Enantioselective determination of a gastroprokinetic drug using amylose tris-(3,5-dimethylphenylcarbamate) as a stationary phase by HPLC

An enantioselective high-performance liquid chromatographic method for determination of enantiomers of mosapride citrate in bulk drugs and pharmaceuticals using UV-vis and polarimetric detectors in series has been developed. Baseline separation with resolution >2.0 was achieved on a column containing amylose tris-(3,5-dimethylphenylcarbamate) as stationary phase using a mobile phase consisting of n-hexane:ethanol:triethylamine (80:20:0.3, v/v/v) at 40 degrees C. The detection was carried out at UV-276 nm and enantiomers were identified by polarimetric detector. The effect of ethanol, 2-propanol, TEA, temperature and mobile phase flow rate on separation of MSP enantiomers was studied and the method was validated with respect to accuracy, precision, linearity and limits of detection and quantification. The linearity of the method was studied between 6.25 and 50 microg/ml and r2 was >0.9997. The recoveries were in the range 99.63-100.22%, the method was suitable not only for process development of mosapride citrate but also for quality assurance of the individual enantiomers in bulk drugs and pharmaceuticals.     

A validated LC method for the determination of vesamicol enantiomers in human plasma using vancomycin chiral stationary phase and solid phase extraction

An enantioseparation high performance liquid chromatographic (HPLC) method was developed and validated to determine D-(+)- and L-(-)-vesamicol in human plasma. The assay involved the use of a solid phase extraction for plasma sample clean up prior to HPLC analysis utilizing a C18 Bond-Elute column. Chromatographic resolution of the vesamicol enantiomers was performed on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol:glacial acetic acid:triethylamine (100:0.1:0.05 (v/v/v)) at a flow rate of 1.0 ml/min and UV detection set at 262 nm. All analyses were conducted at ambient temperature. The method was validated over the range of 1-20 microg/ml for each enantiomer concentration (R2>0.999). Recoveries for D-(+)- and L-(-)-vesamicol enantiomers were in the ranges of 96-105% at 3-16 microg/ml level. The method proved to be precise (within-run precision ranged from 1.3 to 2.7% and between-run precision ranged from 1.5 to 3.4%) and accurate (within-run accuracies ranged from 0.8 to 3.4% and between-run accuracies ranged from 1.7 to 5.0%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 1.0 and 0.5 microg/ml (S/N=3), respectively.     

Cellulose tris(3,5-dichlorophenylcarbamate) immobilised on silica: a novel chiral stationary phase for resolution of enantiomers

CHIRALPAK IC is a new chiral stationary phase (CSP) made by immobilising cellulose tris(3,5-dichlorophenylcarbamate) on silica gel. The chiral selector is distinct from any other commercially available polysaccharide-based CSPs. Apart from its compatibility with the whole series of solvents; this CSP is able to operate under various chromatographic conditions and bring about new characteristics in enantiomeric recognition. It can afford many large and specific enantiomeric separations. It exhibits complementary properties with regard to the existing immobilised chiral packing materials of the same category.     

A validated chiral HPLC method for the enantiomeric separation of Linezolid on amylose based stationary phase

Two chiral HPLC methods namely method A and method B were developed for the separation of enantiomers of Linezolid. The mobile phases containing hexane, 2-propanol and trifluoro acetic acid (TFA) in the ratio (80:20:0.1, v/v/ v); hexane, ethanol and TFA in the ratio (65:35:0.1, v/v/v) were used in method A and method B, respectively. The assay results of the two methods were checked in terms of F-test variance ratio and found to be less than the table value, confirming their good precision. The enantiomeric separation of Linezolid on different chiral stationary phases was investigated. The two enantiomers of Linezoild were well resolved on a Chiralpak AD, an amylose based stationary phase. Preparative chiral HPLC was carried out to obtain pure (+) enantiomer of Linezolid from its racemate. The method A was extensively validated and found to be robust. The chiral assay of Linezolid in bulk and pharmaceutical formulations (tablet) were found to be 100.4 +/- 0.4 and 101.2 +/- 1.4%, respectively at 95% confidence interval. The percentage recovery of (+) enantiomer (chiral impurity) was found to be 99.2 +/- 1.9 at 95% confidence interval. The limit of detection and limit of quantification of (+) enantiomer were found to be 123 and 374 ng/ml, respectively for 10 microl injection volume.     

