of method developmental considerations may be gleaned through a review of several examples of recent chiral separations, a comprehensive document has been provided under a few select headings. The emphasis in this analytical method section has been to focus on certain critical issues and point out certain practices that have been commonly employed. While many issues and considerations are discussed, it is important for analysts to use judgement and discretion when planning a chiral assay procedure for new molecules and/or old molecules. The lack of availability of an assay for an NCE does not mean that elements of method development should begin from scratch. On the contrary, an attempt should be made to determine how the existing literature information can provide some important clues and direction for such an endeavor.
Table 1. Select examples of chromatographic separation of multiple chiral analytes Reference Angelo et al. (1999) No. of analytes 5 Analytes (R)-()-methadone (S)-(+)-methadone (R)-(+)-EDDP (S)()-EDDP Imipramine (IS) Chromatographic conditions/detection system Short analytical LiChrospher RP8 column serially coupled with chiralAGP column UV detection set at 200 nm Mobile phase: 10 mm sodium phosphate buffer (pH 5) acetonitrileDMOA (860:140:0.5 v/v/v) and nal pH adjusted to 6; FR = 0.9 mL/min Validation information Calibration ranges: methadone, 0.068 mol/L (racemate); EDDP, 0.0618 mol/L (racemate) R(2) values >0.99 LOQ: determined to be 0.03 mol/L for both methadone and EDDP enantiomers Accuracy/precision: %dev of the observed values were within 10% of the nominal, while %RSD values were within 10% with the exception of LOQ values for EDDP where the %RSDs were between 16 and 19.5% Specicity/selectivity: generally found to be excellent by cross checking with a previously published GC method; only samples from addicts on ketobemidone were found to show interference and had to be omitted Recovery: the extraction efciency from urine for methadone was about 90% and EDDP was about 80% Calibration ranges: R- and SMDE, 10.1175.9 ng/mL; R- and S-MDA, 1.220.3 ng/mL; R- and S-HME, 16.1332.2 ng/mL Overall method standard deviation was very low, suggesting a good calibration performance for all tested analytes Accuracy and precision of standards were within acceptable norms Recovery: R-MDE, 95.0%; SMDE, 94.8%; R-MDA, 94.4%; S-MDA, 94.3%; R-HME, 95.5%; S-HME, 96.0% Applicable conclusion The inclusion of achiralchiral columns provided more ruggedness for the effective separation of methadone and EDDP enantiomers. Without the inclusion of the achiral component, the chiralAGP column would not have accommodated more than 300 clinical samples. With the inclusion of the two columns in series, the selectivity was imparted for injection of over 1000 samples Enantioselectivity was evident in the urinary excretion of methadone enantiomersthe S/ R enantiomeric urinary ratio for methadone ranged between 0.2 and 0.9. Similarly, the EDDP levels in urine showed stereoselectivity and the S/R ratios ranged between 0.4 and 2.1. The developed assay could be used in pharmacokinetic, metabolism or excretion studies of methadone and its major metabolite EDDP The developed method was readily applied in a clinical study involving six healthy subjects. The duration of pharmacokinetic blood sampling was only for 260 min. Both metabolites of MDE, namely HME and MDA, were readily formed. It appeared that formation of HME preceded that of MDA. All analytes were measurable at the end of sample collection and, since samples were not drawn out for a longer time period, it was not possible to compute the true elimination phases for all the enantiomeric analytes Stereoselectivity was
Copyright 2004 John Wiley & Sons, Ltd. Biomed. Chromatogr. 18: 759784 (2004)
Brunnenberg and Kovar (2001)
R-N-ethyl-3,4methylenedioxyamphetamine (MDE) S-MDE R-3,4-methylenedioxyamphetamine (MDA) S-MDA R-O-glucuronyl-N-ethyl-4hydroxy-3-methoxyamphetamine (HME) S-HME
MDE/MDA separation: two systems were evaluated System 1: ChiralDex column ( -cyclodextrins are covalently bonded on an RP phase) maintained at 20C; mobile phase, 40 mmol potassium dihydrogen phosphate buffer (pH 5.5)acetonitrile (91:9 v/v); FR = 0.8 mL/min System 2: Superspher 60 RP (achiral column) maintained at 20C; mobile phase, 0.01 m -cyclodextrins dissolved in 40 mmol potassium dihydrogen phosphate buffer (pH 3) acetontirle (92:8, v/v); FR = 0.5 mL/min
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Table 1. (continued ) Reference No. of analytes Analytes Chromatographic conditions/detection system Fluoresence detection was employed for MDE and MDA regardless of the two systems; excitation wavelength, 286 nm and the emission wavelength, 320 nm Chiral separation of HME: a chiral CBH (cellobiohydrolase) column was employed and the temperature was kept at 20C; mobile phase, 10 mmol potassium dihydrogen phosphate (pH 7.5) with 50 mol NaEDTApropan2-ol (95:5, v/v); FR = 0.8 mL/min Primary detection: electrochemical detector; quantitation was performed at a cell potential of 650 mV Secondary detection: Fluorescence detector; excitation wavelength, 278 nm and emission wavelength, 320 nm Buechler et al. (2003) 7 (R)- and (S)-N-ethyl-3,4methylenedioxyamphetamine (MDE) (R)- and (S)-N-ethyl-4hydroxy methyoxyamphetamine (HME) (R)- and (S)-3,4methylenedioxyamphetamine (MDA) N-ethyl-3,4-methylene-dioxy benzylamine (IS) Chiral CBH column (cellobiohydrolase) and column temperature was maintained at 17.5C Mobile phase: 20 mm sodium dihydrogenphosphate, 50 mm EDTA disodium salt and isopropanol (7% v/v); the pH was adjusted to 6.44; FR = 0.7 mL/min Fluorescence detector was employed: excitation wavelength, 286 nm; emission wavelength, 322 nm Validation information HME enantiomers could not be separated on the regular cyclodextrin stationary phase because of steric hindrance from the 4-hydroxy-3-methoxy analyte, which prevents interaction between HME and -cyclodextrin. Therefore, CBH stationary phase was employed for HME enantiomers Applicable conclusion demonstrated in the levels of MDE, MDA and HME. The Cmax values for R-MDE (e.g. 180 ng/mL) were much greater than those of S-MDE (e.g. 140 ng/mL). The Cmax values for S-HME (e.g. 450 ng/mL) were much greater than those for RHME (e.g. 160 ng/mL). The Cmax values for S-MDA (e.g. 25 ng/ mL) were much greater than those for R-MDA (e.g. 9 ng/mL)
