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This review highlights recent advancements in using high resolution nuclear magnetic resonance (NMR) spectroscopy as a characterization tool to expedite biologics formulation development, meeting a current need in the biopharmaceutical industry. Conformational changes of protein therapeutics during formulation development can result in various protein-protein and protein-excipient interactions, which can lead to physical aggregation and/or chemical degradation. Innovative analytical techniques that allow studying protein integrity with high specificity during formulation development are urgently needed in order to assess protein formulation stability and mitigate product quality risks. Solution NMR spectroscopy is emerging as a powerful analytical tool for biophysical characterization of protein therapeutics. For instance, one-dimensional (1D) NMR has been employed in high sensitivity monitoring of monoclonal antibody (mAb) structural changes and protein-excipient interactions in parenteral formulations, demonstrating it as a potential tool for formulation screening. 2D NMR, such as ALSOFAST-[

This review presents selective case studies in the literature and in the authors’ laboratories highlighting common degradation pathways that could lead to the formation of genotoxic degradants. It is the intention of this review to spark further discussions in this field, which is apparently still at a juvenile stage, with a goal to provide general guidance that can benefit drug developers and regulatory agencies in the long run.

Ethylnitrilium ion can be generated by protonation of acetonitrile (when used as the LC-MS mobile phase) under the conditions of atmospheric pressure ionizations, including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) as well as atmospheric pressure photoionization (APPI). Ethylnitrilium ion (CH3 C N H and its canonical form CH3 C NH) is shown to efficiently undergo the gas-phase Meerwein reaction with epoxides. This reaction proceeds by the initial formation of an oxonium ion followed by three-to-five-membered ring expansion via an intramolecular nucleophilic attack to yield the Meerwein reaction products. The density functional theory (DFT) calculations at the B3LYP/6-311G(d,p) level show that the gas-phase Meerwein reaction is thermodynamically favorable. Collision-induced dissociation (CID) of the Meerwein reaction products yields the net oxygen-by-nitrogen replacement of epoxides with a characteristic mass shift of 1 Da, providing evidence for the cyclic nature of the gas-phase Meerwein reaction products. The gas-phase Meerwein reaction offers a novel and fast LC-MS approach for the direct analysis of epoxides that might be of genotoxic concern during drug development. Understanding and utilizing this unique gas-phase ion/molecule reaction, the sensitivity and selectivity for quantitation of epoxides can be enhanced. (J Am Soc Mass Spectrom 2010, 21, 1802–1813) © 2010 American Society for Mass Spectrometry

Understanding the origin and fate of organic impurities within the manufacturing process along with a good control strategy is an integral part of the quality control of drug substance. Following the underlying principles of quality by design (QbD), a systematic approach to analytical control of process impurities by impurity fate mapping (IFM) has been developed and applied to the investigation and control of impurities in the manufacturing process of Pazopanib hydrochloride, an anticancer drug approved recently by the U.S. FDA. This approach requires an aggressive chemical and analytical search for potential impurities in the starting materials, intermediates and drug substance, and experimental studies to track their fate through the manufacturing process in order to understand the process capability for rejecting such impurities. Comprehensive IFM can provide elements of control strategies for impurities. This paper highlights the critical roles that analytical sciences play in the IFM process and impurity control. The application of various analytical techniques (HPLC, LC-MS, NMR, etc.) and development of sensitive and selective methods for impurity detection, identification, separation and quantification are highlighted with illustrative examples. As an essential part of the entire control strategy for Pazopanib hydrochloride, analytical control of impurities with 'meaningful' specifications and the 'right' analytical methods is addressed. In particular, IFM provides scientific justification that can allow for control of process impurities up-stream at the starting materials or intermediates whenever possible.

Several mass spectrometry (MS) techniques including accurate MS and MS/MS, as well as hydrogen/deuterium (H/D) exchange, were utilized to characterize a pseudo-oxidative reaction by-product (impurity I) in the pharmaceutical synthesis of S-(thiobenzoyl)thioglycolic acid. The negative ion MS/MS data provided complementary structural information to the positive ion MS/MS data. An understanding of the gas-phase Smiles rearrangement upon collision-induced dissociation (CID) in the negative ion MS/MS mode played an important role in structural elucidation of impurity I. The theoretical calculations by density functional theory (DFT) at the B3LYP/6-311G(d,p) level provided insights into the thermochemistry of the Smiles rearrangement reaction. This pseudo-oxidative impurity is proposed to be generated via the base-catalyzed hydrolysis in solution.

