Effect of the mobile phase on the retention behavior of optical isomers of the mandelic acid derivative methyl 2-phenyl-2-(tetrahydropyranyloxy) acetate by chiral HPLC

Conditions for separation of enantiomers of a mandelic acid derivative, methyl 2-phenyl-2-(tetrahydropyranyloxy) acetate (the analyte) were studied. Because of the presence of two chiral carbons, the analyte consists of four stereoisomers stable at ambient temperature. Chiral HPLC of the analyte resulted in four peaks, using an (S,S)-Whelk-O1 column with the mobile phase consisting of hexane and the t-butyl methyl ether (TBME). It was found that TBME dramatically changed the retention of the isomers, though it produced the best enantioseparation on (S,S)-Whelk-O1. The amount of TBME in the mobile phase influenced the degree of retention shift; 5% (v/v) TBME gave a bigger shift than 8% (v/v) and 10% (v/v). 2-Propanol did not produce the same results. The chiral separation was also tried on cellulose tris (3, 5-dimethyl phenylcarbamate) (CDMPC), but only three peaks were seen, indicating some but not full enantiomer resolution.     

Chiral HPLC separation and CD spectra of the C-2 diastereomers of naringin in grapefruit during maturation

Naringin is the chief flavanone glycoside of grapefruit (Citrus paradisi). It is responsible for part of the bitter taste of the fruit and can cause the inhibition of some cytochrome P450s. The direct separation of (2R)- and (2S)-naringin in the albedo of grapefruits was obtained in normal phase HPLC mode using Chiralcel OD as chiral stationary phase and n-hexane/ethanol with 0.1% of TFA as mobile phase. Chiralpak AD was almost ineffective in the separation. This procedure was used to evaluate the stereochemistry at C-2 during maturation of the grapefruit. The CD curves of (2R)- and (2S)-naringin isolated by semipreparative chiral HPLC were determined and the elution order of the chromatographic peaks was related to the absolute C-2 configuration. Partial resolution of the C-2 diastereomers of narirutin was obtained on Chiralpak AD.     

Possible uses of micellar electrokinetic capillary chromatography and high-performance liquid chromatography for the chiral discrimination of some pyrethroids

Micellar electrokinetic capillary chromatography (MECC) and high-performance liquid chromatography (HPLC) were used for the separation of stereoisomers of the lipophilic uncharged pyrethroids cypermethrin, alphamethrin, permethrin, and fenpropathrin. Different kinds of cyclodextrin (β-cyclodextrin, hydroxypropyl-β-cyclodextrin, dimethyl-β-cyclodextrin, and γ-cyclodextrin), surfactants (sodium dodecyl sulphate [SDS] and cetyltrimethylammonium bromide [CTAB]), and cations of background electrolyte (sodium, ammonium, TRIS, and Ammediol) were tested. Optimized conditions (background electrolyte: 50 mmol/l sodium phosphate, pH ≈ 2.5, 150 mmol/l SDS, 150 mg/ml γ-cyclodextrin) allowed the separation of alphamethrin, the eight cypermethrin stereoisomers being eluted in seven peaks and the separation of two enantiomers of fenpropathrin with resolution R_s = 10 and with n ≃ 500,000 theoretical plates. Different experimental conditions, e.g., mobile phase composition, temperature, injected amount, and flow rate, were also optimized in HPLC experiments. The optimal conditions (stationary phase: ChiraDex, 5 μm; mobile phase: 150 mmol triethylamine/l with H₂SO₄ in water (pH = 3.5) with methanol or acetonitrile; flow rate: 0.8 or 0.6 ml/min; temperature: ambient or 30°, 20°, or 10°C; experimental conditions were modified according to the type of analysis) allow chiral discrimination of alphamethrin enantiomers and analysis of permethrin stereoisomers. MECC offers higher efficiency and shorter analysis time than HPLC, but under tested conditions it was shown that the methods complement each other. Chirality 9:162–166, 1997. © 1997 Wiley-Liss, Inc.     

Indirect chiral separation and analyses in human biological fluids of the stereoisomers of a thienothiopyran-2-sulfonamide (TRUSOPT), a novel carbonic anhydrase inhibitor with two chiral centers in the molecule.