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Calibration ranges: spilt standard curves were used for two of the three enantiomeric analytes Plasma: R- or S-MDE, 5100 ng/ mL and 100800 ng/mL; R- or S-HME, 10100 ng/mL and 1002000 ng/mL; R- or S-MDA, 5100 ng/mL; r > 0.999 for all ranges Urine: R- or S-MDE, 5100 ng/ mL and 100800 ng/mL; R- or S-HME, 10100 ng/mL and 1002000 ng/mL; R- or S-MDA, 5200 ng/mL; r > 0.999 for all ranges Extraction recovery: 92% or higher regardless of matrix Accuracy/precision: %dev and %CV were greater than 94% and 98%, respectively, regardless of analytes or the matrix tested
The developed analytical method allowed monitoring of the enantiomers of MDE, HME and MDA for over a period of 36 h. Stereoselectivity was observed in the plasma levels of MDE (mean Cmax for R-MDE was 134 ng/mL and that for S-MDE was 88 ng/ mL) and was further reected in the elimination half-lives of the two enantiomers of MDE (7.9 and 4.0 h for R- and S-MDE, respectively). The Cmax values of R-enantiomers of the two metabolites (HME and MDA) were greater than the corresponding S-enantiomers. Similarly, urinary levels of MDA and its two metabolites, showed stereoselectivity in the urinary excretion rates
N. R. Srinivas
Table 1. (continued ) Reference Capka and Xu (2001) No. of analytes 7 Analytes d-/l-trihexyphenidyl d-/l-procyclidine d-/l-biperiden Diphenidol (IS) Chromatographic conditions/detection system Cyclobond I 2000 native -CD phase column; variable wavelength UV detector MS detector: electron spray ionization in the +ve mode Mobile phase: acetonitrile methanolacetic acidTEA (95:5:0.5:0.3, v/v); FR, 0.25 mL/min SIM of analytes: procyclidine, m/z 288; trihexyphenidyl, m/z 302; diphenidol, m/z 310; biperiden, m/z 312 MRM data acquision: procyclidine, 288 to m/z 84; trihexyphenidyl, 302 to m/z 98; diphenidol, 310 to m/z 129; biperiden, 312 to m/z 98 Brandsteterova and Wainer (1999) 9 Verapamil; norverapamil; D617; D620; PR24; PR23; PR22; PR25; internal standard ChiralAGP column Fluorescence detection for all analytes: excitation wavelength, 276 nm and emission wavelength, 310 nm Mobile phase: 0.01 m disodium hydrogen phosphate, pH 7acetonitrile (90:10) Typically R-(+)-enantiomers eluted as rst peaks for all analytes followed by S-()enantiomers and maximum separation time was approximately 30 min Generally resolution was close to baseline separation (Rs ranging from 0.93 to 1.57) and a satisfactory value (1.161.44) Gatti et al. (2003) 5 S-uoxetine R-uoxetine S-noruoxetine R-noruoxetine Fluvoxamine (IS) Chiralcel ODR column with temperature controlled at 37C Mobile phase: 100 mm potassium hexauorophosphate and acetonitrile (65:35, v/v); pH of the mobile phase was adjusted to 3.0 using 8.5% phosphoric acide; FR = 0.5 mL/min
Copyright 2004 John Wiley & Sons, Ltd. Biomed. Chromatogr. 18: 759784 (2004) Simultaneous chiral analyses of multiple analytes
Validation information Calibration ranges for all analytes: 11600 ng/mL (r > 0.999) and curve range could be extended up to 1140 ng/mL Detection limit: S/N ratio of 3 was 1 ng/mL for all analytes Inter- and intra-assay precision: RSDs ranged between 4.4 and 10.6 across enantiomeric analytes Absolute recoveries for all analytes enantiomers: 98.4 101.4% with RSDs within 8.5%
Applicable conclusion Although pure enantiomeric reference standards were unavailable, the developed method is capable of monitoring the enantiomeric peaks of various structurally related anticholinergic drugs. Method could be very useful in therapeutic drug monitoring as well as in accidental overdose and/or drug abuse. Since the validated method has a wide dynamic range, good recovery and measures enantiomeric composition, it appears to be well suited for simultaneous analysis in pharmacokinetic, toxicology and clinical pharmacology experiments A method was developed which provides chromatographic conditions for the stereoselective separation of verapamil and all its seven metabolites using a single chiral column and identical conditions. The method could be readily applied for pharmacokinetic studies and/or for therapeutic monitoring Enantioselectivity in the pharmacokinetic disposition of verapamil (R/S serum ratio = 4.3 at 12 h post dosing) and that of norverapamil (R/S serum ratio = 1.8 at 12 h post doing) was demonstrated
Validation was by and large done by the achiral measurement Calibration ranges: verapamil, 20400 ng/mL; norverapamil, 15 300 ng/mL; D617, 2.530 ng/mL; D620, 550 ng/mL; PR22, 1.5 40 ng/mL; PR23, 330 ng/mL; PR24, 440 ng/mL; PR25, 1 10 ng/mL; r > 0.999 for all analytes Accuracy/precision: within 10% dev and within 6% RSD Extraction recoveries (all analytes): ofine SPE, 8893%; online SPE, 9498% Chiral measurements were also performed but validation was not reported
REVIEW
Calibration range: 10 1000 ng/mL for all enantiomeric analytes; all calibration curves produced almost identical slope values following linear regression (0.00340.0037) with r value equal to or greater than 0.992