Many pharmaceutical genotoxic impurities are neutral molecules. Trace level analysis of these neutral analytes is hampered by their poor ionization efficiency in mass spectrometry (MS). Two analytical approaches including chemical derivatization and coordination ion spray-MS were developed to enhance neutral analyte detection sensitivity. The chemical derivatization approach converts analytes into highly ionizable or permanently charged derivatives, which become readily detectable by MS. The coordination ion spray-MS method, on the other hand, improves ionization by forming neutral-ion adducts with metal ions such as Na + , K + , or NH 4 + which are introduced into the electrospray ionization source. Both approaches have been proven to be able to enhance the detection sensitivity of neutral pharmaceuticals dramatically. This article demonstrates the successful applications of the two approaches in the analysis of four pharmaceutical genotoxic impurities identified in a single drug development program, of which two are non-volatile alkyl chlorides and the other two are epoxides.

A novel ion/molecule reaction was observed to occur under electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI) conditions, leading to dimerization of ionized 4-(methyl mercapto)-phenol followed by fast H(*) loss. The reaction is particularly favored during ESI, which suggests that this ion/molecule reaction can occur both in the solution inside the ESI-charged droplets and in the gas-phase environment of most other atmospheric pressure ionization techniques. The dimerization reaction is inherent to the electrolytic process during ESI, whereas it is more by ion/molecule chemistry in nature during APCI and APPI. From the tandem mass spectrometry (MS/MS) data, accurate mass measurements, hydrogen/deuterium (H/D) exchange experiments and density functional theory (DFT) calculations, two methyl sulfonium ions appear to be the most likely products of this electrophilic aromatic substitution reaction. The possible occurrence of this unexpected reaction complicates mass spectral data interpretation and can be misleading in terms of structural assignment as reported herein for 4-(methyl mercapto)-phenol.

Pharmaceutical regulatory agencies are increasingly concerned with trace-level genotoxic impurities in drug substances, requiring manufacturers to deliver innovative approaches for their analysis and control. The need to control most genotoxic impurities in the low ppm level relative to the active pharmaceutical ingredient (API), combined with the often reactive and labile nature of genotoxic impurities, poses significant analytical challenges. Therefore, sophisticated analytical methodologies are often developed to test and control genotoxic impurities in drug substances. From a quality-by-design perspective, product quality (genotoxic impurity levels in this case) should be built into the manufacturing process. This necessitates a practical analysis and control strategy derived on the premise of in-depth process understanding. General guidance on how to develop strategies for the analysis and control of genotoxic impurities is currently lacking in the pharmaceutical industry. In this work, we demonstrate practical examples for the analytical control of five genotoxic impurities in the manufacturing process of pazopanib hydrochloride, an anticancer drug currently in Phase III clinical development, which may serve as a model for the other products in development. Through detailed process understanding, we implemented an analysis and control strategy that enables the control of the five genotoxic impurities upstream in the manufacturing process at the starting materials or intermediates rather than at the final API. This allows the control limits to be set at percent levels rather than ppm levels, thereby simplifying the analytical testing and the analytical toolkits to be used in quality control laboratories.

Derivatization LC/MS methodology has been developed for the determination of a group of commonly encountered alkyl esters of sulfonates or sulfates in drug substances at low ppm levels. This general method uses trimethylamine as the derivatizing reagent for ethyl/propyl/isopropyl esters and triethylamine for methyl esters. The resulting quaternary ammonium derivatization products are highly polar (ionic) and can be retained by a hydrophilic interaction liquid chromatography (HILIC) column and readily separated from the main interfering active pharmaceutical ingredient (API) peak that is usually present at very high concentration. The method gives excellent sensitivity for all the alkyl esters at typical target analyte level of 1-2 ppm when the API samples were prepared at 5mg/mL. The recoveries at 1-2 ppm were generally above 85% for all the alkyl esters in the various APIs tested. The injection precisions of the lowest concentration standards were excellent with R.S.D.=0.4-4%. A linear range for concentrations from 0.2 to 20 ppm has been established with R(2)>or=0.99. This general method has been tested in a number of API matrices and used successfully for determination of alkyl sulfonates or dialkyl sulfates in support of API batch releases at GlaxoSmithKline.

