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.     

Stereoselective determination of propafenone enantiomers in transgenic Chinese hamster CHL cells expressing human cytochrome P450

An enantioselective assay for S(+)- and R(-)-propafenone in transgenic Chinese hamster CHL cells expressing human cytochrome P450 was developed. The method involved extraction of propafenone from the S9s incubates, and formation of propafenone diastereomeric derivatives with the chiral reagent 2,3,4, 6-tetra-O-beta-D-glucopranosyl isothiocyanate. Separation and quantitation of diastereomeric propafenone derivatives were carried out in a reverse-phase-HPLC system with UV detection. The assay was linear from 2 to 200 microg/mL for each enantiomer. The analytical method gave average recoveries of 97.5% and 97.0% for S(+)- and R(-)-propafenone, respectively. The limits of detection and quantitation for the method are 0.1 and 2.0 microg/mL for both S(+)- and R(-)-propafenone, respectively. The reproducibility of the assay was good (RSD <10%). The method allowed study of the depletion of S(+)- and R(-)-propafenone in transgenic Chinese hamster CHL cells expressing human cytochrome P450. The stereoselectivity of propafenone phase I metabolism via cDNA-expressed CYP3A4 was observed.     

A high performance liquid chromatographic method for the determination of albuterol enantiomers in human serum using solid phase extraction and chemical derivatization.

A high performance liquid chromatographic method was developed for the simultaneous assay of R(-)- and S(+)-albuterol in human serum. The assay involves solid phase extraction as a sample clean-up step and derivatization of racemic albuterol to its diastereomeric thioureas with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl isothiocyanate. Chromatographic separation was accomplished under isocratic conditions using an octadecylsilane column and a mobile phase consisting of 29:71 acetonitrile:distilled water containing 0.1% triethylamine, pH 4.0 (adjusted with concentrated phosphoric acid) at a flow rate of 0.8 mL/min. The diastereomers were detected using a fluorescence detector set at 223 nm excitation and no emission filter. Racemic bamethane was used as internal standard. Drug to internal standard peak-height ratios were linear over a 2-20 ng/mL range for each enantiomer. The limit of detection of each analyte was 1.0 ng/mL (S/N = 3).     

Simultaneous determination of disopyramide and mono-N-dealkyldisopyramide enantiomers in human plasma by capillary electrophoresis

In this paper, a rapid method for the enantioselective analysis of the antiarrhythmic drug disopyramide and its main metabolite mono-N-dealkyldisopyramide in human plasma by capillary electrophoresis employing the cyclodextrin-modified electrokinetic chromatography mode is described. Sample clean-up was carried out by alkalinization with sodium hydroxide followed by liquid-liquid extraction with toluene. The complete enantioselective analysis was performed within less than 5 min using 20 mmol/L sodium acetate buffer, pH 5.0, containing 0.2% w/v sulfated beta-cyclodextrin as chiral selector. A 40 cm uncoated fused-silica capillary was used for the analysis, performed at a voltage of 15 kV and at 20 degrees C. The calibration curves were linear over the concentration range of 62.5-1850 ng/mL and 125-1850 ng/mL for each enantiomer of disopyramide and mono-N-dealkyldisopyramide. The mean recoveries for disopyramide and mono-N-dealkyldisopyramide enantiomers were up to 87 and 69%, respectively. All four enantiomers studied could be quantified at three different concentrations (200, 400 and 600 ng/mL) with coefficient of variation and % relative error not higher than 15%. The quantitation limit was 62.5 ng/mL for (+)-(S)-and (-)-(R)-disopyramide and (-)-(R)-mono-N-dealkyldisopyramide and 125 ng/mL for (+)-(S)-mono-N-dealkyldisopyramide, using 1 mL of human plasma.     