The developed method is not only simple and convenient but provides a faster turnaround time compared with other published methods. One of the advantages of the present method relates to a clean separation amidst a host of potential interfering peaks.
773
774
Table 1. (continued ) Reference No. of analytes Analytes Chromatographic conditions/detection system UV variable wavelength detector set at 227 nm The enantioselectivity factor for uoxetine pair of enantiomers was 1.10, while the value for noruoxetine pair of enantiomers was 1.09 Validation information Specicity evaluation: potential for interference from several drug classes, notably antidepressants, anticonvulsants and antipsychotics were evaluated and probable remedies suggested to overcome the interference issues Recovery from plasma: Ruoxetine, 77%; S-uoxetine, 76%; R-noruoxetine, 72%; S-noruoxetine, 66% LOQ: based on a signalnoise ratio of 3, the LOQ was deemed to be 10 ng/mL per enantiomer analyte Accuracy/precision: the accuracy of the predicted values ranged between 90 and 116% of the spiked nominal values, while precision values for both interand intra-assay ranged within 10% CV Stability: autosampler stability for at least 24 h; freezethaw stability for three cycles and freezer stability for 6 months (20C storage) were conrmed for all enantiomeric analytes Calibration ranges: verapamil enantiomers, 0.10125 ng/mL (r 2 = 0.99 or better) Norverapamil enantiomers: 0.12121 ng/mL (r 2 = 0.99 or better) Accuracy/precision: %dev of samples ranged within 16% of the nominal values, while the RSDs were less than 7.8% Stability: both verapamil and norverapamil enantiomers were stable in plasma for up to 5 days when stored at RT Applicable conclusion The specicity evaluation has considered several likely agents that are coadministered with uoxetine. Additionally, the authors have provided specic conditions to overcome such problems of intereference while analyzing samples from the patient population. The method can be easily adopted for single dose pharmacokinetic studies and/or enantioselective monitoring of uoxetine and noruoxetine levels in patient samples
REVIEW
Hedeland et al. (2004)
R-verapamil; S-verapamil, R-norverapamil; Snorverapamil; deuterated (D3)-verapamil
ChiralAGP column with a peak lter A-428X to improve columns shelf-life LC-ESI-MS/MS with selected reaction monitoring (SRM); ESI parameters optimized for sensitivity UV use: diode array detector absorbance at 272 nm Mobile phase: 15% acetonitrile and 85% ammonium acetate buffer (0.02 m; pH 7.4); FR = 0.6 mL/min
A selective and sensitive stereoselective procedure for simultaneous determination of verapamil and norverapamil has been described. Based on the PK disposition, it appears that both S-verapamil and S-norverapamil are rapidly metabolized/cleared from the circulation as compared with their antipodes. Although data are not presented, the utility of this stereoselective assay to a biopharmaceutical in vivo absorption study using
N. R. Srinivas
Table 1. (continued )
Reference
No. of analytes
Analytes
Chromatographic conditions/detection system SRM transitions monitored: verapamil, m/z 455 165; norverapamil, m/z 441 165; D3verapamil, m/z 458 165 For selectivity tests (metabolites were used): D617, m/z 291 151; D620, m/z 277 151; PR23, m/z 441 165
Validation information
Applicable conclusion Loc-I-Gut device has been mentioned. Another advantage of the method is that it precludes the use of achiral column insertion prior to chiral chromatography and/or chemical derivatization scheme
Jansson et al. (2003)
R-/S-mephenytoin; R-/Snirvanol; R-/S-4hydroxymephenytoin internal standard
ChiralAGP column; UV detection set at 205 nm Mobile phase: 2.5% acetonitrile in phosphate buffer; pH 7; ionic strength = 0.1. FR, 0.9 mL/min; autosampler maintained at 8C; analytical columns were maintained at 30 1C
Calibration ranges: a split curve was employed because the intercepts differed from zero when a wide curve range was employed Split ranges were 0250 ng/mL and 2502000 ng/mL for all enantiomeric analytes with r of at least 0.999 LOQ: xed at 10 ng/mL for all analytes Accuracy/precision: generally accuracy of QC samples was within 93110% of the nominal value; while CV was <15% Although selectivity appeared to be good, there was an overlapping peak of tolbutamide co-eluting with R-nirvanol peak Since chiral derivatization scheme was employed (indirect method), optimization of amount of chiral reagent (DIB-Cl), pH requirement, temperature dependence and time of reaction were part of validation Calibration ranges: fenuramine, 55000 ng/mL/enantiomer; phentermine, 22500 ng/mL Norfenuramine was quantitated using fenuramine calibration curves Accuracy/precision: %dev for fenuramine enatiomers and phentermine were <8 while %RSD were <11%. In human plasma, recoveries were as follows: d-fenluramine, 69%; l-fenuramine, 76%; d-norfenuramine, 79%; l-norfenuramine, 84%; phentermine, 59%
The method was applied for stereoselective pharmacokinetic characterization of mephenytoin and its two metabolites in two subjects phenotyped to be extensive and poor metabolizers of CYP2C19 isozyme A dramatic difference in the PK disposition of the various analytes was observed in the two phenotypes The future use of the method would be to employ it as a tool for estimating the basal and altered changes in both CYP2B6 and CYP2C19 activities in humans
Kaddoumi et al. (2001)
d-fenuramine l-fenuramine d-norfenuramine l-fenuramine Phentermine