The indirect chiral separation of the four stereoisomers (1)-(4) of a novel carbonic anhydrase inhibitor with two chiral centers in the molecule is reported. The method is based on chemical derivatization of the secondary amino group of the inhibitor with chiral isocyanate, formation of diastereomeric urea derivatives, each with three chiral centers in the molecule, and their separation under nonchiral HPLC conditions. The attempts to separate racemic mixture (1) + (2) from its diastereomeric counterpart (3) + (4) under nonchiral conditions, and to separate enantiomers (1) and (2) directly on a chiral stationary phase (CSP) are also reported. The indirect method was utilized for the assessment of an in vivo inversion of configuration at either one or both chiral centers of the molecule of (1). Analyses of selected whole blood and urine samples from human subjects after multiple bilateral topical ocular dosing with (1) did not reveal the presence of any of the three possible stereoisomers (2)-(4) of (1) indicating that the inversion of configuration at neither one nor two chiral centers of (1) occurs in vivo.     

Separation of Nadolol Stereoisomers Using Chiralpak IA Chiral Stationary Phase

Chiralpak IA adsorbent is used for both analytical and preparative chromatographic separation of nadolol stereoisomers. The results include a complete screening of the mobile phase composition for both the baseline resolution of all four nadolol stereoisomers (analytical separation) and the simulated moving bed (SMB) pseudo‐binary separation of the most retained stereoisomer. The experimental results show that analytical baseline resolution of nadolol stereoisomers can be achieved using alcohol/hydrocarbon and alcohol/acetonitrile solvent mixtures. The 10%ethanol/90%acetonitrile mixture is presented as the one that presents baseline resolution with lower retention. For the preparative pseudo‐binary separation, pure ethanol, pure methanol, alcohol/acetonitrile, and alcohol/tetrahydrofuran mixtures proved to allow good separation results. The 100%methanol/0.1%diethylamine solvent composition was selected to perform the experimental SMB separation. Using a 10 g/L total feed concentration, the more retained stereoisomer was recovered at the extract outlet stream with 99.5% purity, obtaining a system productivity of 1.98 gL^?1 h^?1 and requiring a solvent consumption of 3.13 L/g of product. Comparing these results with the ones recently presented by Ribeiro et al. (2013), this work shows that the Chiralpak IA chiral adsorbent is an interesting alternative to Chiralpak AD for the separation of nadolol stereoisomers at both analytical and preparative scales. Chirality 28:399–408, 2016. © 2016 Wiley Periodicals, Inc.     

Stereoselective metabolism of metoprolol: enantioselectivity of alpha-hydroxymetoprolol in plasma and urine

Direct stereoselective separation on chiral stationary phase was developed for HPLC analysis of the four stereoisomers of alpha-hydroxymetoprolol in human plasma and urine. Plasma samples were prepared using solid-phase extraction columns and urine samples were prepared by liquid-liquid extraction. The stereoisomers were separated on a Chiralpak AD column at 24 degrees C with fluorescence detection and a mobile phase consisting of a mixture of hexane:ethanol:isopropanol:diethylamine (88:10.2:1.8:0.2) for plasma samples and hexane:ethanol:diethylamine (88:12:0.2) for urine samples. Calibration curves for the individual stereoisomers were linear within the concentration range of 2.0-200 ng/ml plasma or 0.125-25 microg/ml urine. The methods were validated with intra- and interday variations less than 15%. The absolute configuration of the pure stereoisomers were assigned by circular dichroism spectra. The methods were employed to determine the concentrations of alpha-hydroxymetoprolol stereoisomers in a metabolism study of multiple-dose administration of racemic metoprolol to hypertensive patients phenotyped as extensive metabolizers of debrisoquine. We observed stereo-selectivity in the alpha-hydroxymetoprolol formation favoring the new 1'R chiral center from both metoprolol enantiomers (AUC(0-24) (1'R1'S) = 3.02). The similar renal clearances (Cl(R)) of the four stereoisomers demonstrated absence of stereoselectivity in their renal excretion. (-)-(S)-metoprolol was slightly more alpha-hydroxylated than its antipode (AUC(0-24) (2S/2R) = 1.19), suggesting that this pathway is not responsible for plasma accumulation of this enantiomer in humans.     

