Enantioselective analyses of chloroquine and hydroxychloroquine in rat liver microsomes through chiral liquid chromatography–tandem mass spectrometry

An efficient, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) chiral analysis method was established for determination of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes. Effects of polysaccharide chiral stationary phases and basic additives on chiral separations of two analytes were discussed in detail. Amylose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase showed the best separation performance for them with acetonitrile-diethylamine-ethanol-diethylamine mixture (90:0.1:10:0.1, v/v/v/v) among four chiral stationary phases. Then, multiple reaction monitoring mode was selected as the data acquisition for determination of two pairs of enantiomers. The proposed LC–MS/MS chiral analysis method was validated in terms of linearity, accuracy, precision, and specificity. Good linearity with correlation coefficient over 0.998 was obtained in the concentration range of 0.05–5 μM. Limits of quantification for chloroquine and hydroxychloroquine enantiomers were 5.0 and 1.0 nM, respectively. The recoveries ranged from 81.14% to 111.09%. The intra-day and inter-day relative standard deviation were less than 6.5%. Moreover, concentrations of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes were determined through the proposed LC–MS/MS analysis method. After incubated with rat liver microsomes for 10 min, the enantiomeric factor of hydroxychloroquine decreased from 0.50 to 0.45 (p < 0.001). In brief, our developed determination method for chloroquine and hydroxychloroquine enantiomers through LC–MS/MS spectrometry showed the characteristics of high-efficiency, fast speed, and very low detection limit, and would be greatly beneficial for screening and quantitation of them in biological matrices.     

Stereoselective determination of benalaxyl in plasma by chiral high-performance liquid chromatography with diode array detector and application to pharmacokinetic study in rabbits

A chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of benalaxyl (BX) in rabbit plasma. Good separation was achieved at 20 degrees C using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase, a mixture of n-hexane and 2-propanol (97:3) as mobile phase at a flow rate of 1.0 ml/min. The assay method was linear over a range of concentrations (0.25-25 microg/ml) in plasma and the mean recovery was greater than 90% for both enantiomers. The limits of quantification and detection for both enantiomers in plasma were 0.25 and 0.1 microg/ml, respectively. Intra- and interday relative standard deviations (RSDs) did not exceed 10% for three-tested concentrations. The method was successfully applied to pharmacokinetic studies of BX enantiomers in rabbits. The result suggested that the pharmacokinetics of BX enantiomers was stereoselective in rabbits.     

Enantioselective determination of alprenolol in human plasma by liquid chromatography with tandem mass spectrometry using cellobiohydrolase chiral stationary phases

A fast, sensitive, and enantioselective LC-MS/MS bioanalytical method was developed and validated for the direct determination of individual alprenolol enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP) along with supported liquid extraction (SLE) procedures. Complete baseline separation of enantiomeric alprenolol was achieved within 2 min in reversed phase chromatography at 0.9 ml/min. SLE in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.500-500 ng/ml for each alprenolol enantiomer using 0.0500 ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed < or = 7.3% relative standard deviation (RSD) and -6.2 to 8.0% relative error (RE) for both alprenolol enantiomers.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

Cyclodextrins and enantiomeric separations of drugs by liquid chromatography and capillary electrophoresis: basic principles and new developments

Investigation of individual drug enantiomers is required in pharmacokinetic and pharmacodynamic studies of drugs with a chiral centre. Cyclodextrins (CDs) are extensively used in high-performance liquid chromatography as stationary phases bonded to a solid support or as mobile phase additives in HPLC and capillary electrophoresis (CE) for the separation of chiral compounds. We describe here the basis for the liquid chromatographic and capillary electrophoretic resolution of drug enantiomers and the factors affecting their enantiomeric separation. This review covers the use of CDs and some of their derivatives in studies of compounds of pharmacological interest.     