Chiralcel-OD-R Fluorescence detection: excitation wavelength, 325 nm; emission wavelength, 430 nm Mobile phase: mixture of solvent A (acetonitrile0.01 m citrate buffer, pH 2.70.05 m perchlorate buffer, pH 2, 50:25:25 v/v/v) and solvent B (acetonitrile); a third pump was employed to deliver ammonia solution in water (4%); FR = 1 mL/min A gradient system was employed for effective chiral separationa total of seven different changes occurred in the 44 min run time
The developed method was sensitive and selective for the analysis of fenuramine enatiomers; although authors justied use of fenuramine isomers for determining norfenuramine enatiomers, it is not a good practice. Based on the data, it appeared signicant stereoselective disposition occurred for both fenuramine and norfenuramine enantiomers in rat plasma following an intraperitoneal dose of racemic fenuramine. Generally concentrations of the respective d-isomers were at least a log higher than those of l-isomers. Furthermore, the method showed applicability in human plasma measurements of the various enantiomer analytes
REVIEW 775
Table 1. (continued ) Reference Katsuki et al. (2001) No. of analytes 4 Analytes (+)-Lansoprazole ()-Lansoprazole 5-Hydroxylansoprazole Lansoprazole sulfone Isobutyl 4-hydroxybenzoate Chromatographic conditions/detection system Chiralcel OD-R UV detection was performed at 285 nm Mobile phase: methanolwater (75:25, v/v); FR = 0.5 mL/min; column temperature was maintained at 30C Validation information Calibration range: 0.25200 m for lansoprazole enantiomers (r 2 = 0.999) 0.1326 m for the two metabolites (r 2 = 0.999) Precision/accuracy: lansoprazole enantiomers, %dev of QC samples (0.25, 5, 50 m) were within 10.4%, while the %CV for the QC samples were within 5.6% Metabolites: %dev of QC samples (0.13, 2.6, 13 m) were within 5%; while the %CV for the QC samples were within 7.3% LOQ: lansoprazole enantiomers, 0.25 m; hydroxylansoprazole, 0.13 m Lansoprazole sulfone: 0.13 m Recovery: (+)-lansoprazole, 7076%; ()-lansoprazole, 7074%; 5-hydroxylansoprazole, 7074%; lansoprazole sulfone, 7277%; IS, 72% Calibration range: 101000 ng/mL for each enantiomeric analyte and 5 1000 ng/mL for the lansoprazole sulfone (r 2 > 0.9999) LOQ: 10 ng/mL for all enantiomeric analytes; 5 ng/mL for lansoprazole sulfone Recovery: R-lansoprazole, 95 97%; S-lansoprazole, 9598%; R-5-OH-lansoprazole, 9598%; S-5-OH-lansoprazole, 9498%; sulfone, 9498% Precision/accuracy: CV were within 8% and %dev <5% Applicable conclusion The developed method was applied to study the enantioselective metabolism of lansoprazole enantiomer in vitro systems such as human liver microsomal incubations. Following a 2 h incubation, (+)lansoprazole levels were 2-fold greater than those of ()lansoprazole. The intrinsic clearance values were 13.8 L/ min/mg protein and 33.4 L/min/ mg protein for (+)- and ()enantiomer, respectively. The liver microsomes produced both metabolites, lansoprazole sulfone and 5-hydroxylansoprazole. However, the sulfone was formed to a greater extent compared to the other metabolite. The assay could also be readily applied to study the in vivo metabolism aspects of lansoprazole enantiomers and/or simple pharmacokinetic studies The method was applied for quantitation of lansoprazole, 5hydroxy lansoprazole enantiomers and lansoprazole sulfone in renal transplant patients. The plasma levels of the parent/metabolite enantiomers indicated signicant stereoselectivity in the disposition of lansoprazole and its metabolite. PK parameters for the individual enantiomers showed a large intersubject variability. Experiments carried out in conjunction with in vivo studies suggested lack of chiral inversion between lansoprazole isomers in vitro. No artifactual formation of other metabolites were seen when incubated in vitro in plasma
776 REVIEW
Miura (2004)
R-lansoprazole; S-lansoprazole; R-5hydroxylansoprazole; S-5-hydroxylansoprazole; lansoprazole sulfone (nonchiral); internal standard (S)omeprazole
Chiral-CD-phenyl columns and UV detection was performed at 285 nm Mobile phase: 0.5 m sodium chlorateacetonitrilemethanol (6:3:1 v/v/v); FR = 0.5 mL/min; ambient temperature for separation
N. R. Srinivas
Table 1. (continued ) Reference Rodriguez-Rosas et al. (2003a) No. of analytes 6 Analytes R-methadone S-methadone R-2-ethylidene-1,5-dimethyl3,3-diphenyl-pyrrolidine (EDDP) S-EDDP d3-methadone (IS) d3-EDDP (IS) Chromatographic conditions/detection system ChiralAGP analytical column tted with a chiralAGP guard column Mobile phase: acetonitrile ammonium acetate buffer (10 mm, pH 7) in 18:82 v/v proportion. Adjustment of pH was performed using 2% aqueous ammonium hydroxide; FR = 0.9 mL/min; the analytical column temperature was maintained at 25C The enantioselectivity factor for the separation of methadone pair of enantiomers was 1.30 and the corresponding value for EDDP was 1.17 SIM was employed for MS detection and m/z ions were as follows: methadone, m/z 310.20; EDDP, m/z 278.20; d3-methadone, m/z 313.20; d3-EDDP, m/z 281.20
Validation information Salivary matrix was used for validation Calibration ranges: methadone, 5600 ng/mL/enantiomer with r 2 values >0.999; EDDP, 0.515 ng/ mL/enantiomer with r 2 values >0.999 LOQ: methadone = 5 ng/mL; EDDP = 0.5 ng/mL based on accuracy of 80120% of nominal with acceptable precision LOD: based on a signal to noise ratio of 3:1, methadone = 0.1 ng/ mL; EDDP = 0.5 ng/mL Accuracy/precision: %dev from nominal values were within 3%; while both inter- and intra-assay precision values were within 3.9% Extraction recovery: Recoveries for both enantiomers of methadone and EDDP were between 95 and 100%. Generally, higher concentrations gave a few percent improvement in recovery Stability: both analytes were stable while frozen at 80C for 4 months; stability at 20C was conrmed; autosampler stability of extracted sample was conrmed for 24 h while waiting for injection at room temperature Calibration ranges: regardless of analytes, the range was between 1 and 125 ng/mL/enantiomer. The r 2 value was generally 0.9993 or greater LOQ: it was determined to be 1 ng/mL for either ketamine or norketamine enantiomers with an acceptable accuracy/precision LOD: based on a singalnoise ratio of 3, for each enantiomer of the analytes it was determined to be 0.25 ng/mL
