Enantioselective analysis of bambuterol in human plasma using microwave‐assisted chiral derivatization coupled with ultra high performance liquid chromatography and tandem mass spectrometry

In this study, an enantioselective analytical method based on microwave‐assisted chiral derivatization coupled with ultra high performance liquid chromatography and tandem mass spectrometry was developed for the determination of bambuterol enantiomers in human plasma. The chiral derivatization reaction was greatly accelerated by microwave irradiation. Under the optimized conditions, both the derivatization time and separation time on column was only 3 min, and the lower limit of quantification was 2.5 pg/mL. The recoveries were in the range of 90.1–93.0% without significant matrix effect. Compared with the conventional heating chiral derivatization, microwave‐assisted chiral derivatization obtained higher chiral derivatization yields with much shorter time due to the effect of microwave irradiation. Furthermore, the racemization during the derivatization reaction was systematically investigated. The results showed the concentration of acetic acid and the reaction time had significant effects on the racemization, which could be well controlled during microwave‐assisted chiral derivatization for the short reaction time. Finally, this novel approach was demonstrated by determining bambuterol in human plasma of a clinical pharmacokinetic study in eight healthy volunteers. On the basis of the results, microwave‐assisted chiral derivatization coupled with ultra high performance liquid chromatography and tandem mass spectrometry as a simple and effective enantioselective analysis technique for the determination of chiral drugs in complex biological samples showed great promise.     

Validation of an enantioselective LC–MS/MS method to quantify enantiomers of (±)‐OTX015 in mice plasma: Lack of in vivo inversion of (−)‐OTX015 to its antipode

A highly sensitive, specific and enantioselective assay has been validated for the quantitation of OTX015 enantiomers [(+)‐OTX015 and (−)‐OTX015] in mice plasma on LC–MS/MS‐electrospray ionization as per regulatory guidelines. Protein precipitation was used to extract (±)‐OTX015 enantiomers and internal standard (IS) from mice plasma. The active [(−)‐OTX015] and inactive [(+)‐OTX015] enantiomers were resolved on a Chiralpak‐IA column using an isocratic mobile phase (0.2% ammonia/acetonitrile 20 : 80, v /v) at a flow rate of 1.2 mL/min. The total run time was 6.0 min. (+)‐OTX015, (−)‐OTX015 and IS eluted at 3.34, 4.08 and 4.77 min, respectively. The MS/MS ion transitions monitored were m/z 492 → 383 for OTX015 and m/z 457 → 401 for IS. The standard curves for OTX015 enantiomers were linear (r ² > 0.998) in the concentration range 1.03–1030 ng/mL. The inter‐ and intraday precisions were in the range 2.20–13.3 and 8.03–12.1% and 3.80–14.4 and 8.97–13.6% for (+)‐OTX015 and (−)‐OTX015, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (−)‐OTX015 and unequivocally demonstrated that (−)‐OTX015 does not undergo chiral inversion to its antipode in vivo in mice.     

Stereoselective determination of dufulin in watermelon under field conditions using chiral ultra high performance liquid chromatography with high‐resolution mass spectrometry

An effective and sensitive chiral analytical method was established to investigate the stereoselective dissipation of rac‐dufulin in watermelon using ultra high performance liquid chromatography with a superchiral S‐OD chiral column (4.6 × 150 mm i.d., 5 μm) coupled with high‐resolution mass spectrometry. To optimize the pretreatment method for detecting rac‐dufulin in the three matrixes, different extraction solvents, extractant volumes, extraction times, and absorbents were investigated to improve extraction efficiency. Moreover, analysis of variance was used to perform method validation for determination of the two dufulin enantiomers in the three matrixes. Using the optimized method, good linearity was obtained (determination coefficient > 0.999). The limits of detection and quantification of the two dufulin enantiomers in soil, watermelon, and pulp were 0.15 and 0.5 μg/kg, respectively. The average recoveries of the two enantiomers in the three matrixes at four spiked levels ranged from 75.0 to 107.8%, with intra‐ and inter‐day relative standard deviations of 0.4–10.4%. In field trials, the R enantiomer was preferentially dissipated in watermelon. These method validation results confirmed that the developed method was convenient and reliable for the stereoselective determination of enantiomers of rac‐dufulin in watermelon.     