Enantiomeric separation of cizolirtine and metabolites on amylose tris (3,5-dimethylphenyl carbamate) chiral stationary phase

The enantiomeric resolution of (+/-)-cizolirtine, (+/-)-cizolirtine-N-oxide, (+/-)-N-desmethylcizolirtine and (+/-)-5(alpha-hydroxybenzyl)-1-methylpyrazole was achieved on amylose tris (3,5-dimethylphenyl carbamate) chiral stationary phase known as Chiralpak AD using hexane/2-propanol/triethylamine (80:20:0.05, v/v/v) as the mobile phase. The flow rate of the mobile phase used was 1.0 ml/min with UV detection at 230 nm. The values of R(s) of the resolved enantiomers of (+/-)-cizolirtine, (+/-)-cizolirtine-N-oxide, (+/-)-N-desmethylcizolirtine and (+/-)-5(alpha-hydroxybenzyl)-1-methyl pyrazole were 1.20, 0.60, 1.16 and 1.15, respectively.     

键合型直链淀粉手性固定相分离5种质子泵抑制剂对映体

目的使用HPLC手性固定相法实现5种质子泵抑制剂对映体的分离。方法考察手性固定相种类,流动相中有机改性剂的种类和比例,缓冲盐的种类和浓度,碱性添加剂,流速及柱温等因素对分离的影响。结果通过不断优化色谱分离条件,最终确定5种药物的分离条件:色谱柱为Chiralpak ID柱(250 mm×4.6 mm,5μm),柱温为25℃,流速为0.6 mL·min~(-1),分离泮托拉唑、雷贝拉唑对映体的流动相为水-乙腈(60∶40,v/v),分离奥美拉唑对映体的流动相为水-乙醇(20∶80,v/v),分离兰索拉唑对映体的流动相为5 mmol·L~(-1)醋酸铵水溶液-乙腈(60∶40,v/v),分离艾普拉唑对映体的流动相为20 mmol·L~(-1)碳酸氢铵水溶液-乙腈(55∶45,v/v)。在上述条件下,5种质子泵抑制剂对映体均达到完全分离。结论键合型直链淀粉手性固定相对5种质子泵抑制剂对映体均实现完全分离。     

The enantiomeric separation of metipranolol and desacetylmetipranolol on a cellulose tris-3,5-dimethylphenyl-carbamate chiral stationary phase

A high-performance liquid chromatographic (HPLC) method is described for direct separation of the enantiomers of metipranolol and its principal degradation product and main metabolite, desacetylmetipranolol. Separations are performed on a chiral stationary phase of cellulose tris-3,5-dimethylphenyl carbamate (Chiralcel OD), with hexane-propan-2-ol-diethylamine elution and UV detection at 278 nm. The method affords identification and determination of the optical purity of the bulk drug and its formulated ophthalmic products.     

Chiral separation of tamsulosin isomers by HPLC using cellulose tris (3,5-dimethhylphenylcarbamate) as a chiral stationary phase

A high-performance liquid chromatographic (HPLC) method was developed for the chiral separation of an antagonist of alpha1A adrenoceptors, tamsulosin and its S-isomer. Baseline separation of the isomers was achieved within 35 min on a CHIRALCEL OD-RH column with a binary solvent mixture of 50 mmol l(-1) KPF6-acetonitrile (v/v (70:30), pH 5.0) as the optimized mobile phase. The detection limits and quantification limits of both R-isomer and S-isomer were 0.11 and 0.44 ng, respectively. The R.S.D. values of peak-area for the two isomer were 0.42% (of peak-height: 0.77%) for R-isomer and 0.64% (of peak-height:0.92%) for S-isomer (n = 5).     

A validated chiral LC method for the enantioselective analysis of Levetiracetam and its enantiomer R-alpha-ethyl-2-oxo-pyrrolidine acetamide on amylose-based stationary phase