Applicable conclusion The method was applied to analyze the concentrations of both methadone and EDDP enantiomers in clinical samples obtained from patients Enantioselectivity was evident in the concentrations of methadone enantiomers (R/S ratio in saliva samples ranged from 1.31 to 2.21) The concentrations of EDDP did not reveal the presence of any degree of enantioselectivity (R/S ratio in saliva samples ranged from 0.75 to 1.17) The authors noted very high variability in the levels of both enantiomers of methadone at the two dose levels tested in the studyalmost 10-fold variation in the levels were observed between individuals. However, EDDP levels did not appear to show such a large variation regardless of the dose or the enantiomer
Rodriguez-Rosas et al. (2003b)
S-ketamine R-ketamine S-norketamine R-norketamine Bromoketamine
ChiralAGP analytical column equipped with a chiralAGP guard column Mobile phase: 2-propanol ammonium acetate buffer (10 mm, pH 7.6) in a 6:94 proportion v/v; pH adjustment was done using ammonium hydroxide solution; FR = 0.5 mL/min and the analytical column temperature was maintained at 25C Detailed optimization of chromatographic conditions including selection of organic solvents, buffer systems, pH effects to strike a balance
The method was found to have adequate sensitivity and selectivity for the routine measurement of both ketamine and norketamine enantiomers in the management of pain in patients. In the illustrative reported in this work, the disposition of ketamine appears to show no stereoselectivity, while that of the metabolite appears to show mild stereoselectivity Application to large sample analysis from clinical studies of racemic ketoprofen has been documented
REVIEW 777
778
Table 1. (continued ) Reference No. of analytes Analytes Chromatographic conditions/detection system between retention time and good resolution has been described The enantioselectivity factors were: ketamine, 1.09; norketamine, 1.46; bromoketamine, 1.22 Mass spectral detection: full scan in the positive ion mode with a scan range between m/z 100 and m/z 600 was employed SIM was employed for individual enantiomeric analytes Ketamine, m/z 238.1 Norketamine, m/z 224.1 Bromoketamine, m/z 284.0 Several MS detector parameters were optimized to obtain an optimized condition to improve sensitivity of the signals of the analytes. These parameters included: fragmentation voltage (5080 V), capillary voltage (10003000 V), nebulizer pressure (4055 p.s.i.g), drying gas temperature (200350C) Srinivas et al. (1989) 10 Separation condition 1: methoxyphenamine, P1 and P2 enantiomers; NDMP, P1 and P2 enantiomers; amantidine Separation condition 2: ODMP, P1 and P2 enantiomers; 5HMP, P1 and P2 enantiomers; d-ephedrine Separation condition 1: OV-225 capillary column; column oven temperature 220C, injector temperature 280C, detector temperature 300C; carrier gas 5% argon in methane with column ow rate 1 mL/min; make-up gas (5% argon in methane) ow rate 60 mL/min; split vent ow rate 32 mL/min; septum vent ow rate 2 mL/min; column head pressure 105 kPa Separation condition 2: OV-225 capillary column; similar conditions to the above with the following modications: initial column oven temperature 240C, held for 4 min, thereafter Calibration ranges: methoxyphenamine, 0.252 g/ mL/enantiomer; NDMP, 0.094 0.75 g/mL/enantiomer; ODMP, 0.252 g/mL/enantiomer; 5HMP, 0.1881.5 g/mL/ enantiomer; r 2 values were 0.99650.9994 Accuracy/precision: %dev and CV% were within 10% Extraction recovery: a simple comparison of peak heights between extracted and neat suggested a recovery of at least 80% for all enantiomeric analytes A method was described for the enantioselective determination of methoxyphenamine and its oxidative metabolites (NDMP, ODMP, 5HMP) in human urine. Application of the assay to urinary samples revealed a signicant enantioselectivity in the urinary excretion of methoxyphenamine and two of its metabolites (NDMP and 5HMP). However, urinary excretion of ODMP metabolite was found to be nonstereoselective Approximately 12% of the dose was accounted by methoxyphenamine enantiomers in the urine (P1P2 ratio Validation information Applicable conclusion
REVIEW N. R. Srinivas