A novel GC-MS method which requires small injection volumes was developed for fast and selective determination of headspace oxygen in pharmaceutical packages. This method does not require a specific GC column for separation of oxygen from other permanent gases such as nitrogen; instead it offers the advantage of using co-eluting nitrogen as the internal standard for quantifying oxygen in the headspace under electron ionization (EI, 70 eV) conditions. The relative ionization efficiency of oxygen to nitrogen, termed as ionization efficiency correction factor (IECF), can be measured using a control sample with known composition of oxygen and nitrogen such as the standard dry air used in this study. To avoid contamination, it is necessary to flush the syringe with pure helium. The measurements by the method are independent of the variations of sampling volumes. The determined headspace oxygen contents (R.S.D.

Oxidation of 1,1'-bis(diphenylphosphino)-ferrocene (DPPF) was found to occur when it served as the ligand for Pd(II)(CH 3 COO) 2 in a Heck reaction. This oxidative impurity of DPPF, referred to as DPPF(O), was identified by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) and exact mass measurements. Protonated DPPF(O) exhibited unique fragmentation pathways in the gas phase. Hydrogen/deuterium (H/D) exchange experiments provided important insights into the dissociation mechanisms of protonated DPPF(O), suggesting the existence of isomeric structures of the product ions by retaining or losing a proton (or deuteron) upon collision-induced dissociation (CID). The specific fate of the proton (or deuteron) upon CID is postulated to be dependent on the distance between the exchangeable proton (or deuteron) and the sites of bond cleavage. Density functional theory (DFT) calculations at the B3LYP/LANL2DZ level of theory showed that oxygen in DPPF(O) plays a pivotal role in invoking π-cation interactions between the p-type lone pair electrons (n π ) in oxygen and the anti-bonding orbital of Fe(II), accounting for the major fragmentation pathways of protonated DPPF(O). Facile formation of organometallic distonic ions in dissociation of protonated DPPF(O), and especially of protonated DPPF, could be useful for further exploration of their chemical properties by gas-phase ion/molecule reactions.

Structural elucidation of pharmaceutical impurities in drug substances and drug products is an important task in pharmaceutical analysis in various phases of drug development. Liquid chromatography-mass spectrometry (LC-MS) technologies play a key role in this task owing to their general attributes of superior selectivity, sensitivity and speed. Full scan and product ion scan analysis, providing molecular weight information and fragmentation data, respectively, offer rich structural information and allow proposal of candidate structures rather quickly. However, these proposed structures often lack certainty especially when dealing with structural isomers. On-line hydrogen/deuterium (H/D) exchange by LC-MS using D2O as the mobile phase component is a powerful tool for identifying active hydrogen atoms, thus constituting a simple strategy for distinguishing between isomeric structures which are sometimes difficult by product ion spectral data or accurate mass data. This review describes the typical experimental setup we use routinely in the laboratories for performing H/D exchange LC-MS experiments in conjunction with representative applications of the strategy in structural elucidation of pharmaceutical impurities.

Fragmentation mechanisms of trans-1,4-diphenyl-2-butene-1,4-dione were studied using a variety of mass spectrometric techniques. The major fragmentation pathways occur by various rearrangements by loss of H2O, CO, H2O and CO, and CO2. The other fragmentation pathways via simple alpha cleavages were also observed but accounted for the minor dissociation channels in both a two-dimensional (2-D) linear ion trap and a quadrupole time-of-flight (Q-TOF) mass spectrometer. The elimination of CO2 (rather than CH3CHO or C3H8), which was confirmed by an exact mass measurement using the Q-TOF instrument, represented a major fragmentation pathway in the 2-D linear ion trap mass spectrometer. However, the elimination of H2O and CO becomes more competitive in the beam-type Q-TOF instrument. The loss of CO is observed in both the MS2 experiment of m/z 237 and the MS3 experiment of m/z 219 but via the different transition states. The data suggest that the olefinic double bond in protonated trans-1,4-diphenyl-2-butene-1,4-dione plays a key role in stabilizing the rearrangement transition states and increasing the bond dissociation (cleavage) energy to give favorable rearrangement fragmentation pathways. Copyright © 2006 John Wiley & Sons, Ltd.