Enantiomeric separation of imidazolinone herbicides using chiral high-performance liquid chromatography

Chiral high-performance liquid chromatography (HPLC) is one of the most powerful tools to prepare enantiopure standards of chiral compounds. In this study, the enantiomeric separation of imidazolinone herbicides, i.e., imazethapyr, imazapyr, and imazaquin, was investigated using chiral HPLC. The enantioselectivity of Chiralpak AS, Chiralpak AD, Chiralcel OD, and Chiralcel OJ columns for the three analytes was compared under similar chromatographic conditions. Chiralcel OJ column showed the best chiral resolving capacity among the test columns. The resolved enantiomers were distinguished by their signs of circular dichroism detected at 275 nm and their structures confirmed with LC-mass spectrometric analysis. Factors affecting the chiral separation of imidazolinones on Chiralcel OJ column were characterized. Ethanol acted as a better polar modifier than the other alcohols including 2-propanol, 1-butanol, and 1-pentanol. Although the acidic modifier in the mobile phase did not influence chiral recognition, it was necessary for reducing the retention time of enantiomers and suppressing their peak tailing. Thermodynamic evaluation suggests that enantiomeric separation of imidazolinones on Chiralcel OJ column is an enthalpy-driven process from 10 to 40 degrees C. This study also shows that small amounts of pure enantiomers of imidazolinones may be obtained by using the analytical chiral HPLC approach.     

Enantiomeric separation and recognition mechanism of 2-arylpropionic acid derivatives by high-performance liquid chromatography using a chiral column

An optical resolution of the amide derivatives of ibuprofen and the carbamate-alkylester derivatives of the trans-alcohol metabolite of loxoprofen and an analogous compound, CS-670, was studied by chiral high-performance liquid chromatography (HPLC). The chiral columns SUMIPAX OA-4000 and OA-4100 were used to investigate the enantiomeric separation behavior of these derivatives using both reversed and normal mobile phases. A better separation factor (α) of the amide and the carbamate ester derivatives was obtained in the normal mobile phase than in the reversed mobile phase HPLC. In addition, the recognition mechanisms of both amide and carbamate ester enantiomers were investigated by ¹H-nuclear magnetic resonance (NMR). It is suggested that the important driving forces for the enantiomeric separation are the formation of hydrogen bonding and the charge transfer complex between these derivatives and an active site of the chiral stationary phase. © 1995 Wiley-Liss, Inc.     

Direct Enantioseparation of Nitrogen‐heterocyclic Pesticides on Amylose‐tris‐(5‐chloro‐2‐methylphenylcarbamate) by Reversed‐Phase High‐Performance Liquid Chromatography

In this study, 11 nitrogen‐heterocyclic pesticides were stereoselectively separated on amylose‐tris‐(5‐chloro‐2‐methylphenylcarbamate) chiral stationary phase, using reversed‐phase high‐performance liquid chromatography with diode array detector and optical rotation detector at 426 nm. The effects of mobile phase composition and column temperature (5–40 °C) on separation were investigated. When acetonitrile and water were used as mobile phase, satisfactory separations were obtained on amylose‐tris‐(5‐chloro‐2‐methylphenylcarbamate) for four pesticides with elution orders of (+)/(?)‐simeconazole (1) , (?)/(+)‐nuarimol (3) , (?)/(+)‐carfentrazone‐ethyl (4) , and (?)/(+)/(?)/(+)‐bromuconazole (9) and part separations for three with elution orders of (?)/(+)‐famoxadone (6) , (+)/(?)‐fenbuconazole (10) , and (?)/(+)‐triapenthenol (11) . Only two chromatographic peaks on diode array detector were obtained for diclobutrazol (2) , cyproconazole (5) , etaconazole (7) , and metconazole (8) , although they should have four stereoisomers in theory because of presences of two chiral centers in molecules. The stereoisomeric optical signals of all pesticides did not reverse with temperature changes but would reverse with different solvent types for some pesticides. These results will be useful to prepare and analyze individual enantiomers of chiral pesticides. Chirality 24:1031–1036, 2012. © 2012 Wiley Periodicals, Inc.     

Diphenylethanediamine (DPEDA) as chiral selector: VII. Efficient HPLC resolution of a series of underivatized diarylcarbinols on a chiral stationary phase based on C5-amide-bonded N-3,5-dinitrobenzoyl-DPEDA

Fast and efficient baseline separation of asymmetrically substituted diarylmethanols and 1,1-diarylethanols was achieved on an endcapped, amide-linked N-3,5-dinitrobenzoylated, (R, R)-1,2-diphenyl-1,2-ethanediamine-derived chiral stationary phase (CSP). Optimal enantioselectivities on this CSP were obtained using 1% 2-propanol in n-heptane as the mobile phase. Enantiorecognition was found to be governed by π-basicity and the substitution pattern of the aromatic substituents. © 1996 Wiley-Liss, Inc.     