The direct determination of the enantiomers of ketorolac and parahydroxyketorolac in plasma and urine using enantioselective liquid chromatography on a human serum albumin-based chiral stationary phase

An enantioselective assay has been developed for the determination of the enantiomers of ketorolac and its metabolite p-hydroxyketorolac in plasma and urine. The analytical method utilizes a coupled achiral–chiral HPLC system where the initial separation of ketorolac from p-hydroxyketorolac and matrix interferences was achieved on a C₁₈-stationary phase and the enantioselective separations of the two target solutes were accomplished on a human serum albumin-based chiral stationary phase. The two columns were attached in sequence and the assay was carried out without the necessity of column-switching techniques. The method has been validated for use in pharmacokinetic and metabolic studies and represents the initial report of the determination of ketorolac and p-hydroxyketorolac enantiomers in urine. The results of the study indicate that after the administration of racemic ketorolac there was an enantioselective distribution of ketorolac enantiomers in plasma [(R)-ketorolac: (S)-ketorolac = 3.89 ± 0.93 (n = 6) and urine (R)-ketorolac: (S)-ketorolac = 1.26 ± 0.09 (n = 7)]. The mean ratio of the p-hydroxyketorolac enantiomers was 1.77 ± 0.46 (n = 7). Both ketorolac and p-hydroxyketorolac are glucuronized in the acyl carboxyl moiety and the results of this study indicate that this process is not enantiospecific. © 1994 Wiley-Liss, Inc.     

Stereoselective pharmacokinetics of flobufen in rats

Stereoselectivity of the pharmacokinetics of the nonsteroidal anti-inflammatory drug flobufen, 4-(2', 4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in male Wistar rats after intravenous administration. Pharmacokinetic parameters and chiral inversion of flobufen enantiomers were studied after a bolus injection of the racemate and individual enantiomers (5 mg/kg). Determinations of the enantiomers in rat plasma were performed using chiral HPLC (terguride column). After i.v. administration of flobufen racemate, plasma levels of R-enantiomer decreased more rapidly. The S-/R-enantiomer ratio of AUCs after rac-flobufen was 13.3. The total plasma clearance value of S-flobufen was more than 10-fold lower than R-flobufen. The other pharmacokinetic parameters of the enantiomers were also significantly different. While only traces of R-enantiomer (less than 1%) were detected in rat plasma after S-flobufen administration, considerable conversion to the S-enantiomer was found after injection of R-flobufen (R-enantiomer AUC/S-enantiomer AUC = 0.52). The results indicate substantial stereoselectivity in the disposition of flobufen enantiomers in the rat, which is, at least in part, attributed to chiral bioconversion.     

Stereoselective HPLC analysis of tertatolol in rat plasma using macrocyclic antibiotic chiral stationary phase

Enantiomeric resolution of teratolol was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase known as Chirobiotic V with UV detection set at 220 nm. The polar ionic mobile phase (PIM) consisted of methanol-glacial acetic acid-triethylamine (100:0.01:0.015, v/v/v) has been used at a flow rate of 0.8 ml min(-1) . The calibration curves in plasma were linear over the range of 5-500 ng ml(-1) for each enantiomer with detection limit of 2 ng ml(-1) . The proposed method was validated in compliance with the international conference on harmonization (ICH) guidelines. The developed method applied for the trace analyses of tertatolol enantiomers in plasma and for the pharmacokinetic study of tertatolol enantiomers in rat plasma. The assay proved to be suitable for therapeutic drug monitoring and chiral quality control for tertatolol formulations by HPLC.     

Bioanalytical chiral chromatographic technique and docking studies for enantioselective separation of meclizine hydrochloride: Application to pharmacokinetic study in rabbits

Enantiomeric resolution and molecular docking studies of meclizine hydrochloride on polysaccharide-based chiral stationary phase comprising cellulose tris(4-methylbenzoate) chiral selector (150 × 4.6 mm, 3.0 μm) were presented. The mobile phase used was acetonitrile:10mM ammonium bicarbonate (95:05, v/v). The developed technique was used to perform the enantioselective assay of meclizine hydrochloride in its marketed formulation. The elution order of meclizine hydrochloride enantiomers was determined by docking studies. Target compound was extracted from rabbit plasma using protein precipitation technique, followed by development of bioanalytical chiral separation method using the same matrix. Application of the method to determine pharmacokinetic parameters of meclizine hydrochloride enantiomers was performed using Phoenix WinNonlin 8.1 software. The results demonstrated stereoselective disposition of meclizine hydrochloride enantiomers in rabbits.     