Strategies using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with techniques such as chemical derivatization and on-line hydrogen/deuterium (H/D) exchange for structural elucidation of drug metabolites in crude samples are reviewed. Useful mass spectrometric scan techniques discussed include product ion scan, constant neutral-loss scan, precursor ion scan, multistage MS(n), and accurate mass measurements. In biological systems, xenobiotics are transformed into metabolites, which usually involves introduction of one or more polar functional groups or removal or blockage of such structural moieties. Therefore, chemical derivatization strategies for determination of functional groups and on-line H/D exchange approaches for probing number of exchangeable hydrogens are powerful tools for structural elucidation of drug metabolites in drug metabolism studies. More importantly, these experiments can be carried out on crude samples in microscale, providing sufficient material for LC-MS/MS analysis. Therefore, labor intensive and technically challenging purification of low levels of drug metabolites from complex biological matrices can be avoided. It is the authors' conclusion that strategies such as chemical derivatization and on-line H/D exchange should be used more routinely in drug metabolism studies in order to facilitate metabolite identification.

A new type of quadrupole linear ion trap mass spectrometer, Q TRAP™ LC/MS/MS system (Q TRAP™), was evaluated for its performance in two studies: firstly, the in vitro metabolism of gemfibrozil in human liver microsomes, and, secondly, the quantification of propranolol in rat plasma. With the built-in information-dependent-acquisition (IDA) software, the instrument utilizes full scan MS in the ion trap mode and/or constant neutral loss scans as survey scans to trigger product ion scan (MS2) and MS3 experiments to obtain structural information of drug metabolites ‘on-the-fly’. Using this approach, five metabolites of gemfibrozil were detected in a single injection. This instrument combines some of the unique features of a triple quadrupole mass spectrometer, such as constant neutral loss scan, precursor ion scan and multiple reaction monitoring (MRM), together with the capability of a three-dimensional ion trap. Therefore, it becomes a powerful instrument for metabolite identification. The fast duty cycle in the ion trap mode allows the use of full product ion scan for quantification. For the quantification of propranolol, both MRM mode and full product ion scan in the ion trap mode were employed. Similar sensitivity, reproducibility and linearity values were established using these two approaches. The use of the product ion scan mode for quantification provided a convenient tool in selecting transitions for improving selectivity during the method development stage. Copyright © 2003 John Wiley & Sons, Ltd.

An Erratum for this article has been published in Rapid Communications in Mass Spectrometry 17(3) 2003, 264 Biotransformation studies performed on an investigational compound (I, represented by R1-CH(NH2)-CO-N(R2)-CH2-S-R3) led to the identification of five metabolites (M1–M5). Based on LC/MS (liquid chromatography/mass spectrometry) analysis which included the use of H2O and D2O in the mobile phases, they were identified as the sulfoxide (M1), sulfone (M2), carbamoyl glucuronide (M3), N-glucuronide (M4), and N-glucoside (M5) metabolites, respectively. The structure of M3, a less commonly seen carbamoyl glucuronide metabolite, was established using on-line H/D (hydrogen/deuterium) exchange experiments conducted by LC/MS. H/D exchange experiments were also used to distinguish the S-oxidation structures of M1 and M2 from hydroxylation. Herein, the application of deuterium oxide as the LC/MS mobile phase for structural elucidation of drug metabolites in biological matrices is demonstrated. Copyright © 2001 John Wiley & Sons, Ltd.