Ketopinic acid and diisoproylideneketogulonic acid as chiral ion-pair selectors in capillary electrophoresis. Enantiomeric impurity analysis of S-timolol and 1R,2S-ephedrine

1S,4R-(+)-ketopinic acid [(+)-KPA] has been introduced as a chiral selector for the separation of pharmacologically active amines by non-aqueous capillary electrophoresis (NACE). (+)-KPA gave enantioresolution for most of the compounds previously separated by 2R,3S,4R,5S-(-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid [(-)-DIKGA], but with a reversed migration order. A complete enantioresolution (Rs=4.2) was obtained for timolol, a compound that could not be resolved using (-)-DIKGA as the selector. Thus, (+)-KPA was evaluated for the enantiomeric purity determination of S-timolol. A method based on pre-concentration by transient isotachophoresis (tITP) provided a limit of detection (LOD) of 0.2% R-timolol in S-timolol samples. Because of the lack of enantioresolution of ephedrine when (+)-KPA was used as the selector, a method with (-)-DIKGA has been developed and validated for determination of the enantiomeric purity of the 1R,2S enantiomer. The method gave good precision and accuracy with an LOD (S/N=3) of 0.033% for the enantiomeric impurity 1S,2R-ephedrine.     

Capillary electrophoretic enantioselective determination of zopiclone and its impurities

A capillary electrophoretic enantioselective method with UV detection was developed and validated for the simultaneous quantification of zopiclone enantiomers and its impurities, zopiclone-N-oxide enantiomers, and 2-amino-5-chloropyridine, in tablets. The analytes were extracted from the tablets using ACN and were separated in an uncoated fused-silica capillary (50 μm, 42 cm effective length, 50 cm total length) using 80 mM sodium phosphate buffer pH 2.5 and 5 mM carboxymethyl-β-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A voltage of 27 kV was applied and the capillary temperature was maintained at 25°C. All enantiomers were analyzed within 8 min and linear calibration curves over the concentration range of 0.4-0.8 mg mL?1 for each zopiclone enantiomer, 0.8-1.6 μg mL?1 for 2-amino-5-chloropyridine and 0.4-0.8 μg mL?1 for each zopiclone-N-oxide enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of zopiclone under stress conditions. This application demonstrated the importance of a stability-indicating assay method for this drug.     

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.     

1,5-Benzoxathiepin derivatives. III. Optical resolution of methyl (+/-)-cis-3-hydroxy-4-[3-(4-phenyl-1-piperazinyl)propyl]-3,4-dihydro- 2H-1,5-benzoxathiepin-4-carboxylate hydrochloride ((+/-)-CV-5197) with selective 5-hydroxytryptamine2(5-HT2)-antagonistic activity

The selective 5-HT2-receptor antagonist, methyl (+/-)-cis-3-hydroxy-4-[3-(4-phenyl-1-piperazinyl)propyl]-3,4-dihydro-2H- 1,5-benzoxathiepin-4-carboxylate hydrochloride ((+/-)-CV-5197) was resolved in high optical purity using (R)-(-)- and (S)-(+)-1,1'-binaphthyl-2,2'-diyl hydrogen phosphates ((R)-(-)- and (S)-(+)-BNP). The absolute configuration of (+)-CV-5197 was determined to be 3S,4R by X-ray crystallographic analysis. In the binding assay, it was demonstrated that (+)-CV-5197 was a more active isomer (IC50 = 23 nM +/- 6.3) for 5-HT2 receptor binding than the (-)-enantiomer (IC50 = 1600 nM +/- 82). (+)-CV-5197 completely inhibited the 5-HT-induced contraction of the isolated pig coronary artery at a concentration of 3 x 10(-7) M, whereas (-)-CV-5197 showed little antagonistic activity, even at 3 x 10(-4) M. Thus, the agreement between the results of the binding assays and the biological activities for the 3S,4R enantiomer of CV-5197 suggests that its physiological activity is probably exerted through 5-HT2-receptor antagonism.     

