Stereoselective high-performance liquid chromatographic determination of ketoprofen, ibuprofen and fenoprofen in plasma using a chiral alpha 1-acid glycoprotein column

The effect of mobile phase composition, pH and temperature on the chiral resolution and retention of some 2-arylpropionic acids using the chiral alpha 1-acid glycoprotein column EnantioPac is described. Furthermore, a direct stereoselective high-performance liquid chromatographic assay to determine the enantiomers of ketoprofen, ibuprofen and fenoprofen in plasma is presented. Detection was at 260, 220 and 220 nm for ketoprofen, ibuprofen and fenoprofen, respectively. The limit of detection was 0.1 micrograms/ml for the enantiomers of ketoprofen and ibuprofen, and 0.25 micrograms/ml for the enantiomers of fenoprofen. The method was demonstrated to be applicable for stereoselective pharmacokinetic studies of ketoprofen, ibuprofen and fenoprofen after administration under clinical conditions.     

Direct enantiomeric resolution of disopyramide and its metabolite using chiral high-performance liquid chromatography. Application to stereoselective metabolism and pharmacokinetics of racemic disopyramide in man

A method for the simultaneous determination of disopyramide and mono-N-desisopropyldisopyramide enantiomers extracted from human plasma and urine is presented. Separation and quantitation were carried out using two columns coupled in series, and UV detection at 254 nm. First, the racemates of the two compounds were separated using a reversed-phase column, and then the enantiomers were separated using a stereoselective column packed with human alpha 1-acid glycoprotein. The mobile phase was 8 mM phosphate buffer, pH 6.20-2-propanol (92:8, v/v). The coefficients of variation (%) for the plasma daily determination were 6.7% for R(-)- and S(+)-disopyramide at drug levels of 1.5 micrograms/ml, and 8.5% and 7.7% for R(-)- and S(+)-mono-N-desisopropyldisopyramide, respectively, at drug levels of 0.375 micrograms/ml. The method has allowed the study of stereoselective metabolism and pharmacokinetics of disopyramide after oral administration as a racemate.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C18 column (3 microns) with a mobile phase of acetonitrile-sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (lambda ex = 227 nm, lambda em = 310 nm) was used. When 100 microliters of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.     

Direct injection analysis of ketoprofen enantiomers in plasma using column-switching high-performance liquid chromatography system

A high-performance liquid chromatography system was developed for the stereoselective determination of ketoprofen enantiomers in human plasma following direct sample injection. The system comprised of a pretreatment column and a chiral separation column connected in a series via a switching valve. When a 200 microliter portion of human plasma containing a therapeutic level of ketoprofen was directly applied to the system, ketoprofen was adsorbed in the pretreatment column, while plasma proteins were excluded. After the elution of proteins from the pretreatment column, the valve was switched and ketoprofen was desorbed and transferred to the chiral separation column where the enantiomers were separated and determined by ultraviolet-absorption. The mobile phase conditions for the pretreatment and chiral separation were optimized, which enabled rapid and complete recovery followed by satisfactory separation of the enantiomers. The calibration line for each enantiomer showed good linearity in the range of 0.25-5 micrograms/ml with a detection limit of 0.02 micrograms/ml (signal to noise ratio (S/N) greater than 3), which was sufficient for practical demands. The precision test indicated that the coefficient of variation for five repeated determinations of (-) ketoprofen was 5.4% at 0.1 microgram/ml and 1.4% at 1 microgram/ml.     

Stereoselective analysis of the enantiomers of mexiletine by high-performance liquid chromatography using fluorescence detection and study of their stereoselective disposition in man