HPLC with polysaccharide chiral stationary phase in polar‐organic phase mode: Application to the asymmetric epoxidation of allylic alcohols

A simple and rapid HPLC method using a polysaccharide‐based chiral stationary phase (Chiralpak AD‐H) in polar‐organic phase mode has been developed for direct resolution of glycidyl nitrobenzoate (GNB) and 2‐methyl glycidyl nitrobenzoate (MGNB) enantiomers. ACN and methanol were used as mobile phase and the effects of the addition of ethanol and 2‐propanol as organic modifier in the mobile phase, flow rate and the column temperature were tested. The optimized conditions were: methanol/ethanol (80:20) at a flow rate of 0.9 mL/min and 40°C. Analysis time was ?13 min and the chiral resolution was ?2. The method was validated and resulted to be selective, precise and accurate. The method was found to be linear in 2–300 μg/mL range (R² >0.999) with an LOD nearly 0.5 μg/mL for four enantiomers. GNB and MGNB enantiomers were obtained by asymmetric epoxidation of allyl alcohol and 2‐methyl allyl alcohol, respectively, using chiral titanium–tartrate complexes as catalyst and dichloromethane as solvent after in situ derivatization of the intermediate glycidols derivatives. The quite simple and rapid validated method was applied successfully for direct determination of the enantiomeric excess (?90%) and yield obtained in real samples of asymmetric epoxidation of allylic alcohols without further purification, workup or solvent removal. The method provides a useful and value‐added tool for controlling the enantiomeric purity of the synthesized epoxides.     

Separation and determination of the enantiomers of lactic acid and 2‐hydroxyglutaric acid by chiral derivatization combined with gas chromatography and mass spectrometry

Lactic acid and 2‐hydroxyglutaric acid are chiral metabolites that have two distinct d‐ and l ‐enantiomers with distinct biochemical properties. Perturbations of a single enantiomeric form have been found to be closely related to certain diseases. Therefore, the ability to differentiate the d and l enantiomers is important for these disease studies. Herein, we describe a method for the separation and determination of lactic acid and 2‐hydroxyglutaric acid enantiomers by chiral derivatization (with l‐ menthol and acetyl chloride) combined with gas chromatography and mass spectrometry. The two pairs of above‐mentioned enantiomers exhibited linear calibration curves with a correlation coefficient (R²) exceeding 0.99. The measured data were accurate in the acceptable recovery range of 88.17–102.30% with inter‐ and intraday precisions (relative standard deviations) in the range of 4.23–17.26%. The limits of detection for d‐ lactic acid, l‐ lactic acid, d‐ 2‐hydroxyglutaric acid, and l‐ 2‐hydroxyglutaric acid were 0.13, 0.11, 1.12, and 1.16 μM, respectively. This method was successfully applied to analyze mouse plasma. The d‐ lactic acid levels in type 2 diabetes mellitus mouse plasma were observed to be significantly higher (P < 0.05, t‐test) than those of normal mice, suggesting that d‐ lactic acid may serve as an indicator for type 2 diabetes mellitus.     