Determination of enantiomers of atenolol and propranolol in pharmaceutical formulations by HPLC

A validated HPLC-UV method was developed for the determination of R(-), S(+)-atenolol and R(-), S(+)-propranolol in pharmaceutical formulations. The proposed method required no elaborate sample preparation and was found to be selective, linear, and repeatable within the established ranges. Atenolol and propranolol isomers were separated using a Chirex 3022 (S) column with the mobile phases hexane-dichloromethane-methanol-trifluoroacetic acid (35 + 35 + 5 + 0.25, v/v/v/v) and hexane-dichloromethane-ethanol-trifluoroacetic acid (55 + 40 + 5 + 0.25, v/v/v/v), respectively. The LOD values of R(-) and S(+)-atenolol were 12.3 and 9.86 microg/mL, respectively, and 0.61 and 0.89 microg/mL, respectively, for R(-) and S(+)-propranolol. Retention times of R(-)-propranolol and S(+)-propranolol were 12.4 and 14.3 min, respectively, and 29.06 and 32.71 min, respectively, for (R)-atenolol and (S)-atenolol. The proposed method was applied to the determination of enantiomers in pharmaceutical formulations, and no interference from any excipients was found.     

Enantioselective analysis of ibuprofen in human plasma by anionic cyclodextrin-modified electrokinetic chromatography

This paper reports the development of a rapid method for the enantioselective analysis of the nonsteroidal anti-inflammatory drug ibuprofen in human plasma by capillary electrophoresis employing the anionic cyclodextrin-modified electrokinetic chromatography mode. Sample cleanup was carried out by acidification with HCl followed by liquid-liquid extraction with hexane:isopropanol (99:1 v/v). The complete enantioselective analysis was performed within 10 min, using 100 mmol L(-1) phosphoric acid/triethanolamine buffer, pH 2.6, containing 2.0% w/v sulfated beta-cyclodextrin as chiral selector; fenoprofen, another nonsteroidal anti-inflammatory drug, was used as internal standard. The calibration curves were linear over the concentration range of 0.25-125.0 microg mL(-1) for each enantiomer of ibuprofen. The mean recoveries for ibuprofen enantiomers were up to 85%. The enantiomers studied could be quantified at three different concentrations (0.5, 5.0 and 50.0 microg mL(-1)) with a coefficient of variation and relative error not higher than 15%. The quantitation limit was 0.2 microg mL(-1) for (+)-(S)- and (-)-(R)-ibuprofen using 1 mL of human plasma. The plasma endogenous compounds and other drugs did not interfere with the present assay. The analysis of real plasma samples obtained from a healthy volunteer after administration of 600 mg of racemic ibuprofen showed a maximum plasma level of 29.6 and 39.9 microg mL(-1) of (-)-(R)- and (+)-(S)-ibuprofen, respectively, and the area under plasma concentration-time curve AUC(0-infinity) (+)-(S)/AUC(0-infinity) (-)-(R) ratio was 1.87.     

Validated LC-MS/MS method for quantification of gabapentin in human plasma: application to pharmacokinetic and bioequivalence studies in Korean volunteers

A sensitive validated liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for gabapentin (GB) in human plasma has been developed and applied to pharmacokinetic (PK) and bioequivalence (BE) studies in human. In a randomized crossover design with a 1-week period, each subject received a 300 mg GB capsule. The procedure involves a simple protein precipitation with acetonitrile and separated by LC with a Gemini C(18) column using acetonitrile-10 mm ammonium acetate (20:80, v/v, pH 3.2) as mobile phase. The GB and internal standard [(S)-(+)-alpha-aminocyclohexanepropionic acid hydrate] were analyzed using an LC-API 2000 MS/MS in multiple reaction monitoring mode. The ionization was optimized using ESI(+) and selectivity was achieved using MS/MS analysis, m/z 172.0 --> 154.0 and m/z 172.0 --> 126.0 for GB and IS, respectively. The assay exhibited good linearity over a working range of 20-5000 ng/mL for GB in human plasma with a lower limit of quantitation of 20 ng/mL. No endogenous compounds were found to interfere with the analysis. The accuracy and precision were shown for concentrations over the standard ranges. This method was successfully applied for the PK and BE studies by analysis of blood samples taken up to 36 h after an oral dose of 300 mg of GB in 24 healthy volunteers.     