Enantiomeric separation of prothioconazole and prothioconazole‐desthio on chiral stationary phases

Prothioconazole is a type of broad‐spectrum triazole thione fungicide developed by the Bayer Company. Prothioconazole‐desthio is the main metabolite of prothioconazole in the environment. In our study, enantiomeric separation of prothioconazole and prothioconazole‐desthio was performed on various chiral stationary phases (CSPs) by high‐performance liquid chromatography (HPLC). It was found that polysaccharide CSPs showed better ability than brushing CSPs in enantiomeric separation. The successful chiral separation of prothioconazole could be achieved on self‐made Chiralcel OD, commercialized Chiralcel OJ‐H and Lux Cellulose‐1. Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralcel OD, Chiralpak AY‐H, Chiralpak AZ‐H, and Lux Cellulose‐1 realized the baseline separation of prothioconazole‐desthio enantiomers. Simultaneous enantiomeric separation of prothioconazole and prothioconazole‐desthio was performed on Lux Cellulose‐1 using acetonitrile (ACN) and water as mobile phase. In most cases, low temperature favored the separation of two compounds. The influence of the mobile phase ratio or type was deeply discussed. We obtained larger Rs and longer analysis time with a smaller proportion of isopropanol (IPA) or ethanol and more water content at the same temperature. The ratio of ACN and water had influences on the outflow orders of prothioconazole‐desthio enantiomers. This work provides a new approach for chiral separation of prothioconazole and prothioconazole‐desthio with a discussion of chiral separation mechanism on different CSPs.     

Direct chiral separations of the enantiomers of phenylpyrazole pesticides and the metabolites by HPLC

The enantiomeric separation of the enantiomers of three phenylpyrazole pesticides (fipronil, flufiprole, ethiprole) and two fipronil metabolites (amide‐fipronil and acid‐fipronil) were investigated by high‐performance liquid chromatography (HPLC) with a CHIRALPAK^® IB chiral column. The mobile phase was n‐ hexane or petroleum ether with 2‐propanol or ethanol as modifier at a flow rate of 1.0 mL/min. The influences of mobile phase composition and column temperature between 15 and 35°C on the separations were studied. All the analytes except ethiprole obtained complete enantiomeric separation after chromatographic condition optimization. Fipronil, flufiprole, amide‐fipronil, and acid‐fipronil obtained complete separation with the best resolution factors of 2.40, 3.40, 1.67, and 16.82, respectively, but ethiprole showed no enantioselectivity under the optimized conditions. In general, n‐ hexane with 2‐propanol gave better separations in most cases. The results showed decreasing temperature and content of modifier in the mobile phase resulted in better separation and longer analysis time as well. The thermodynamic parameters calculated according to linear the Van't Hoff equation indicated the chiral separations in the study were enthalpy‐driven. Fipronil and its two chiral hydrolyzed metabolites obtained baseline separation simultaneously under optimized conditions.     

Enantioseparation of chiral perfluorooctane sulfonate (PFOS) by supercritical fluid chromatography (SFC): Effects of the chromatographic conditions and separation mechanism

Perfluorooctane sulfonate (PFOS) is one of the most frequently detected perfluoroalkyl substances in environmental and human samples. Previous studies have shown that nonracemic PFOS in biological samples can be used as a marker of PFOS exposure sources. In recent years, supercritical fluid chromatography (SFC) has emerged as a powerful method to separate chiral compounds. In this study, a method of perfluoro‐1‐methylheptane sulfonate (1 m‐PFOS) enantioseparation by SFC was established. The optimal separation was obtained using a Chiralpak QN‐AX column with CO₂/2‐propanol (70/30, v/v) as the mobile phase with a flow rate of 1 mL/min, column temperature was 32°C, and BPR pressure was 1800 psi. The resolution (Rs) and retention time were 0.88 and 130 minutes, respectively. This method is more economic and greener than HPLC. Modifier pH and column temperature were determined to be significant factors of SFC chiral separation. Modifier pH is negatively correlated with the retention factors and Rs. Adsorption thermodynamics were used to explain the influence of temperature change, and it was concluded that the transfer of two enantiomers from the mobile phase to the stationary phase is enthalpy‐driven. Enantioseparation of 1 m‐PFOS by SFC follows the same rules of ion exchange as those for the chiral separation by HPLC.     