A sensitive, stereoselective high-performance liquid chromatographic assay was developed for the resolution of the enantiomers of mexiletine as their 2-naphthoyl derivatives on a Pirkle type 1A chiral phase column. Detection of the derivatives was accomplished with a fluorescent detector. Maximum recovery of the enantiomers from plasma was 83% and was observed when plasma proteins were precipitated with a mixture of barium hydroxide-zinc sulphate. The calibration curve in plasma was linear over the concentration range 5-750 ng/ml for each enantiomer (r2 = 0.999) and in urine the linear range was 0.25-7.5 micrograms/ml (r2 = 0.999) for each enantiomer. The minimum detectable quantity of each enantiomer in plasma was 5 ng/ml at a signal-to-noise ratio of 5:1, representing 100 pg injected. A preliminary pharmacokinetic study was undertaken in one healthy male volunteer following an oral dose of 300 mg of racemic mexiletine hydrochloride. The apparent elimination half-lives determined from the plasma data were 12.1 and 14.1 h for the R(-) and S(+) enantiomers, respectively. The cumulative urinary excretion amounts of R(-)- and S(+)-mexiletine were found to be 8.01 and 10.46 mg, respectively. The plasma data indicated that a cross-over of the enantiomer ratios occurred at approximately 8 h. The urinary excretion of the enantiomers was consistent with the pattern found in plasma.     

Stereospecific high-performance liquid chromatographic assay of pirprofen enantiomers in rat plasma and urine.

A stereospecific high-performance liquid chromatographic method was developed for the assay of pirprofen enantiomers in rat plasma and urine. Following addition of internal standard (ketoprofen) and acidifier (L-ascorbic acid) to biological fluids, pirprofen was extracted into an isopropanol-isooctane (5:95) mixture. Diastereomers of pirprofen enantiomers, which were formed using L-leucinamide, were separated on a reversed-phase column with ultraviolet detection at 275 nm using 0.06 M KH2PO4-acetonitrile-triethylamine (64:36:0.1) as mobile phase. The limit of quantitation was 0.1 microgram/ml for each enantiomer, based on 100 microliters of rat plasma. No spontaneous oxidation of pirprofen to its pyrrole metabolite occurred during sample preparation and analysis. In three female rats which were dosed with 10 mg/kg racemic pirprofen orally, plasma concentrations of the enantiomers could be followed for 24 h. Pirprofen enantiomers in plasma were virtually unconjugated, and negligible concentrations of pyrrole metabolites were observed. Less than 10% of the total dose was recovered in urine as intact drug and its glucuroconjugates. The assay was found suitable for the study of the pharmacokinetics of pirprofen enantiomers in the rat.     

A stereospecific HPLC method and its application in determination of pharmacokinetics profile of two enantiomers of naringenin in rats

In this present study, a stereospecific HPLC method that could separate two enantiomers (R/S epimer) of naringenin in bottom base has been developed and validated, and then further applied it to determine stereoselective pharmacokinetics of naringenin in rats. In this method, a normal phase column (Chiralpak AD-H) was used and mobile phase consisting of n-hexane, isopropanol, and trifluoroacetic acid with a gradient elution program. The ultraviolet detection wavelength was at 288 nm and injection volume was 20 μL. The column temperature was at 30 °C, and flow rate was 0.8 mL/min. The validation of the method showed that the calibration curves in plasma were linear ranging from 0.05 to 20 μg/mL for each enantiomer with correlation coefficients of 0.9993 and 0.9997, respectively. The inter-day assay and intra-day assay accuracies (%) for both enantiomers were between 100.89-108.33%, while the inter- and intra-day assay precisions (%RSD) were between 1.99-7.71%. Extraction recovery, elution, and stability of both enantiomers in plasma were evaluated too, and the results showed that the method was reliable. The method was applied to determine the pharmacokinetic profile of two enantiomers of naringenin in rats after intravenous and oral administration. Naringenin has a bioactive effect as antioxidant, anti-inflammatory and immune system modulator and study on its pharmacological potency is becoming popular. The variety of naringenin's bioactivity might be due to its feature of chiral structure according to some reports, the availability of a stereospecific analytical method will be of great help to interpret the findings in different areas.     