A method with parallel extraction columns and parallel analytical columns (PEC-PAC) for on-line high-flow liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for simultaneous quantification of a drug candidate and its six metabolites in dog plasma. Two on-line extraction columns were used in parallel for sample extraction and two analytical columns were used in parallel for separation and analysis. The plasma samples, after addition of an internal standard solution, were directly injected onto the PEC-PAC system for purification and analysis. This method allowed the use of one of the extraction columns for analyte purification while the other was being equilibrated. Similarly, one of the analytical columns was employed to separate the analytes while the other was undergoing equilibration. Therefore, the time needed for re-conditioning both extraction and analytical columns was not added to the total analysis time, which resulted in a shorter run time and higher throughput. Moreover, the on-line column extraction LC/MS/MS method made it possible to extract and analyze all seven analytes simultaneously with good precision and accuracy despite their chemical class diversity that included primary, secondary and tertiary amines, an alcohol, an aldehyde and a carboxylic acid. The method was validated with the standard curve ranging from 5.00 to 5000 ng/mL. The intra- and inter-day precision was no more than 8% CV and the assay accuracy was between 95 and 107%. Copyright © 2001 John Wiley & Sons, Ltd.

The present work demonstrates, for the first time, the application of the mass spectrometric kinetic method for quantitative chiral purity determination by automatic flow-injection MS/MS. The particular compound analyzed is GSK2251052A, a novel boron-containing systemic antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections. Chiral recognition and quantitation of GSK2251052A was achieved based on the competitive dissociation kinetics of the Cu(II)-bound trimeric complex [Cu(II)(A)(ref*)2-H](+) (A = GSK2251052A or its R-enantiomer, ref* = L-tryptophan) that gives rise to Cu(II)-bound dimeric complexes. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow chiral purity determination of pharmaceutical compounds during enantioselective synthesis. By using flow-injection MS/MS, enantiomeric quantitation of GSK2251052A by the kinetic method proved to be fast (2 min for analysis of each sample) and to have accuracy comparable to chiral LC-MS/MS and LC-UV methods as well as the method using chiral derivatization followed by LC-MS/MS analysis. This flow-injection MS/MS method represents an alternative approach to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful for rapid screening of chiral drugs during pharmaceutical development.

A gas-phase derivatization strategy is reported by using the gas-phase Meerwein reaction for rapid and direct LC-MS analysis of epoxides, which are potential genotoxic impurities (GTIs) in active pharmaceutical ingredients (APIs). This class-selective ion/molecule reaction occurs between epoxides and the ethylnitrilium ion (CH(3)-C≡NH↔CH(3)-C=NH) that is generated by atmospheric pressure ionizations (when acetonitrile is used as the mobile phase). Density functional theory (DFT) calculations at the B3LYP/6-311+G(d,p) level show that the gas-phase Meerwein reaction is thermodynamically favorable. Commonly used atmospheric pressure ionization techniques including ESI, APCI and APPI were evaluated for optimal formation of the Meerwein reaction products. APCI appears to be the method of choice since it offers better sensitivity and more robust detection under typical LC-MS instrumentation conditions. Quantitative analysis of epoxides can be achieved by either single ion monitoring (SIM) or multiple reaction monitoring (MRM) of the Meerwein reaction products. We demonstrate herein quantitative analysis of two potential GTIs of SB797313 and SB719133 in APIs. The validated methods afford excellent linearity (r(2)≥0.999), sensitivity (LOD≤1 ppm by w/w in 10 mg/mL APIs) and recovery (ranging from 92% to 102%), as well as accuracy (≤2.8% difference) and precision (≤2.2% RSD) based on injections of six prepared standards. This novel strategy is particularly useful when a target analyte is difficult to be directly analyzed by LC-MS (e.g. due to poor ionization) or unstable in the course of solution-phase derivatization.

Elesclomol (N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide)), which is a novel anticancer agent, can form chelating complexes with Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) in the gas phase during electrospray ionization (ESI) mass spectrometry. In the solution phase with acidic medium during chromatographic separation, however, only Cu(II) and Ni(II) to a lesser degree favor the formation of chelating complexes with elesclomol. The Cu(II)-chelating complex [Cu(II)+elesclomol-H]+· exhibits more complicated MS/MS fragmentation pathways than the Ni(II)-chelating complex [Ni(II)+elesclomol-H]+. One significant difference is the ready occurrence of the electron transfer upon collision-induced dissociation (CID) of [Cu(II)+elesclomol-H]+·. This leads to the reduction of Cu(II) to Cu(I). However, such phenomenon was not observed upon CID of [Ni(II)+elesclomol-H]+. On the basis of the density functional theory (DFT) calculations at the B3LYP/6-31+G(d)/LANL2DZ level, the Cu(II)- and Ni(II)-chelating complexes of elesclomol exist in the keto-form with tetra-coordinated trapezoid geometry in the gas phase but at different levels of distortion. As compared to the Ni(II)-elesclomol complex, the Cu(II)-elesclomol complex is more stable (by -55.25 kcal/mol). This relative stability of the chelating complexes of elesclomol is consistent with the Irving-Williams series of bindings to ligands.