Enantiomeric separation of dansyl amino acids using macrocyclic antibiotics as chiral mobile phase additives by narrow-bore high-performance liquid chromatography

Seven macrocyclic antibiotics were evaluated as chiral selectors for the enantiomeric separation of 11 dansyl amino acids using narrow-bore high-performance liquid chromatography (HPLC). The macrocyclic antibiotics were incorporated as mobile phase additives to determine the enantioselective effects on the chiral analytes. The resolution and capacity factor (k') of each analyte were assessed while varying the structure of macrocyclic antibiotic and the mobile phase buffer pH. The selectivity of the chiral selectors was measured as a function of changes in these parameters. All 11 dansyl amino acids were separated by at least one of the chiral selectors. Three-dimensional computer modeling of the more effective chiral selectors illustrated the importance of macrocyclic antibiotic structure concerning stereospecific analyte interaction.     

Direct enantioselective HPLC monitoring of lipase-catalyzed kinetic resolution of tiaprofenic acid in nonstandard HPLC organic solvents

The first straightforward lipase-catalyzed enantioselective access to enantiomerically enriched tiaprofenic acid as a versatile method in chiral separation of racemates is demonstrated. The latter was directly monitored by enantioselective HPLC using a 3,5-dimethylphenylcarbamate derivative of cellulose-based chiral stationary phase namely Chiralpak IB (the immobilized version of Chiralcel OD). Non-standard HPLC organic solvents were used as diluent to dissolve the "difficult to dissolve" enzyme substrate (the acid) and as eluent for the simultaneous enantioselective HPLC baseline separation of both substrate and product in one run without any further derivatization. The existence of a non-standard HPLC organic solvent (e.g., methyl tert-butyl ether) in the mobile phase composition is mandatory to accomplish the simultaneous enantioselective HPLC baseline separation of both substrate and product.     

Enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors,potential adrenergic ligands,by analytical and preparative liquid chromatography on amylose chiral stationary phases and characterization of the enantiomers

To obtain milligram amounts of the enantiomers of benzoxazolinone derivatives to be tested for binding to adrenergic sites, analytical HPLC methods using derivatized amylose chiral stationary phases were developed for the direct enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors, derivatives with one or two chirals centers. The separations were made using normal phase methodology with a mobile phase of n‐hexane‐alcohol (ethanol, 1‐propanol, or 2‐propanol) in various proportions, and silica‐based amylose (tris‐3, 5‐dimethylphenylcarbamate) Chiralpak AD and (tris‐(S)‐1‐phenylethylcarbamate) Chiralpak AS columns. The effects of concentration of various aliphatic alcohols in the mobile phase were studied. The best separation was achieved on Chiralpak AS, so preparative HPLC was set up with this chiral stationary phase using a mobile phase consisting of n‐hexane‐alcohol using isocratic conditions and multiple repetitive injections. Physicochemicals properties of enantiomers were reported The effect of structural features of the solutes on discrimination between the enantiomers was examined. Limit of detection (LD) and limit of quantification (LQ) were determined using both ultra‐violet (UV) and evaporative light‐scattering detection (ELSD). Chirality, 2009. © 2008 Wiley‐Liss, Inc.     

The stereochemical resolution of the enantiomers of aspartame on an immobilized alpha-chymotrypsin HPLC chiral stationary phase: the effect of mobile-phase composition and enzyme activity

The enantioselective and diastereoselective resolutions of the stereoisomers of N alpha-aspartyl-phenylalanine 1-methyl ester (APME) have been accomplished on an HPLC chiral stationary phase based upon alpha-chymotrypsin (the ACHT-CSP) with observed enantioselectivities (alpha 1) for the DL-/LD-enantiomer of as high as 29.17 and for the DD-/LL-enantiomers of as high as 28.97. In addition, the effect on the chromatographic retention of the APME stereoisomers of the activity of the ACHT and the composition of the mobile phase--structure of the anionic component, molarity, and pH--have been studied. The results of this study suggest that the aspartyl moiety and/or the aspartyl-phenylalanine amide linkage play key roles in the observed enantioselectivity; the APME stereoisomers containing L-phenylalanine, i.e., DL- and LL-APME, bind at a different site in the ACHT molecule (the L-Phe site) than the APME stereoisomers containing D-phenylalanine (the D-Phe site); and the observed enantioselectivity is a measure of the difference in the binding affinities at the two sites rather than the consequence of differential affinities at a single site.     