Enantioselective LC‐ESI‐MS/MS determination of dropropizine enantiomers in rat plasma and application to a pharmacokinetic study

We developed and validated a simple, sensitive, selective and reliable LC–ESI‐MS/MS method for direct quantitation of dropropizine enantiomers namely levodropropizine (LDP) and dextrodropropizine (DDP) in rat plasma without the need for derivatization as per regulatory guidelines. Dropropizine enantiomers and carbamazepine (internal standard) were extracted from 50 μL rat plasma using ethyl acetate. LDP and DDP resolved with good baseline separation (R_s = 4.45) on a Chiralpak IG‐3 column. The mobile phase consisted of methanol with 0.05% diethylamine pumped at a flow rate of 0.5 mL/min. Detection and quantitation were done in multiple reaction monitoring mode following the transitions m/z 237 → 160 and 237 → 194 for dropropizine enantiomers and the internal standard, respectively, in the positive ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 3.23–2022 ng/mL for each enantiomer. The intra‐ and inter‐day precisions were in the ranges of 3.38–13.6 and 5.11–13.8 for LDP and 4.19–11.8 and 8.89–10.1 for DDP. Both LDP and DDP were found to be stable under different stability conditions. The method was successfully used in a stereoselective pharmacokinetic study of dropropizine enantiomers in rats following oral administration of racemate dropropizine at 100 mg/kg. The pharmacokinetic results indicate that the disposition of dropropizine enantiomers is not stereoselective and chiral inversion does not occur in rats.     

Analysis of metoprolol enantiomers in human serum by liquid chromatography on a cellulose-based chiral stationary phase

Metoprolol is a lipophilic, cardioselective beta-adrenergic blocking agent commercially available as a racemic compound. A normal phase high-performance liquid chromatographic method was developed to directly determine individual enantiomeric concentrations of metoprolol in human serum. Separation of the enantiomers was accomplished by a cellulose-tris(3,5-dimethylphenylcarbamate) chiral stationary phase. Metoprolol enantiomers were detected by means of fluorescence with excitation and emission wavelengths of 275 and 315 nm, respectively. Standard curves were linear over the concentration range 12.5-400 ng/ml for each enantiomer. Within-day coefficient of variation was less than 15% at all concentrations and the between-day coefficient of variation ranged from 4.1 to 11.2%. The limit of detection was determined to be 5 ng/ml for each enantiomer and the stereoselective resolution (alpha) of R- and S-metoprolol was 3.08. The assay was employed to determine enantiomeric serum concentrations of metoprolol in healthy male volunteers.     

Enantioselective LC–ESI–MS/MS method for quantitation of (−)-lumefantrine and (+)-lumefantrine in mice plasma and application to a pharmacokinetic study

We developed and validated a simple, sensitive, selective, and reliable LC–MS/MS–ESI method for the direct quantitation of lumefantrine (LFN) enantiomers [(−)-LFN and (+)-LFN] in mice plasma as per regulatory guideline. LFN enantiomers and carbamazepine (internal standard) were extracted from mice plasma using Strata X SPE (solid-phase extraction) cartridges. Good resolution between enantiomers was achieved on a Chiralpak IA-3 column using an isocratic mobile phase (0.1% of diethyl amine in methanol), which was delivered at a flow rate of 0.8 mL/min. Detection and quantitation were performed using multiple reaction monitoring mode following the transitions m/z 530.27 → 512.30 and 237.00 → 194.00 for LFN enantiomers and the internal standard, respectively, in the positive-ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 2.39–895 ng/mL for each enantiomer. The intra- and inter-day precisions were in the range of 1.03–6.14 and 6.36–8.70 and 2.03–4.88 and 5.82–11.5 for (−)-LFN and (+)-LFN, respectively. Both (−)-LFN and (+)-LFN were found to be stable under different stability conditions. The method was successfully used to delineate stereoselective pharmacokinetics of LFN enantiomers in mice after an oral administration of rac-LFN (20 mg/kg). The pharmacokinetic results indicated that the disposition of LFN enantiomers was stereoselective in mice.     

Determination of the R-(-) and S-(+) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma by enantioselective high-performance liquid chromatography.

An enantioselective liquid chromatographic assay for the simultaneous determination of the S-(+) and R-(-) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma is described. The metabolite is the active principle. The method is based on the extraction of plasma with diethyl ether-dichloromethane (2:1, v/v), separation of the organic phase, evaporation of the solvent and dissolution of the residue in the mobile phase. The two enantiomers were resolved on a Chiralcel OD (250 mm x 4.6 mm I.D.) high-performance liquid chromatographic column. The separation was achieved by isocratic elution with n-hexane-2-propanol (77:23, v/v). The flow-rate of the mobile phase was 1.0 ml/min and the two enantiomers were detected by ultraviolet absorbance at 210 nm. The analytical method is suitable for the quantitative and simultaneous determination of the two enantiomers in plasma at concentrations down to 0.4 mumol/l after administration of oxcarbazepine.     