Enantioselective liquid chromatographic-electrospray mass spectrometric assay of beta-adrenergic blockers: application to a pharmacokinetic study of sotalol in human plasma

An enantioselective high performance liquid chromatographic-electrospray ionization mass spectrometric (HPLC-ESI-MS) method for the direct determination of several beta-adrenergic blockers was developed and validated. The method is based on the direct separation of the enantiomers of drugs on a laboratory-made chiral stationary phase (CSP) containing covalently bonded teicoplanin (TE) as chiral selector. Detection of the effluent was performed by electrospray ionization mass spectrometry, run in the selected-ion recording (SIR) mode. The method was applied to the pharmacokinetic monitoring of sotalol (STL) in the plasma of five young healthy volunteers, dosed with racemic drug. The limits of quantitation (LOQ) reached 4 ng/ml for both sotalol enantiomers. Such a method, fully validated, offers a novel, fast and very efficient tool for the direct determination of sotalol enantiomers in human plasma, and can be generally applied to the beta-adrenergic blockers stereoselective pharmacokinetics.     

Enantioselective LC/MS method for the determination of an antimalarial agent Fenozan B07 in dog plasma

A chiral liquid chromatography/mass spectrometry (LC/MS) bioanalytical procedure has been developed for the analysis of the antimalaric agent Fenozan B07 in dog plasma. Normal-phase chromatography involving a phenylcarbamate derivative of cellulose coated on silica gel as the chiral stationary phase was used to resolve (-)-(S,S)-B07 from (+)-(R,R)-B07. The enantiomers were detected by a mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operated in the negative ion mode. A mass spectrum, characterized by a base peak of m/z 285, was obtained for each enantiomer. The m/z 285 ion was very specific for the analysis of both enantiomers in the plasma. The selected ion monitoring analysis of the plasma samples was therefore performed at m/z 285 for quantitative purposes. The enantiomers were extracted from the plasma in a basic medium and purified by solid-phase extraction using a hydrophilic-lipophilic balanced sorbent. A lower limit of quantification of 2 ng/mL in plasma was achieved for both enantiomers. The quantitative procedure reported in this study was highly specific and sensitive, and was validated according to the FDA guidance on bioanalytical method validation.     

Enantioselective high performance liquid chromatographic assay of acebutolol and its active metabolite diacetolol in human serum

A stereoselective direct liquid chromatographic method for assay of the enantiomers of the beta-adrenergic blocker acebutolol (AC) and its active metabolite, diacetolol (DC), in human serum was developed. The assay is based on extraction with ethyl acetate and separation of enantiomers on an amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase (Chiralpak AD) column. The method was validated and proved useful for the determination of the enantiomers in serum samples of patients suffering from hypertension and chronically treated with racemic AC. The results were compared and found similar with an indirect assay based on derivatization of the enantiomers with (+)-(S)-1-(1-naphthyl)ethyl isocyanate (NEIC).     

Determination of MK-0767 enantiomers in human plasma by normal phase LC-MS/MS

A sensitive and selective analytical method for the enantioselective determination of MK-0767, a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist, in human plasma has been developed and validated. The chromatography is based on normal-phase chiral separation on a Kromasil, 5 microm, CHI-DMB 250 mm x 4.6 mm column. The detection involves the direct introduction of the normal phase eluent into MS/MS without the addition of a post-column reagent. Atmospheric pressure chemical ionization (APcI) mode was selected as the ion source in this method. With proper sample handling and processing procedures, ex vivo interconversion of the enantiomers was kept to minimum during sample collection, preparation and short term storage of frozen human plasma samples. The method was successfully utilized to determine the concentrations of MK-0767 enantiomers in human plasma to support pharmacokinetic investigation in man.     