Biosurfactant trehalose lipid‐enhanced ultrasound‐assisted micellar extraction and determination of the main antioxidant compounds from functional plant tea

Hydrosoluble trehalose lipid (a biosurfactant) was employed for the first time as a green extraction solution to extract the main antioxidant compounds (geniposidic acid, chlorogenic acid, caffeic acid, and rutin) from functional plant tea (Eucommia ulmoides leaves). Single‐factor tests and response surface methodology were employed to optimize the extraction conditions for ultrasound‐assisted micellar extraction combined with ultra‐high‐performance liquid chromatography in succession. A Box‐Behnken design (three‐level, three‐factorial) was used to determine the effects of extraction solvent concentration (1–5 mg/mL), extraction solvent volume (5–15 mL), and extraction time (20–40 min) at a uniform ultrasonic power and temperature. In consequence, the best analyte extraction yields could be attained when the trehalose lipid solution concentration was prepared at 3 mg/mL, the trehalose lipid solution volume was 10 mL and the extraction time was set to 35 min. In addition, the recoveries of the antioxidants from Eucommia ulmoides leaves analyzed by this analytical method ranged from 98.2 to 102%. These results indicated that biosurfactant‐enhanced ultrasound‐assisted micellar extraction coupled with a simple ultra‐high‐performance liquid chromatography method could be effectively applied in the extraction and analysis of antioxidants from Eucommia ulmoides leaf samples.     

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.     

Enantioseparation of N‐acetyl‐glutamine enantiomers by LC–MS/MS and its application to a plasma protein binding study

A novel chiral method was developed and validated to determine N‐acetyl‐glutamine (NAG) enantiomers by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Enantioseparation was achieved on a Chiralpak QD‐AX column (150 × 4.6 mm i.d., 5 μm) using methanol–water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 μL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N‐acetyl‐l ‐glutamine and N‐acetyl‐d ‐glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02–20 μg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within‐run and between‐run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.     

Simultaneous determination of 103 pesticide residues in tea samples by LC‐MS/MS

A simple and sensitive method was developed and validated for the simultaneous determination of 103 pesticide residues in tea by LC‐MS/MS. For the analysis of the pesticide with polarity, thermal lability or low volatility, this LC‐MS/MS method has an advantage over GC. In this work, residual pesticides were extracted from the tea sample with ACN and then purified using Carb‐NH₂ SPE cartridges. Using the multiple reaction monitoring mode, the pesticides were quantified and identified by the most abundant and characteristic fragment ions. The recoveries obtained for each pesticide ranged between 65 and 114% at three spiked concentration levels. The intra‐day precisions were lower than 19.6%. Good linear relationships were observed with the correlation coefficients r² >0.996 for all analytes. The established method was successfully applied to the determination of pesticide residues in real tea samples.     

Enantioseparation of nornicotine in tobacco by ultraperformance convergence chromatography with tandem mass spectrometry

Nornicotine, an alkaloid constituent of tobacco, is a precursor to the carcinogen N‐nitrosonornicotine that is produced during the curing and processing of tobacco. Accumulating evidence reveals that nornicotine enantiomers have different neurochemical and behavioral effects. In the present study, an accurate and rapid method was developed for the enantioseparation of (R )‐(+)‐nornicotine and (S )‐(−)‐nornicotine enantiomers in tobacco by ultra‐performance convergence chromatography with tandem mass spectrometry. Chromatographic conditions were investigated to achieve the optimal resolution of two enantiomers. Results indicated that (R )‐(+)‐nornicotine and (S )‐(−)‐nornicotine could be separated within 5 min when ammonium hydroxide was added into the cosolvent, and the best resolution (R _s = 4.76) was achieved on a immobilized cellulose tris‐(3,5‐dichlorophenylcarbamate) chiral stationary phase. The proposed method was validated and was finally applied to analyze the compositions of (R )‐(+)‐nornicotine and (S )‐(−)‐nornicotine in three typical types of tobaccos (flue‐cured, burley, and oriental). It was found that, enantiomer fraction of nornicotine (the proportion of (S )‐(−)‐nornicotine in the nornicotine pool) in burley tobacco samples was relatively high and constant compared with flue‐cured and oriental tobaccos. The effective and rapid enantioseparation of nornicotine may help the understanding of alkaloid metabolites in different tobacco varieties and may also benefit pharmacological studies of alkaloid enantiomers.     