Development and validation of a stereoselective HPLC method for the determination of the in vitro transport of nateglinide enantiomers in rat intestine

A simple stereoselective high performance liquid chromatographic method was developed for the determination of the in vitro transport of the enantiomers of nateglinide (N-(trans-4-isopropylcyclohexyl-carbonyl)-phenylalanine) in the rat intestine using a Chiralcel OJ-RH column (150 x 4.0 mm, 5 microm). The effects of the mobile phase composition, pH, the flow rate, and the temperature on the chromatographic separation were investigated. The enantioseparation was achieved at 33 degrees C using a mobile phase containing 100 mM potassium dihydrogen phosphate, pH 2.5, and ACN (32:68 v/v) delivered at a flow rate of 1 mL/min. The analytes were monitored at 210 nm and linearity (r >0.99) was obtained for a concentration range of 0.5-50 microg/mL. The LOD and LOQ were 0.2 and 0.5 microg/mL for the R-enantiomer and 0.2 and 0.8 microg/mL for the S-enantiomer, respectively. Both, the intra- and interday accuracy and precision of the calibration curves were determined. The method was successfully applied to estimate the in vitro passage of the enantiomers and the racemate of nateglinide in duodenum, jejunum, and ileum of rats. Generally, higher concentrations of nateglinide and the S-enantiomer were observed when the racemate was administered compared to administration of the individual enantiomers of nateglinide.     

Liquid chromatographic analysis of propafenone enantiomers in human plasma

A convenient and sensitive high-performance liquid chromatographic method for analysis of the enantiomers of propafenone (PPF) in human plasma was developed. Racemic propafenone and (-)-ephedrine (internal standard) were first extracted from plasma samples into a mixture of hexane-2-propanol-heptafluorobutanol (95:5:1.25, v/v). After evaporation of the organic layer, the samples were derivatized with R(-)-naphthylethyl isocyanate. The derivatization reached its maximum within 30 s at room temperature with an efficiency of 93.9 +/- 2.8% (mean +/- S.D.). The formed diastereomers were subsequently separated on a silica column with a mobile phase of hexane-2-propanol-isobutanol (96:2:2, v/v) at a flow-rate of 1.5 ml/min. The ultraviolet detection wavelength was set at 220 nm. Using 1 ml plasma, the detection limit was 6.25 ng/ml for the propafenone enantiomers. The assay was successfully employed to measure propafenone enantiomers in plasma samples of a healthy subject after oral administration of a single 150-mg dose of the racemate.     

Determination of zopiclone enantiomers in plasma by liquid chromatography using a chiral cellulose carbamate column.

The enantiomers of zopiclone were determined in human plasma using a sequential achiral-chiral liquid chromatographic method. Zopiclone was separated from the biological matrix and quantified on an achiral silica column. The limit of detection was 5 ng/ml. The eluent fraction containing zopiclone was collected, evaporated, reconstituted with the mobile phase and injected onto a chiral cellulose carbamate column where the enantiomeric ratio was calculated. This validated method, applied to a pilot study, suggests that pharmacokinetics of zopiclone is stereoselective.     

Direct chiral resolution of cloquintocet-mexyl and its application to in vitro degradation combined with clodinafop-propargyl

A simple chiral high‐performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed and validated for measuring Cloquintocet‐mexyl (ClM) enantiomers and clodinafop‐propargyl (CP) using cellulose tris‐(3,5‐dimethylphenylcarbamate) (CDMPC) as chiral stationary phase (CSP). The effects of mobile phase composition and column temperature on the ClM enantiomer separation were investigated. Good separation was achieved by using a mixture of n‐hexane and n‐propanol as mobile phase. Based on the chiral HPLC method, enantioselective quantitative determination analysis methods for this herbicide combined with CP in diluted plasma were developed and validated. The assay method was linear over a range of concentrations (0.5–100 µg/mL) in diluted plasma and the mean recovery was greater than 80% for both enantiomers and CP. The limits of quantification and detection for both ClM enantiomers and CP were 0.5 and 0.2 µg/mL, respectively. Intra‐ and interday relative standard deviations did not exceed 10% for three tested concentrations. The result suggested that the degradation of ClM enantiomers was stereoselective in rabbit plasma, and both rac‐ClM and CP degraded quickly in plasma, showing that the main existing forms with biological effect in animals are their metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

Stereoselective determination of R(-)- and S(+)-MK-571, a leukotriene D4 antagonist, in human plasma by chiral high-performance liquid chromatography.