A new, simple chiral HPLC method was developed for the enantiomeric separation of Levetiracetam, [(S)-alpha-ethyl-2-oxo-pyrrolidine acetamide], an antiepileptic drug in pharmaceutical formulations and in bulk materials. Enantiomeric separation was achieved on a chiralpak AD-H column using a mobile phase consisting of hexane and isopropanol in the ratio (90:10, v/v) at a flow rate of 1.0 ml/min. The resolution between the enantiomers was found to be not less than 7 in the optimized method. Interestingly, unwanted enantiomer, namely R-alpha-ethyl-2-oxo-pyrrolidine acetamide ((R)-enantiomer), was eluted prior to its mirror image in the developed method. The developed method was found to be selective in the presence of related impurities of Levetiracetam, namely N-(1-carbamoyl-propyl)-4-chloro-butyramide (Imp-1) and 1-ethyl-2-oxo-1-pyrrolidine acetic acid (Imp-2), and also under exposed conditions of UV light and 60 degrees C. The limit of detection (LOD) and limit of quantification (LOQ) of (R)-enantiomer were found to be 900 and 2250 ng/ml, respectively, for 10 microl injection volume. The method precision for (R)-enantiomer at limit of quantification level was within 8% R.S.D. Calibration curve for (R)-enantiomer was linear over the studied ranges (2250-9000 ng) with correlation coefficient greater than 0998. The active pharmaceutical ingredient was extracted from its finished dosage form (tablet) using isopropanol. The percentage recoveries of (R)-enantiomer were ranged from 94.2 to 102.6 and from 93.5 to 104.1 in spiked bulk and formulation samples of Levetiracetam, respectively. Levetiracetam sample solution and mobile phase are found to be stable for at least 48 h. The developed method was found to be rugged and robust. The proposed method was found to be suitable and accurate for the quantitative determination of (R)-enantiomer in bulk drugs and commercial formulations. Chiralcel OD-H column can also be used as an alternative column for the above purpose.     

Enantiomeric separation of beta-blockers by HPLC using (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid as chiral stationary phase

Direct liquid chromatographic separations of the enantiomers of metoprolol and bisoprolol have been developed, using (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid as chiral stationary phase (CSP). The separations were achieved in a normal phase system employing a mobile phase containing n-hexane, 1,2-dichloroethane and methanol. Column efficiency was strongly dependent on the composition of the mobile phase. The eluent contents of methanol and of 1,2-dichloroethane were optimized, and so was flow-rate and column temperature. Under the optimal conditions, linear responses for (R)-metoprolol and (S)-metoprolol are obtained in the range of 0.079–1.38 and 0.015–5.80 mg/ml, with detection limits of 0.008 and 0.002 mg/ml, respectively. As for bisoprolol, the linear ranges of (R)-isomer and (S)-isomer are 0.05–1.31 and 0.02–1.00 mg/ml with detection limits of 0.001 and 0.008 mg/ml, respectively. The relative standard deviation (R.S.D.) of each enantiomer did not exceed 0.90%. The method has been successfully applied to the determination of enantiomers in pharmaceuticals.     

Simultaneous determination of propafenone and 5-hydroxypropafenone enantiomers in plasma by chromatography on an amylose derived chiral stationary phase

An enantioselective liquid chromatography method was developed for the simultaneous determination of propafenone (PPF) and 5-hydroxypropafenone (PPF-5OH) enantiomers in plasma. After liquid liquid extraction with dichloromethane, the enantiomers were resolved on a Chiralpak AD column using hexane-ethanol (88:12, v/v) plus 0.1% diethylamine as the mobile phase and monitored at 315 nm. Under these conditions the enantiomeric fractions of the drug and of its metabolite were analysed within 20 min. The extraction procedure resulted in absolute recoveries of 62.9 and 61.3% for (R)- and (S)-PPF, respectively, and of 57.6 and 56.5% for (R)- and (S)-PPF-5OH, respectively. This procedure was efficient in removing endogenous interferents as well the interference of an other PPF metabolite, N-despropylpropafenone (PPF-NOR). The calibration curves were linear over the concentration range 25-1250 ng/ml. Low values of the coefficients of variation were demonstrated for both within-day and between day assays. The method described in this paper allows the determination of PPF and PPF-5OH enantiomers at plasma levels as low as 25 ng/ml and can be used in clinical pharmacokinetic studies.     

A validated chiral CE method for Frovatriptan, using cyclodextrin as chiral selector

A cyclodextrin modified capillary zone electrophoretic method has been developed for the evaluation of chiral purity of Frovatriptan using sulfobutyl ether beta cyclodextrin (SB-beta-CD) as the chiral selector. The method is highly specific, accurate and reproducible. The method was optimized with a systematic method development approach by optimizing the pH of electrolyte, attempting the separation in different classes of chiral selectors and modifying parameters such as cyclodextrin concentration and the organic modifier type and concentration. The optimized method was validated for specificity, precision, linearity, accuracy and stability in solution using Imidazole as the internal standard. The limit of detection (LOD) and limit of quantification (LOQ) were 1.0 microg/mL and 5.0 microg/mL respectively for each isomer. The method was applied for estimating the chiral purity of various batches of Frovatriptan.     