Development and validation of a sensitive assay for the quantification of imatinib using LC/LC‐MS/MS in human whole blood and cell culture

We developed and validated a semi‐automated LC/LC‐MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back‐flushed onto the analytical column. Ion transitions [M + H]⁺ of imatinib (m/z = 494.3 → 394.3) and its internal standard trazodone (372.5 → 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter‐day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi‐automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative determination of β,β‐dimethylacrylshikonin (DASK) in rat whole blood by liquid chromatography–tandem mass spectrometry with pre‐column derivation and its pharmacokinetic application

A sensitive and selective liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of β,β‐dimethylacrylshikonin (DASK) in rat whole blood. DASK was pretreated using pre‐column derivatization with 2‐mercaptoethanol followed by liquid–liquid extraction with cyclohexane. Detection was performed on Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer by selected reaction monitoring mode via electrospray ionization source. The linear range for the determination of DASK spiked in rat whole blood (0.25 mL) was 3–3000 ng/mL. The accuracy was within 9%. Intra‐ and inter‐day precisions were no more than 16.1 and 13.3%, respectively. The validated LC‐MS/MS method was successfully applied to the preliminary pharmacokinetic study in rats. After DASK administration (60 mg/kg, p.o.) in rats, pharmacokinetic parameters were obtained, where the area under the drug concentration–time curve was 2393.7 ± 224.4 ng h/mL and the elimination half‐life was 27.6 ± 5.3 h. Copyright © 2008 John Wiley & Sons, Ltd.     

Enantioselective separation and determination of the dinotefuran enantiomers in rice,tomato and apple by HPLC

An effective chiral analytical method was developed for the resolution and determination of dinotefuran enantiomers in rice, tomato and apple samples. Dinotefuran enantiomers were baseline‐separated and determined on a novel chiral column, ChromegaChiral CCA, with n‐hexane–ethanol–methanol (85:5:10, v/v/v) as the mobile phase at a flow rate of 1.0 mL/min with UV detection at 270 nm. The resolution of dinotefuran enantiomers was about 1.8. The first eluted enantiomer was (+)‐dinotefuran and the second eluted one was (?)‐dinotefuran. The effects of mobile‐phase composition and column temperature on the enantioseparation were evaluated. The method was validated for linearity, repeatability, accuracy, LOD and LOQ. LOD was 0.15 mg/kg in rice and tomato, 0.05 mg/kg in apple, with an LOQ of 0.5 mg/kg in rice and tomato, 0.2 mg/kg in apple. The average recoveries of the pesticide from all matrices ranged from 75.8 to 92.9% for all fortification levels The precision values associated with the analytical method, expressed as RSD values, were <16.5% for the pesticide in all matrices. The methodology was successfully applied for the enantioselective analysis of dinotefuran enantiomers in real samples, indicating its efficiency in investigating the environmental stereochemistry of dinotefuran in food matrix.     

Indirect enantioseparation of fluoxetine in mouse serum by derivatization with 1R‐(–)‐menthyl chloroformate followed by ultra high performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry

Here we describe a simple and sensitive analytical method for the enantioselective quantification of fluoxetine in mouse serum using ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry. The sample preparation method included a simple deproteinization with acetonitrile in 50 μL of serum, followed by derivatization of the extracts in 50 μL of 2 mM 1R‐(–)‐menthyl chloroformate at 45ºC for 55 min. These conditions were statistically optimized through response surface methodology using a central composite design. Under the optimized conditions, neither racemization nor kinetic resolution occurred. The derivatized diastereomers were readily resolved on a conventional sub‐2 μm C₁₈ column under a simple gradient elution of aqueous methanol containing 0.1% formic acid. The established method was validated and found to be linear, precise, and accurate over the concentration range of 5.0–1000.0 ng/mL for both R and S enantiomers (r² > 0.993). Stability tests of the prepared samples at three different concentration levels showed that the R‐ and S‐fluoxetine derivatives were relatively stable for 48 h. No significant matrix effects were observed. Last, the developed method was successfully used for enantiomeric analysis of real serum samples collected at a number of time points from mice administered with racemic fluoxetine.     