Table 1. (continued ) Reference No. of analytes Analytes Chromatographic conditions/detection system increased at a rate of 10C/min to a nal temperature of 280C which was held for 10 min Indirect method was employed following precolumn derivatization with Nheptauorobutyryl-l-prolyl chloride. The derivatization conditions were optimized with regard to the amount of chiral reagent as well as the time of reaction Detection was by either electroncapture detector or MS Yanagihara et al. (2000) 5 R-ketamine S-ketamine R-norketamine S-norketamine Bromoketamine (IS) Chiralcel OD column UV detection at 215 nm Mobile phase: n-hexane: 2-propanol (98:2, v/v); FR = 0.8 mL/min; the column temperature was maintained at 35C Calibration ranges: ketamine/norketamine, 10500 ng/mL/enantiomer LOQ (employing a 10:1 S/N ratio): ketamine, 5 ng/mL/ enantiomer; norketamine, 10 ng/ mL/enantiomer Accuracy/precision: %dev of QCs were within 10% of nominal and the CV% ranged between 2.9 and 10.7% Recovery from plasma: R-ketamine, 88.1%; S-ketamine, 87.4%; R-norketamine, 91.3%; S-norketamine, 86.1% Ulrich (2003) 6 R-uoxetine S-uoxetine R-noruoxetine S-noruoxetine Fluvoxamine Nisoxetine Two-dimensional capillary gasliquid chromatography with hydrodex- -6-TBDM fused-silica capillary column coated with (50% heptakis-(2,3-di-O-methyl6-O-tert-butyldimethyl-silyl)- cyclodextrin in 14% cyanopropylphenyl86% dimethylpolysiloxane Hydrogen (1.45 mL/min at 170C and 1.20 mL/min at 201C, 140 kPa) as the carrier gas. Septum vent was set at 0.2 mL/ min. Detector gases FR were air, Calibration ranges: for all enantiomeric analytes: 25 250 ng/mL (r = 0.994 to 0.995), standard curves for all enantiomers had negative intercepts suggesting that it may overestimate concentrations at the lower end of the curve range LOD: using an S/N ratio of 3, the detection limits were 1.5 ng/ mL for uoxetine enantiomers and 6 ng/mL for noruoxetine enantiomers A satisfactory stereoselective method for the determination of uoxetine and noruoxetine enantiomers is described. The results of the investigation of clinical samples in one patient were in agreement with previously published data of both analyte enantiomers in patients. The plasma concentrations of the R-enantiomers of both uoxetine and noruoxetine were dramatically lower as compared to the corresponding S-antipodes.
Applicable conclusion was 0.57); while approximately another 10% of the dose was contributed by all three oxidative metabolites (P1P2 ratios of NDMP, 5HMP and ODMP were 0.23, 1.98 and 1.09, respectively). The developed analytical assay should aid a detailed stereoselective investigation of methoxyphenamine and its metabolites. Since methoxyphenamine is a drug showing genetic polymorphism, the tool of stereoselective assay should be of a great value in probing the metabolism aspects of methoxyphenamine A sensitive and reliable method was described for stereoselective determination of ketamine and norketamine in human plasma. Method had the desired sensitivity to be applicable for single dose PK studies as well as to monitor levels of the analytes in pharmacodynamic (PD; i.e. analgesia) studies The PK disposition of both ketamine and norketamine enantiomers appeared to be nonstereoselective
REVIEW 779
780
Table 1. (continued ) Reference No. of analytes Analytes Chromatographic conditions/detection system 120 mL/min; hydrogen, 1.3 mL/ min; auxillary helium gas, 13 mL/ min. Injector temperature was at 230C and the detector temperature was set at 300C. Nitrogenphosphorous selective detector was employed at a baseline of about pA. A program temperature run chromatography was followed: initial temperature of 170C maintained for 7 min; nal temperature of 201C, ramping was done at 1C/min, a total run time of 38 min Validation information Selectivity test: interference tests included several tricyclic and tetracyclic antidepressant drugs (amitriptyline, imipramine, maprotiline) and phenothiazine anti-psychotic agents (chlorpromazine and promethazine) Accuracy/precision: %dev of QCs were within 5% of the nominal values; while CV% were <13% for both intra- and inter-assay runs Absolute recovery was very poor for all enantiomeric analytes. Extraction yields were about 50 and 34% for uoxetine and noruoxetine enantiomers, respectively Calibration ranges: MHD/DHD, 1150 g/mL/enantiomer in human urine (due to the lack of availability of reference standards of enantiomers of DHD, they were referred to as Diol 1 and Diol 2 peaks in both linear regression parameters and as well as in the assessment of accuracy and precision) Regardless of analytes, the slope and intercept values were consistent within the individual calibration curve ranges resulting in a small standard error of the estimates Accuracy/precision: the accuracy of all standards for all analytes was within 94.8109% of the nominal values, while the precision for all analytes was found to be within 5.1% RSD Recovery: extraction efciciency for all analytes was over 90% with RSD not exceeding 5% Applicable conclusion The author speculated that the low R/S ratio observed for both uoxetine and noruoxetine may reect the phenotype status of the patient to be in line with a poor metabolizer of CYP2D6. As compared with other various chiral capillaries the hydrodex- TBDM-capillary appeared to provide the best performance
REVIEW
Volosov et al. (2000)
(R)-10-hydroxycarbazepine (MHD) S-MHD Enantiomers of carbamazepine-10,11-transdihydrodiol (DHD) Oxcarbazepine oxime (IS)
Chiralcel OD column and UV detection was at 215 nm Sample preparation: urine was alkanized with KOH solution (2 m) + IS was added. Extraction carried out with tert-butylmethyl etherdichloromethane (2:1, v/v) mixture. Organic layer separated and evaporated to dryness residue was taken up in the mobile phase and injected on to chiral column Mobile phase: n-hexaneethanol 2-propanol (18:2:1, v/v/v) mixture, with addition of 0.1% of glacial acetic acid; FR = 1.0 mL/min
The developed assay procedure for simultaneous enantioselective determination of MHD and DHD was found suitable for application to pharmacokinetic and/or metabolic studies involving oxcarbazepine and/or carbamazepine (CBZ). Since CBZ has been previously shown to undergo metabolism via CYP3A4 and CYP2C8, the developed assay may be useful in assessing the metabolic fate of CBZ since DHD is a primary metabolite of CBZ
N. R. Srinivas
Table 1. (continued ) Reference No. of analytes Analytes Chromatographic conditions/detection system