新型键合纤维素手性固定相拆分氯西加酮对映体

目的:建立一种用新型键合纤维素手性固定相拆分氯西加酮对映异构体的高效液相色谱方法。方法:使用Chiralpak IB(250 mm×4.6 mm,5μm)色谱柱,流动相为正己烷-无水乙醇(90︰10),流速0.8 mL.min-1,检测波长230 nm,柱温30℃;通过对比氯西加酮和添加了苏式氯西加酮的氯西加酮色谱图,判断先流出物的构型。结果:氯西加酮对映体在新型键合直链纤维素衍生化合物手性固定相上能够完全分离,分离度为1.79;先流出物为苏式氯西加酮。结论:本方法可方便地实现氯西加酮对映体的分离。     

Evaluation of six chiral stationary phases in LC for their selectivity towards drug enantiomers.

Six chiral stationary phases (CSP) were evaluated for their enantioselectivity towards a series of 45 drugs with different acidic, basic or neutral properties. These CSPs were: a polyacrylamide phase, Chiraspher; two polysaccharide-based phases, cellulose tris-3,5-dimethyl phenylcarbamate (Chiralcel OD) and the S-naphthylethylcarbamate derivative of beta-cyclodextrin (SN-beta-CD; Cyclobond I SN); and three protein-based CSPs--alpha 1-acid glycoprotein (Chiral-AGP), ovomucoid (OVM) and cellulase. A total of 28 different mobile phases were involved. Chiral-AGP, OVM and Chiralcel OD appeared to be the most promising CSPs for the enantio separation of the series of structurally different compounds evaluated. Cellulase and Chiralcel OD show particularly high enantioselectivity towards the group of beta-blocker drugs. The different protein-based CSPs were used in their usual reversed-phase mode. The other phases were used in combination with apolar mobile phases, except for SN-beta-CD, which was evaluated in both modes. Formal optimization strategies were not adopted, although the effect of organic modifier and eluent pH on enantioselectivity was briefly examined for the protein-based phases.     

A validated chiral LC method for the determination of zolmitriptan and its potential impurities

A new, accurate and reliable chiral HPLC method was developed for the determination of Zolmitriptan, (4S)-4-[[3-[2-(dimethylamino)ethyl]-1H-indol-5-yl] methyl]-2-oxazolidinone an antimigraine agent and its potential impurities namely (4R)-4-[[3-[2-(dimethylamino)ethyl]-1H-indol-5-yl] methyl]-2-oxazolidinone [(R)-enantiomer] and (4S)-4-(4-aminobenzyl)-2-oxazolidinone (Imp-1) in pharmaceutical formulations and in bulk drugs. HPLC separation was carried out by normal phase chromatography with a mobile phase composed of hexane:isopropanol:methanol:diethylamine in the ratio (75:10:15:0.1, v/v/v/v) pumped at a flow rate of 1.0 ml/min on a Chiralpak AD-H column. Zolmitriptan and its potential impurities were baseline resolved in the optimized method. The presence of diethylamine in the mobile phase has played a key role in achieving chromatographic resolution between the enantiomers and also in enhancing chromatographic efficiency. The developed method was also found to be selective under exposed conditions UV light and 60 degrees C. The developed method was completely validated and proved to be robust. The values of the limit of detection (LOD) and limit of quantification (LOQ) of (R)-enantiomer and Imp-1 were 100, 250 ng/ml and 30, 1000 ng/ml, respectively, for 10 microl injection volume. The validated method yielded good results regarding selectivity, linearity, precision, accuracy and ruggedness. Zolmitriptan sample solution and mobile phase are found to be stable for at least 24 h. The proposed method was found to be suitable and accurate for the quantitative determination of Zolmitriptan and its impurities namely (R)-enantiomer and Imp-1 in bulk drugs and commercial formulations.     

Chiral resolution of the enantiomers of new selective CB(2) receptor agonists by liquid chromatography on amylose stationary phases

Analytical HPLC methods using derivatized amylose chiral stationary phases, Chiralpak AD-H and Chiralpak AS, were developed for the direct enantioseparation of eight substituted 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives with one stereogenic center. Baseline separation (R_s > 1.5) was always achieved on amylose based Chiralpak AD-H column to the difference with Chiralpak AS. Using UV detection, a linear response was observed within a 180–420 μmol L⁻¹ concentration range (r² > 0.991) for three racemic compounds 1, 3 and 4 with best pharmacological potentials; repeatability, limit of detection (LD) and quantification (LQ) were also determined: LD varied, for the solutes, from 0.36 to 2.56 μmol L⁻¹. Finally, the enantiopurity of these compounds was determined. Additionally, the effect of temperature variations upon isomer separations was investigated.     