Identification of regioisomers and enantiomers of triacylglycerols in different yeasts using reversed‐ and chiral‐phase LC–MS

LC with atmospheric pressure chemical ionization (ACPI) MS with RP and chiral phase was used for separation of triacylglycerols (TAGs) from yeasts of the genera Candida, Kluyveromyces, Rhodotorula, Saccharomyces, Torulospora, Trichosporon, and Yarrowia. Chiral LC–APCI‐MS is based on using two columns in series packed with a 3,5‐dimethylphenyl carbamate modified β‐cyclodextrin chiral phase. All regioisomers and enantiomers of TAGs containing one to five double bonds were separated. Molecular species of TAGs, i.e. regioisomers and enantiomers, were identified and quantified by MS/MS. Among the 94 identified TAGs, the most abundant were triolein, oleopalmitoleoolein, and dipalmitoleoolein. In strains producing palmitoleic acid in amounts >25% of total fatty acids (FAs), this acid, or unsaturated FA is bound in sn‐1. In strains containing palmitoleic acid at 10–25% total FAs this acid is mainly bound in sn‐3, saturated FA being bound in sn‐1. Strains containing <10% palmitoleic acid form preferentially symmetrical TAGs.     

Enantioselective determination of ketamine in dog plasma by chiral liquid chromatography–tandem mass spectrometry

Ketamine is an N‐methyl‐d ‐aspartate receptor antagonist that is usually used clinically as a racemic mixture. Its two enantiomers exhibit different pharmacological activities. To determine whether the enantiomers have different pharmacokinetic profiles, a chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of ketamine enantiomers in dog plasma. The enantiomers of ketamine were extracted from 50 μL of plasma by methyl tert‐butyl ether. Adequate chromatographic retention and baseline resolution of the enantiomers were achieved within a runtime of 5 min on a chiral column coated with polysaccharide derivatives, using a gradient mobile phase of acetonitrile and 10 mm ammonium bicarbonate aqueous solution. Ketamine enantiomers were detected by mass spectrometry with multiple reaction monitoring mode using the transitions of m/z 238.3 → 125.9 for the analytes and m/z 237.1 → 194.1 for carbamazepine (internal standard). The method was linear over the concentration range from 0.5 to 500 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) for each enantiomer was 0.5 ng/mL. The intra‐ and inter‐day precision was <7.3% and 8.5% for R‐ and S‐ketamine, respectively. The accuracy was 92.9–110.4% for R‐ketamine and 99.8–102.4% for S‐ketamine. The method was successfully applied to characterize the stereoselective pharmacokinetic profiles of ketamine in beagle dogs.     

Enantiomeric separation of glycidyl tosylate by CE: Application to the study of catalytic asymmetric epoxidation of allyl alcohol

A CE method using CDs as chiral selectors was developed and validated to achieve the separation of glycidyl tosylate enantiomers originated by in situ derivatization of glycidol enantiomers obtained in asymmetric epoxidation of allyl alcohol with chiral titanium‐tartrate complexes as catalysts. The effects of the nature, pH and concentration of the buffer, the nature and concentration of chiral selector, the addition of SDS, methanol, ethanol or 2‐propanol, the capillary temperature, the effective capillary length and the applied voltage on the chiral resolution of glycidyl tosylate enantiomers were investigated. The best separation conditions were achieved using a Tris‐borate buffer mixture (50 and 25 mM, respectively) at pH=9.3 with a dual CD system consisting of 2.5% succinyl‐β‐CD and 1.0% β‐CD w/v at 15°C. A baseline separation (resolution～2.0) of the glycidyl tosylate enantiomers was obtained in a relatively short time (less than 12 min). Satisfactory results were obtained in terms of linearity (r>0.99) and intermediate precision (RSD below 8.5%). The LOD and LOQ were 3.0 and 10.0 mg/L, respectively, and the recoveries ranged from 99.8 to 108.8%. Finally, the method was applied to the determination of the enantiomeric excess and the yield obtained in the asymmetric epoxidation of allyl alcohol employing chiral titanium‐tartrate complexes as catalysts after an in situ derivatization of glycidol enantiomers to glycidyl tosylate.     