Validation information LOQ: MHD, the values were 0.2 mg/L, while the values for Diol 1 and Diol 2 were 0.4 mg/L Stability: all analytes had conrmed stability when stored in solution for at least 1 month under refrigeration. The enantiomeric stability was conrmed for both MHD and DHD, following incubation of spiked urine samples at 37C with and without the presence of -glucuronidase enzyme
Applicable conclusion
Zhang et al. (1999)
R-()-salmeterol S-(+)-salmeterol R--hydroxysalmeterol S--hydroxysalmeterol
Chiral CBH analytical column (based on cellobiohydralase) with an identical guard column An coulometric electrochemical detector was employed comprising a guard cell (operating at 1000 mV) and a dual-electrode analytical cell with detectors 1 and 2 set at 300 and 700 mV, respectively Mobile phase: 15% 2-propanol in 25 mm sodium phosphate buffer (pH 6) with 50 m disodium EDTA. FR = 0.9 mL/min and system maintained at ambient temperature
Live microsomal incubation mixtures were used in validation experiments Calibration ranges: salmeterol, 2.540 m (r > 0.999); -Hydroxysalmeterol 0.54 m (r > 0.999). All intercepts were not signicantly different from zero
This is the rst method reported for the simultaneoius analysis of the enantiomers of salmeterol and its hydroxy metabolite. Since salmeterol is a highly potent molecule, small therapeutic doses are administered, making it difcult to perform in vivo metabolism-related work. Therefore, the current method was needed to understand stereoselective metabolism aspects in vitro using human liver microsomes. Following incubation of pure isomers of salmoterol in human liver microsomes, the Km and Vmax values of the enzyme for the formation of R-hydroxy salmeterol were identical with those for the formation of S-hydroxysalmeterol Following liver microsomal incubation with racemic salmeterol, Km and Vmax remained identical for the formation of hydroxy metabolites, indicating that the metabolic pathway is nonstereoselective. Based on the Km values obtained in the study, the authors concluded that in clinical setting a drugdrug interaction potential affecting the formation of hydroxyl metabolite is unlikely
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a Conjugates were analyzed by indirect approachesconversion into respective parent entities; blockade of glucuronidase with the use of specic inhibitor such as saccharo-1,4-lactone allows the differential quantitation of sulfate conjugates contribution.
N. R. Srinivas
Method was useful to monitor plasma concentrations of multiple analytes after a single oral dose administration of PPF (300 mg). The method would be useful for a detailed stereoselective evaluation in the PK disposition of PPF and its phase I metabolite, 5-OHPPF and phase II metabolites (glucuronic acid and sulfate conjugates)
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Applicable conclusion
Calibration ranges: PPF, 20 1600 ng/mL/enantiomer (r > 0.99); 5-OHPPF, 20500 ng/mL/ enantiomer (r > 0.99)
No. of analytes
Table 1. (continued )
Reference
Zhong and Chen (1999)
R-/S-propafenone (PPF); R-/ S-5-hydroxy PPF; glucuronic acid/sulfate conjugatesa of PPF and 5-hydroxy PPF
Analytes
ChiralAGP column; SRM via electrospray ionization (ESI) Mobile phase PPF: 10 mm ammonium acetate buffer (pH 5.96)1 propanol (100:9 v/v); FR, 0.5 mL/min. SRM transitions, m/z 342 to m/z 324 for PPF Mobile phase 5-OHPPF: 10 mm ammonium acetate buffer (pH 4.1)2 propanol (100:0.9 v/v); FR, 0.6 mL/min. SRM transitions, m/z 358 to m/z 340 for 5-OHPPF
Chromatographic conditions/detection system
Accuracy/precision: for both analytes were acceptable (within 11%dev and/or CV)
LOQ: PPF, 20 ng/mL/ enantiomer; 5-OHPPF, 20 ng/ mL/enantiomer
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BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr. 18: 759784 (2004) Published online in Wiley InterScience (www.interscience.wiley.com).