Chiral HPLC analysis of milnacipran and its FMOC-derivative on cellulose-based stationary phases

The HPLC enantioseparation of the last generation antidepressive drug milnacipran (+/-)-1 was investigated on different cellulose-based chiral stationary phases (CSPs). On carbamate-type columns, Chiralcel OD and OD-H (+/-)-1 could be separated with alpha value about 1.20 but the resolution was quite low because of the tailing of the peaks. Direct determination of (+/-)-1 with high selectivity and resolution was obtained on Chiralcel OJ in normal phase mode elution. Precolumn derivatization of milnacipran with Fmoc-Cl gave compound (+/-)-2 which was enantioseparated on all the investigated CSPs and allowed enhanced UV or fluorimetric detection. The Chiralpak IB, that could be considered the immobilized version of Chiralcel OD-H, was found completely ineffective in the chiral recognition of (+/-)-1 and moderately efficient in the separation of (+/-)-2.     

Liquid chromatographic retention behavior and enantiomeric separation of three chiral center beta-blocker drug (nadolol) using heptakis (6-azido-6-deoxy-2, 3-di-O-phenylcarbamolyted) beta-cyclodextrin bonded chiral stationary phase

Nadolol, a beta-blocker used in the management of hypertension and angina pectoris, has three chiral centers and is currently marketed as an equal mixture of its four stereoisomers. Enantiomeric separation of nadolol by high-performance liquid chromatography was studied on a column packed with novel heptakis (6-azido-6-deoxy-2, 3-di-O-phenylcarbamolyted) beta-cyclodextrin bonded chiral stationary phase. The retention behavior and resolution of nadolol enantiomers were investigated and discussed with respect to the mobile phase composition and flow rate, pH, ionic strength, and temperature. The optimal separation condition was found; the mobile phase contained 80% buffer solution (1% triethylamine acetate, pH 5.5) and 20% methanol with 0.3 ml/min mobile phase flow rate at a temperature of 20 degrees C. At the optimal conditions, resolution of three stereoisomers of nadolol was obtained with a complete separation of the most active enantiomer, (RSR)-nadolol. Thermodynamic properties including enthalpy and entropy change of binding to the CSP for the enantiomeric separation were also determined.     

Reversed-phase chiral liquid chromatography on polysaccharide-based stationary phase coupled with tandem mass spectrometry for simultaneous determination of four stereoisomers of MK-0974 in human plasma

MK-0974 (1a), N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifuoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo-[4,5-B] pyridine-1-yl)piperidine-1-carboxamide, is a novel calcitonin gene-related peptide (CGRP) receptor antagonist with two chiral centers. Direct separation of its four stereoisomers (1a-d) was achieved using a cellulose chiral stationary phase, a Chiralcel OJ-RH column (150 mm x 4.6 mm), under reversed-phase condition, following the extraction of 0.2 mL plasma on Oasis muElution HLB 96-well solid-phase-extraction (SPE) plate. The tandem mass spectrometric detection was conducted in the positive-ion mode with a turbo-ion-spray (TIS) interface using multiple-reaction-monitoring on a Sciex API3000. Addition of ammonium trifluoroacetate to low-organic mobile phase improved detection sensitivity by more than 30-fold. The simultaneous quantification of the four stereoisomers in human plasma was validated over the ranges of 0.5-5000 nM for 1a and 0.5-500 nM for its three isomers (1b-d). Intraday validation, conducted with five lots of human control plasma, resulted in <12.4% (% coefficient of variation, CV) precision and 96.3-105.4% accuracy for all four stereoisomers. Further evaluation indicated that the assay was specific, the samples were stable after three freeze/thaw cycles, the recovery was reasonable (above 65%) and no matrix effect was observed for all four isomers. Investigation on the chiral integrity of 1a indicated that the diastereomer 1c, inversion at azepinone-3 carbon, was the only isomer observed in the post-dose clinical samples and accounted for 2.4-5.2% of MK-0974 exposure in the circulatory system. The possibility of inversion during blood collection, plasma storage and sample preparation was ruled out, while inversion was observed in the clinical formulation accounting for approximately 0.12% of 1a in a 100-mg capsule.