Investigating interactions between chloroquine/hydroxychloroquine and their single enantiomers and angiotensin-converting enzyme 2 by a cell membrane chromatography method

Chloroquine and hydroxychloroquine have been studied since the early clinical treatment of SARS-CoV-2 outbreak. Considering these two chiral drugs are currently in use as the racemate, high-expression angiotensin-converting enzyme 2 cell membrane chromatography was established for investigating the differences of two paired enantiomers binding to angiotensin-converting enzyme 2 receptor. Molecular docking assay and detection of SARS-CoV-2 spike pseudotyped virus entry into angiotensin-converting enzyme 2-HEK293T cells were also conducted for further investigation. Results showed that each single enantiomer could bind well to angiotensin-converting enzyme 2, but there were differences between the paired enantiomers and corresponding racemate in frontal analysis. R-Chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine. Moreover, each single enantiomer was proved effective compared with the control group; compared with S-chloroquine or the racemate, R-chloroquine showed better inhibitory effects at the same concentration. As for hydroxychloroquine, R-hydroxychloroquine showed better inhibitory effects than S-hydroxychloroquine, but it slightly worse than the racemate. In conclusion, R-chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability and inhibitory effects compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine (racemate), while the effect of preventing SARS-CoV-2 pseudovirus from entering cells was weaker than R-hydroxychloroquine/hydroxychloroquine (racemate).     

Enantiomeric determination of praziquantel and its main metabolite trans-4-hydroxypraziquantel in human plasma by cyclodextrin-modified micellar electrokinetic chromatography

A capillary electrophoresis method for the simultaneous quantitation of praziquantel and its main metabolite trans-4-hydroxypraziquantel enantiomers in human plasma was developed and validated using cyclodextrin-modified micellar electrokinetic chromatography. Sample clean-up involved a single-step liquid-liquid extraction of plasma with toluene after the addition of NaCl. The complete enantioselective analysis was obtained in less than 7 min using 2% w/v sulfated beta-cyclodextrin as chiral selector and 20 mmol/L sodium deoxycholate as surfactant, in 20 mmol/L sodium borate buffer, pH 10. A 50 microm x 42 cm uncoated fused-silica capillary was used for the analysis, performed at a voltage of 18 kV and at 20 degrees C. The calibration curves were linear over the 125-625 ng/mL concentration range. The mean recoveries for praziquantel and trans-4-hydroxypraziquantel were up to 96 and 71%, respectively, with good precision. All four enantiomers were quantified at two concentration levels (200 and 600 ng/mL) with precision and accuracy below 15%. The quantitation limit was 50 ng/mL for (-)-(R)- and (+)-(S)-praziquantel and 62.5 ng/mL for (-)-(R)- and (+)-(S)-trans-4-hydroxypraziquantel, using 1 mL of human plasma.     

Development and Validation of a Chiral Liquid Chromatographic Assay for Enantiomeric Separation and Quantification of Verapamil in Rat Plasma: Stereoselective Pharmacokinetic Application

A novel, fast and sensitive enantioselective HPLC assay with a new core–shell isopropyl carbamate cyclofructan 6 (superficially porous particle, SPP) chiral column (LarihcShell-P, LSP) was developed and validated for the enantiomeric separation and quantification of verapamil (VER) in rat plasma. The polar organic mobile phase composed of acetonitrile/methanol/trifluoroacetic acid/triethylamine (98:2:0.05: 0.025, v/v/v/v) and a flow rate of 0.5 mL/min was applied. Fluorescence detection set at excitation/emission wavelengths 280/313 nm was used and the whole analysis process was within 3.5 min, which is 10-fold lower than the previous reported HPLC methods in the literature. Propranolol was selected as the internal standard. The S-(−)- and R-(+)-VER enantiomers with the IS were extracted from rat plasma by utilizing Waters Oasis HLB C18 solid phase extraction cartridges without interference from endogenous compounds. The developed assay was validated following the US-FDA guidelines over the concentration range of 1–450 ng/mL (r² ≥ 0.997) for each enantiomer (plasma) and the lower limit of quantification was 1 ng/mL for both isomers. The intra- and inter-day precisions were not more than 11.6% and the recoveries of S-(−)- and R-(+)-VER at all quality control levels ranged from 92.3% to 98.2%. The developed approach was successfully applied to the stereoselective pharmacokinetic study of VER enantiomers after oral administration of 10 mg/kg racemic VER to Wistar rats. It was found that S-(−)-VER established higher C_max and area under the concentration-time curve (AUC) values than the R-(+)-enantiomer. The newly developed approach is the first chiral HPLC for the enantiomeric separation and quantification of verapamil utilizing a core–shell isopropyl carbamate cyclofructan 6 chiral column in rat plasma within 3.5 min after solid phase extraction (SPE).     