Simultaneous determination of sulforaphane and its major metabolites from biological matrices with liquid chromatography-tandem mass spectroscopy

A simple, sensitive and specific LC-MS/MS method for the simultaneous determination of sulforaphane (SFN) and its major metabolites, the glutathione (SFN-GSH) and N-acetyl cysteine conjugates (SFN-NAC) from biological matrices was developed and validated. The assay procedure involved solid-phase extratcion of all three analytes from rat intestinal perfusate using C2 extraction cartridges, whereas from rat plasma, metabolites were extracted by solid-phase extraction and SFN was extracted by liquid-liquid extraction with ethyl acetate. Chromatographic separation of SFN, SFN-GSH and SFN-NAC was achieved on a C8 reverse phase column with a mobile phase gradient (Mobile Phase A: 10mM ammonium acetate buffer, pH: 4.5 and Mobile Phase B: acetonitrile with 0.1% formic acid) at a flow rate of 0.3 mL/min. The Finnigan LCQ LC-MS/MS was operated under the selective reaction monitoring mode using the electrospray ionization technique in positive mode. The nominal retention times for SFN-GSH, SFN-NAC and SFN were 8.4, 11.0, and 28.2 min,, respectively. The method was linear for SFN and its metabolites with correlation coefficients >0.998 for all analytes. The limit of quantification was 0.01-0.1 microm depending on analyte and matrix, whereas the mean recoveries from spiked plasma and perfusate samples were approximately 90%. The method was further validated according to U.S. Food and Drug Administration guidance in terms of accuracy and precision. Stability of compounds was established in a battery of stability studies, i.e., bench top, auto-sampler and long-term storage stability as well as freeze/thaw cycles. The utility of the assay was confirmed by the analysis of intestinal perfusate and plasma samples from single-pass intestinal perfusion studies with mesenteric vein cannulation in rats.     

Simultaneous quantification of the enantiomers of verapamil and its N-demethylated metabolite in human plasma using liquid chromatography-tandem mass spectrometry

A stereoselective bioanalytical method for the simultaneous quantification of the enantiomers of verapamil and its active main metabolite norverapamil in human plasma has been developed and validated. The samples were analysed by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) in the Selected Reaction Monitoring (SRM) mode using a deuterated internal standard. The stationary phase used for the chiral separation was a Chiral-AGP. The enantiomers of verapamil were selectively detected from those of norverapamil by the mass spectrometer due to different molecular masses, although there was a chromatographic co-elution. Thus, time-consuming procedures like achiral preseparation or chemical derivatisation could be avoided. Higher detection sensitivity than earlier published methods based on fluorescence detection was obtained, although a mobile phase of high water-content and high flow-rate was introduced into the electrospray interface (85% aqueous ammonium acetate pH 7.4 +15% acetonitrile at 0.6 ml/min). The enantiomers of verapamil and norverapamil could be quantified at levels down to 50 pg and 60 pg/500 microl plasma sample, respectively, with R.S.D. in the range of 3.6-7.8%. The presented method was successfully applied to an in vivo intestinal absorption and bioavailability study in humans, using the Loc-I-Gut method.     

Enantiomeric separation of β‐blockers and tryptophan using heparin as stationary and pseudostationary phases in capillary electrophoresis

The separation methods of the enantiomers of two β‐blockers and tryptophan were studied using capillary electrochromatography with heparin covalently as well as non‐covalently, bonded onto the capillary inner wall as stationary phase and electrokinetic chromatography with heparin as pseudostationary phase. In the case of heparin, used as a stationary phase, the method was unable to resolve enantiomers in both cases β‐blockers and tryptophan. On the other hand, when heparin was used as a pseudostationary phase, the resolution of the enantiomers was obtained only with 3‐aminopropyltriethoxysilane which were immobilised onto the inner phase of the capillary. The results of this study let us infer that the electrostatic, hydrophobic, and steric interactions were involved in the separation mechanisms. The separation was achieved in less than 10 minutes under the optimized conditions: 30 mM phosphate buffer (pH 2.5) with the adding of 15 mg/mL of heparin at 15°C and 10 kV. The usefulness of heparin as a chiral selector both in electrokinetic chromatography using 3‐aminopropyltriethoxysilane attached to the capillary was demonstrated for the first time. The developed method was powerful, sensitive, and fast, and it could be considered an important alternative to conventional methods used for chiral separation.     