Simultaneous chiral analyses of multiple analytes DOI: 10.1002/bmc.447

REVIEW REVIEW 759

THERAPY MARKERSDIFFERENTIATION FROM DRUG ABUSE AND/OR APPLICABILITY IN FORENSICS

Simultaneous chiral analyses of multiple analytes

ROLE IN PHARMACOGENETIC/POLYMORPHIC EVALUATION

MONITORING OF PARENT AND ACTIVE METABOLITE(S) IN CLINICAL AND PRECLINICAL INVESTIGATIONS

Simultaneous chiral analyses of multiple analytes

UNDERSTANDING THE ROLE CHIRALITY PLAYS IN DELINEATING A PECULIAR TOXIC EFFECT

METHOD DEVELOPMENT CONSIDERATIONS

Simultaneous chiral analyses of multiple analytes

Simultaneous chiral analyses of multiple analytes

Simultaneous chiral analyses of multiple analytes

Copyright 2004 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 18: 759784 (2004)

Simultaneous chiral analyses of multiple analytes

Brunnenberg and Kovar (2001)

R-N-ethyl-3,4methylenedioxyamphetamine (MDE) S-MDE R-3,4-methylenedioxyamphetamine (MDA) S-MDA R-O-glucuronyl-N-ethyl-4hydroxy-3-methoxyamphetamine (HME) S-HME

Hedeland et al. (2004)

R-verapamil; S-verapamil, R-norverapamil; Snorverapamil; deuterated (D3)-verapamil

Jansson et al. (2003)

R-/S-mephenytoin; R-/Snirvanol; R-/S-4hydroxymephenytoin internal standard

Kaddoumi et al. (2001)

d-fenuramine l-fenuramine d-norfenuramine l-fenuramine Phentermine

Rodriguez-Rosas et al. (2003b)

S-ketamine R-ketamine S-norketamine R-norketamine Bromoketamine

Volosov et al. (2000)

(R)-10-hydroxycarbazepine (MHD) S-MHD Enantiomers of carbamazepine-10,11-transdihydrodiol (DHD) Oxcarbazepine oxime (IS)

Table 1. (continued ) Reference No. of analytes Analytes Chromatographic conditions/detection system

Zhang et al. (1999)

R-()-salmeterol S-(+)-salmeterol R--hydroxysalmeterol S--hydroxysalmeterol

Copyright 2004 John Wiley & Sons, Ltd.

Zhong and Chen (1999)

Chromatographic conditions/detection system

LOQ: PPF, 20 ng/mL/ enantiomer; 5-OHPPF, 20 ng/ mL/enantiomer

Copyright 2004 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 18: 759784 (2004)

Copyright 2004 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 18: 759784 (2004)

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