Quantification of propranolol enantiomers in small blood samples from rats by reversed-phase high-performance liquid chromatography after chiral derivatization

A high-performance liquid chromatographic (HPLC) technique is described for quantification of R(+)- and S(-)-propranolol from 100-microliters rat blood samples. The procedure involves chiral derivatization with tert.-butoxycarbonyl-L-leucine anhydride to form diastereomeric propranolol-L-leucine derivatives which are separated on a reversed-phase HPLC column. The method as previously reported has been modified for assaying serial blood microsamples obtained from the rat for pharmacokinetic studies. An internal standard, cyclopentyldesisopropylpropranolol, has been incorporated into the assay and several derivatization parameters have been altered. Standard curves for both enantiomers were linear over a 60-fold concentration range in 100-microliters samples of whole rat blood (12.5-750 ng/ml; r = 0.9992 for each enantiomer). Inter- and intra-assay variability was less than 12% for each enantiomer at 25 ng/ml. No enantiomeric interference or racemization was observed as a result of the derivatization. No analytical interference was noted from endogenous components in rat blood samples. Preliminary data from two male Sprague-Dawley rats given a 2.0 mg/kg intravenous dose of racemic propranolol revealed differential disposition of the two enantiomers. R(+)-Propranolol achieved higher initial concentration but was eliminated more rapidly than S(-)-propranolol. Terminal half-lives of R(+)- and S(-)-propranolol were 19.23 and 51.95 min, respectively, in one rat, and 14.50 and 52.07 min, respectively, in the other.     

Enantiomeric separation and determination of the enantiomeric impurity of armodafinil by capillary electrophoresis with sulfobutyl ether-β-cyclodextrin as chiral selector

A selective capillary electrophoresis method using sulfobutyl ether-β-cyclodextrin as a chiral selector was developed and validated for the determination of the enantiomeric impurity of (R)-modafinil, i.e., armodafinil. Several parameters were optimized for a satisfactory enantioresolution, including the type and concentration of chiral selector and organic modifier, pH of background electrolyte (BGE), capillary temperature. The finally adopted condition was: 20 mmol/L phosphate buffer at pH 7.5, containing 20 mmol/L sulfobutyl ether-β-cyclodextrin and 20% methanol, at temperature of 25 °C. A good resolution of 3.3 for the two enantiomers of modafinil was achieved by applying the optimal conditions. The limit of detection (LOD) and limit of quantification (LOQ) of (S)-modafinil were 1.25 μg/mL and 2.50 μg/mL, respectively. The established method was also proven to display good selectivity, repeatability, linearity and accuracy. Finally, the method was used to investigate the enantiomeric purity of armodafinil in bulk samples.     

Development and validation of a capillary electrophoresis method for the determination of escitalopram and sensitive quantification of its enantiomeric impurity in formulations

A rapid capillary zone electrophoresis method has been developed capable of quantifying 0.05% of R-enantiomer and assaying the main component in escitalopram formulations. Many parameters influencing enantioseparation were investigated, which include chiral selectors, buffer composition and pH, applied voltage, capillary length, temperature, and rinsing procedure. Optimal separation conditions were obtained by using a 25 mM phosphate buffer at pH 7.0, containing 1.6% (w/v) sulfated-β-cyclodextrin with short-end injection at 0.5 psi for 5 s. Online UV detection was performed at 205 nm. A voltage of -20 kV was applied and the capillary temperature was kept at 25°C. Separation was achieved in less than 2 min. The method was further validated, including robustness, stability of the solution, selectivity, linearity (escitalopram from 0.25 μg/mL to 600 μg/mL, y = 1528.3 × +1812.9; R2 = 0.9999), LOD and LOQ (0.08 and 0.25 μg/mL, respectively), precision and accuracy. The proposed method was then applied to the quality control of the bulk sample and tablets of escitalopram (10 mg).     