Enantiomeric separation of malathion and malaoxon and the chiral residue analysis in food and environmental matrix

Malathion is a widely used chiral phosphorus insecticide, which has a more toxic chiral metabolite malaoxon. In this work, the enantiomers of malathion and malaoxon were separated by high-performance liquid chromatography-mass/mass (HPLC-MS/MS) with chiral columns using acetonitrile/water or methanol/water as mobile phase, and the chromatographic conditions were optimized. Based on the chiral separation, the chiral residue analysis methods for the enantiomers in soil, fruit, and vegetables were set up. Two pairs of the enantiomers were better separated on CHIRALPAK IC chiral column, and baseline simultaneous separations of malathion and malaoxon enantiomers were achieved with acetonitrile/water (40/60, v/v) as mobile phase at a flow rate of 0.5 mL/min. The elution orders were −/+ for both malathion and malaoxon measured by an optical rotation detector. The chiral residue analysis in soil, fruit, and vegetables was validated by linearity, recovery, precision, limit of detection (LOD), and limit of quantification (LOQ). The LODs and LOQs for the enantiomers of malathion were 1 μg/kg and 3–5 μg/kg and 0.08 μg/kg and 0.20–0.25 μg/kg for malaoxon enantiomers. Good linear calibration curves for each enantiomer in the matrices were obtained within the concentration range of 0.02–12 mg/L. The mean recoveries of the enantiomers of malathion and malaoxon ranged from 82.26% to 109.04%, with RSDs of 0.71–8.63%.The results confirmed that this method was capable of simultaneously determining the residue of malathion and malaoxon in food and environmental matrix on an enantiomeric level.     

Reliable HPLC separation,vibrational circular dichroism spectra,and absolute configurations of isoborneol enantiomers

Resolution of chiral compounds has played an important role in the pharmaceutical field, involving detailed studies of pharmacokinetics, physiological, toxicological, and metabolic activities of enantiomers. Herein, a reliable method by high‐performance liquid chromatography (HPLC) coupled with an optical rotation detector was developed to separate isoborneol enantiomers. A cellulose tris(3, 5‐dimethylphenylcarbamate)‐coated chiral stationary phase showed the best separation performance for isoborneol enantiomers in the normal phase among four polysaccharide chiral packings. The effects of alcoholic modifiers and column temperature were studied in detail. Resolution of the isoborneol racemate displayed a downward trend along with an increase in the content of ethanol and column temperature, indicating that less ethanol in the mobile phase and lower temperature were favorable to this process. Moreover, two isoborneol enantiomers were obtained via a semipreparative chiral HPLC technique under optimum conditions, and further characterized by analytical HPLC, and experimental and calculated vibrational circular dichroism (VCD) spectroscopy, respectively. The solution VCD spectrum of the first‐eluted component was consistent with the Density Functional Theory (DFT) calculated pattern based on the SSS configuration, indicating that this enantiomer should be (1S , 2S , 4S )‐(+)‐isoborneol. Briefly, these results have provided reliable information to establish a method for analysis, preparative separation, and absolute configuration of chiral compounds without typical chromophoric groups.     

High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of D-(-) and L-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and DL-(+/-)-lactic acids in calf feces and DL-(+/-)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 microm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-L-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2-14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for D- and L-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for DL-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.     

Enantioselective determination of triazole fungice tetraconazole by chiral high-performance liquid chromatography and its application to pharmacokinetic study in cucumber, muskmelon, and soils

A simple chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of tetraconazole enantiomers in cucumber, muskmelon, and soils. Good separation was achieved at 20°C using cellulose tris-(4-methylbenzoate) as chiral stationary phase, a mixture of n-hexane and ethanol (90:10) as mobile phase at a flow rate of 0.8 ml/min. The assay method was linear over a range of concentrations (0.5-50 μg/ml) and the mean recoveries in all samples were more than 85% for the two enantiomers. The limits of detection for both enantiomers in plant and soil samples were 0.06 and 0.12 μg/g, respectively. Then, the proposed method was successfully applied to the study of enantioselective degradation of rac-tetraconazole in cucumber, muskmelon, and soils. The results showed that the degradation of two enantiomers of tetraconazole followed first-order kinetics and significantly stereoselective behavior was observed in cucumber, muskmelon, and Beijing soil. The preferential absorption and degradation of (-)-S-tetraconzole resulted in an enrichment of the (+)-R-tetraconazole residue in plant samples, whereas the (+)-R-tetraconazole showed a faster degradation in Beijing soil and the stereoselectivity might be caused by microorganisms. No stereoselective degradation was observed in Heilongjiang soil.