Trace analysis of unstable and reactive pharmaceutical genotoxic impurities (GTIs) is a challenging task in pharmaceutical analysis. Many method issues such as insufficient sensitivity, poor precision, and unusual (too high/low) spiking recovery are often directly related to analytes' instability. We report herein a matrix deactivation approach that chemically stabilizes these analytes for analytical method development. In contrast to the conventional chemical derivatization approach where the analytes are transformed into stable detectable species, the matrix deactivation approach chemically deactivates the hypothetical reactive species in the sample matrix. The matrix deactivation approach was developed on the premise that the instability of certain analytes at trace level is caused by reactions between the analytes and low level reactive species in the sample matrix. Thus, quenching the reactivity of the reactive species would be a key to stabilizing the unstable and reactive analytes. For example, electrophilic alkylators could be destabilized by nucleophiles or bases through either nucleophilic substitution or elimination reactions. One way to mask those reactive species is via protonation by adding acids to the diluent. Alternatively, one can use nucleophile scavengers to deplete reactive unknown species in the sample matrix completely, in analogy to the use of antioxidants and metal chelators to prevent oxidation in the analysis of compounds liable to oxidation. This paper reports the application of the matrix deactivation to the analyses of unstable and reactive pharmaceutical genotoxic impurities. Some of the methods have been used to support development of manufacturing processes for drug substances and a recent regulatory filing.

Genotoxic impurities (GTIs) in pharmaceuticals at trace levels are of increasing concerns to both pharmaceutical industries and regulatory agencies due to their potentials for human carcinogenesis. Determination of these impurities at ppm levels requires highly sensitive analytical methodologies, which poses tremendous challenges on analytical communities in pharmaceutical RD. Practical guidance with respect to the analytical determination of diverse classes of GTIs is currently lacking in the literature. This article provides an industrial perspective with regard to the analysis of various structural classes of GTIs that are commonly encountered during chemical development. The recent literatures will be reviewed, and several practical approaches for enhancing analyte detectability developed in recent years will be highlighted. As such, this article is organized into the following main sections: (1) trace analysis toolbox including sample introduction, separation, and detection techniques, as well as several 'general' approaches for enhancing detectability; (2) method development: chemical structure and property-based approaches; (3) method validation considerations; and (4) testing and control strategies in process chemistry. The general approaches for enhancing detection sensitivity to be discussed include chemical derivatization, 'matrix deactivation', and 'coordination ion spray-mass spectrometry'. Leveraging the use of these general approaches in method development greatly facilitates the analysis of poorly detectable or unstable/reactive GTIs. It is the authors' intent to provide a contemporary perspective on method development and validation that can guide analytical scientists in the pharmaceutical industries.

An online sample extraction chiral bioanalytical method was developed and validated for the quantification of terbutaline, a β2-selective adrenoceptor agonist, spiked into human plasma by using two extraction columns and a chiral stationary phase (CSP) in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). In this method, two Oasis HLB extraction columns were used in parallel for plasma sample purification and a Chirobiotic T CSP was used for enantiomeric separation. Atmospheric pressure chemical ionization MS/MS was employed in multiple reaction monitoring mode for the detection and quantification. Subsequent to the addition of an internal standard solution, the plasma samples were directly injected onto the system for extraction and analysis. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for reconditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a shorter run time and higher throughput. A lower limit of quantification of 1.0 ng/mL was achieved using only 50 microliter of human plasma. The method was validated with a dynamic range of 1.0–200 ng/mL. The intra- and interday precision was no more than 11% CV and the assay accuracy was between 94–106%. Chirality 14:742–749, 2002. © 2002 Wiley-Liss, Inc.