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(-)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D4 antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine-acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine-pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r2 greater than 0.999) for concentrations of enantiomers of 1 from 0.05 micrograms/ml, the lowest quantitation limit, up to 2.5 micrograms/ml. Intra-day coefficients of variation at 0.05 microgram/ml were 2.4% for the R(-)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(-)- and S(+)-1 were 2.6 and 3.6%, respectively. The utility of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.     

Determination of the enantiomers of a novel 20,21-dinoreburnamenine derivative in rat plasma and brain by high-performance liquid chromatography using a chiral stationary phase.

A high-performance liquid chromatographic method with solid-phase extraction was developed for the assay of the enantiomers of a novel 20,21-dinoreburnamenine derivative (RU 49041) in rat plasma and brain using a chiral stationary phase (Nucleosil Chiral 2) and ultraviolet detection. The limit of detection was 10 ng/ml (or ng/g) in both tissues and the intra-assay precision was satisfactory (plasma, ca. 5%; brain, ca. 1%). The pharmacokinetic profiles of the two enantiomers were determined following oral administration of the racemate (10 mg/kg). The results show that their pharmacokinetics are very different: whereas both enantiomers appear in the brain, only the 3 alpha,16 beta-enantiomer is detected in plasma.     

Stereoselective high-performance liquid chromatographic determination of the enantiomers of ketamine and norketamine in plasma.

An enantioselective high-performance liquid chromatographic assay for the quantitation of the enantiomers of ketamine and its major metabolite norketamine in human plasma is described (assay I). The procedure involved extraction of the compounds from alkalized plasma into cyclohexane. Stereoselective separation was achieved with a prepacked alpha 1-acid glycoprotein column without any derivatization procedure. A second assay using a conventional reversed-phase column to determine total (racemic) ketamine and norketamine is also described. Because of interfering plasma peaks (assay II) the cyclohexane solution was reextracted into 1 M hydrochloric acid. The detection wavelength was 215 nm for all substances. The limit of quantification of the method was ca. 40 ng/ml in plasma. The assays were sensitive and reproducible. The method was demonstrated to be sensitive for stereoselective pharmacokinetic studies of ketamine after clinical doses.     

Liquid-phase microextraction combined with capillary electrophoresis,a promising tool for the determination of chiral drugs in biological matrices

A disposable device for liquid-phase microextraction (LPME) based on porous polypropylene hollow fibres has recently been introduced. In the present paper, LPME was combined with capillary electrophoresis (CE) and the combination was for the first time evaluated for chiral determination of drugs in biological matrices. The chiral antidepressant drug mianserin was selected as model compound. The mianserin enantiomers were extracted from 0.5 ml of plasma added internal standard and made alkaline with 0.25 ml of 2 M NaOH. The unionised analytes were extracted into di-n-hexyl ether impregnated in the pores of the hollow fibre, and into an acidic solution inside the hollow fibre. This resulted in a three-phase system where the extracts were aqueous, and hence directly compatible with the CE system. Efficient sample clean-up was seen and the extraction recovery was 80% for both enantiomers. Discrimination between the enantiomers in the extraction system was not observed. The limit of quantitation (S/N= 10; 12.5 ng/ml for both enantiomers) and the limit of detection (S/N=3; 4 ng/ml for both enantiomers) were below the therapeutic range for mianserin. The method was validated and successfully applied to determine R- and S-mianserin in plasma samples from seven patients treated with mianserin, indicating that LPME-CE is a promising combination for analysis of racemic drugs present in low concentrations in biological matrices.     

Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography.

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane-isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound alpha 1-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.