Simple and sensitive determination of malondialdehyde in human urine and saliva using UHPLC–MS/MS after derivatization with 3,4‐diaminobenzophenone

Malondialdehyde has been used as a biomarker for lipid peroxidation in biological samples. An ultra‐high performance liquid chromatography with tandem mass spectrometry method was developed to determine the levels of malondialdehyde in human urine and saliva samples. To select the optimum derivatization reagent from four diamino compounds, the reactivity and sensitivity of their derivatives were compared, and 3,4‐diaminobenzophenone was selected. The optimum reaction conditions for malondialdehyde with 3,4‐diaminobenzophenone were as follows: a reagent dosage of 50 mg/L, pH of 4, and reaction for 30 min at 50°C. The formed derivative product was analyzed using ultra‐high performance liquid chromatography with tandem mass spectrometry without additional extraction or concentration steps. In the optimal conditions, the method was used to determine malondialdehyde concentration in human urine and saliva samples. The limits of quantification for malondialdehyde in biological samples were over a concentration range of 0.1–0.3 μg/L. Additionally, the calibration curve showed a linearity greater than r ² = 0.997. The method was used to analyze 14 human urine and saliva samples from healthy volunteers. Malondialdehyde was detected in the concentration range of 1.7–33.6 μg/g creatinine in all human urine samples and 0.1–1.3 μg/L in all human saliva samples.     

Resolution and isolation of enantiomers of (±)‐isoxsuprine using thin silica gel layers impregnated with l‐glutamic acid,comparison of separation of its diastereomers prepared with chiral derivatizing reagents having l‐amino acids as chiral auxiliaries

Thin silica gel layers impregnated with optically pure l ‐glutamic acid were used for direct resolution of enantiomers of (±)‐isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l ‐alanine, l ‐valine and S‐benzyl‐l ‐cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography on a C₁₈ column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin‐layer chromatography (TLC) on reversed phase (RP) C₁₈ plates. Diastereomers prepared with enantiomerically pure (+)‐isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)‐isoxsuprine. The elution order in the experimental study of RP‐TLC and RP‐HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1–0.09 µg/mL in TLC while it was in the range of 22–23 pg/mL in HPLC and 11–13 ng/mL in RP‐TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a method to measure methadone enantiomers and its metabolites without enantiomer standard compounds for the plasma of methadone maintenance patients

A liquid chromatography–photodiode array (LC‐PDA) method using a chiral analytical column was developed to determine the plasma levels of enantiomers of methadone and its chiral metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), without the standard compounds of R‐form or S‐form enantiomers. This method was established by the characteristics of recombinant cytochrome P‐450 (CYP) isozymes, where CYP2C19 prefers to metabolize R‐methadone and CYP2B6 prefers to metabolize S‐methadone. We incubated the racemic methadone standard with either enzyme for 24 h. We identified the retention times of R‐ and S‐methadone to be around 10.72 and 14.46 min, respectively. Furthermore, we determined the retention times of R‐ and S‐EDDP to be approximately 6.76 and 7.72 min, respectively. No interferences were shown through the retention times of morphine, buprenorphine and diazepam. With the high recovery rate of a solid‐phase extraction procedure, this method was applied in analyzing plasma concentrations of seven methadone maintenance patients where R‐ and S‐methadone and R‐ and S‐EDDP were 233.4 ± 154.9 and 185.9 ± 136.3 ng/mL and 84.4 ± 99.4 and 37.6 ± 22.9 ng/mL, respectively. These data suggest that the present method can be applied for routine assay for plasma methadone and EDDP concentrations for patients under treatment. Copyright © 2009 John Wiley & Sons, Ltd.