Enantioselective determination of arotinolol in human plasma by HPLC using teicoplanin chiral stationary phase

A sensitive enantioselective high-performance liquid chromatography (HPLC) method was developed and validated to determine S-(+)- and R-(-)-arotinolol in human plasma. Baseline resolution was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar organic mobile phase consisting of methanol:glacial acetic acid:triethylamine, 100:0.1:0.1, (v/v/v) at a fl ow rate of 0.8 mL/min and UV detection set at 317 nm. Human plasma was spiked with stock solution of arotinolol enantiomers and labetalol as the internal standard. The assay involved the use of liquid-liquid extraction procedure with ethyl ether under alkaline condition for human plasma sample prior to HPLC analysis. Recoveries for S-(+)- and R-(-)-arotinolol enantiomers were in the range 93-103% at 200-1400 ng/mL level. Intra-day and inter-day precision calculated as %RSD was in the ranges 1.3-3.4 and 1.9-4.5% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percentage error were in the ranges 1.2-3.5 and 1.5-6.2% for both enantiomers, respectively. Linear calibration curves in the concentration range 100-1500 ng/mL for each enantiomer showed a correlation coefficient (r) of 0.9998. The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 and 50 ng/mL (S/N = 3), respectively.     

Enantioselective determination of trantinterol in rat plasma by ultra performance liquid chromatography-electrospray ionization mass spectrometry after derivatization

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of trantinterol enantiomers in rat plasma. Diphenhydramine was employed as the internal standard. The plasma samples were prepared using liquid-liquid extraction with n-hexane-dichloromethane-isopropanol (20:10:1, v/v/v) as the extractant. Trantinterol enantiomers after pre-column derivatization using diacetyl-l-tartaric anhydride (DATAAN) were separated on a C18 column using a gradient solvent programme. The mobile phase was composed of 3 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curve for each enantiomer was obtained in the concentration range of 1-80 ng/mL, with limit of quantification (LOQ) of 1 ng/mL. The intra- and inter- precision (R.S.D.) values were below 9.6% and accuracy (R.E.) was from −2.4 to 6.2% at all quality control (QC) levels. The developed method was applied to the enantioselective pharmacokinetic study of trantinterol in rats.     

Liquid chromatographic method for enantiopurity control of alaptide using polysaccharide stationary phases

Separation of veterinary drug alaptide ((S)-8-methyl-6,9-diazaspiro(4,5)decane-7,10-dione) from a chiral impurity (R-enantiomer) was developed. Five chiral columns (three amylose and two cellulose type) were evaluated in a reversed-phase system. Three of them offered satisfactory enantiomeric resolution. Finally, three methods were validated and proved to be applicable for the determination of a chiral impurity content below 0.1% (method A: 3-AmyCoat column, tris-[3,5-dimethylphenyl]carbamoyl amylose; mobile phase: water/methanol/propan-2-ol/butan-2-ol=75:20:3.5:1.5 v/v, flow rate: 0.40 mL/min; column temperature: 30 °C; method B: Chiralpak AS-3R, tris-[1-phenylethyl]carbamoyl amylose; water/acetonitrile=80:20 v/v, 0.40 mL/min; 40 °C; method C: Chiralcel OZ-3R, tris-[3-chloro-4-methylphenyl] carbamoyl cellulose; water/acetonitrile=80:20 v/v, 0.40 mL/min; 40 °C). Some decrease in efficiency with repeated sample injections was observed for the 3-AmyCoat column. The resistance to mass transfer in the stationary phase increased probably due to the change in chiral selector conformation. This effect was considerably suppressed by propan-2-ol or to a greater extent by butan-2-ol added to a mobile phase. Simple regeneration was also suggested to recover efficiency of the column.