Development and validation of a LC method for the enantiomeric purity determination of S-ropivacaine in a pharmaceutical formulation using a recently commercialized cellulose-based chiral stationary phase and polar non-aqueous mobile phase

Ropivacaine is the first enantiomerically pure long-acting local anaesthetic used for surgical anaesthesia and post-operative pain relief.     

Enantioselective LC analysis of synephrine in natural products on a protein-based chiral stationary phase

An enantioselective LC method with photodiode array detection (PAD) was developed for the enantioseparation of (±)-synephrine from C. aurantium L. var. amara fruits and phytotherapic derivatives by using a protein-based chiral stationary phase with cellobiohydrolase as the chiral selector (Chiral-CBH). Analyses were carried out on a Chiral-CBH column (100 × 4.0 mm i.d., 5 μm), with a mobile phase consisting of 2-propanol (5%, w/w) in sodium phosphate buffer (pH 6.0; 10 mM) and disodium EDTA (50 μM). The flow rate was 0.8 mL/min. Detection was set at 225 nm. To identify the order of elution, the racemate was resolved by the preparation of suitable diastereoisomeric salts with antipodes of appropriate organic acids.

Isolation of synephrine from C. aurantium fruits and phytoproducts was performed by solid-phase extraction (SPE) with a strong cation-exchange phase.

The method developed was validated and was found to be linear in the 0.40–40.14 μg/mL range (r² = 1.000, P < 0.0001) for both synephrine enantiomers. The limit of detection (LOD) for each enantiomer was 0.04 μg/mL. The limit of quantification (LOQ) for each enantiomer was 0.13 μg/mL. Intra-day precision (calculated as %R.S.D.) ranged from 0.03 to 0.24% for (−)-synephrine and from 0.03 to 0.35% for (+)-synephrine. Inter-day precision (calculated as %R.S.D.) ranged from 0.07 to 1.45% for (−)-synephrine and from 0.06 to 1.26% for (+)-synephrine. Intra- and inter-day accuracies (calculated as %recovery) were in the ranges of 97.4–100.6 and 98.0–101.6% for (−)-synephrine, and in the ranges 97.0–101.5 and 98.1–102.8% for (+)-synephrine.

The results of the application of the method to the analysis of C. aurantium samples showed that (−)-synephrine was the main component. (+)-Synephrine was not detected in C. aurantium fruits and was present in low concentration in the phytoproducts.     

Fundamentals of stationary phase optimized selectivity LC (SOS-LC)

A new procedure (SOS-LC) is described for the optimization of the HPLC stationary phase, using serially connected columns and the principle of the "PRISMA" model. In the course of the base measurements the retention factors (k) of the analytes were determined on three different base stationary phases; other parameters were fixed. By use of these data the k values were predicted in the cases of all theoretically combined stationary phases. These predictions resulted in numerous intermediate theoretical separations but only the optimal one was compiled and tested applying other parameters fixed as in the case of the base measurements. The overall selectivity of this separation was better than that of any individual stationary phase. SOS-LC is independent of the mechanism and the scale of separation and will be very advantageous in the case of hyphenated techniques (LC-MS and LC-NMR) due to stable mobile phase background.     

Direct enantiomeric resolution of some cardiovascular agents using synthetic polymers imprinted with (-)-S-timolol as chiral stationary phase by thin layer chromatography

Molecular imprinted polymers (MIPs) of S-timolol were prepared as chiral stationary phases (CSPs) in thin layer chromatography (TLC). The resolution of the enantiomers of some cardiovascular drugs, including propranolol, atenolol, timolol, nadolol, nifedipine and verapamil were investigated on these CSPs. A mobile phase system of either methanol or acetonitrile was used and the effects of acetic acid content of the mobile phases were also investigated. The best resolution was achieved for enantioseparation of propranolol, timolol and atenolol on plates based on MIP of (-)-S-timolol using methacrylic acid as functional monomer (alpha = 1.52, 1.6, 1.59) respectively, using acetonitrile containing 5% acetic acid and (alpha = 1.47, 1.52, 1.5) in methanol containing 1% acetic acid as mobile phases. The results obtained show that TLC based on MIPs could be applied in the direct separation of several beta-adrenergic drugs. As the side chains on beta-blockers are similar, it is possible that this method could also be used for the resolution of other racemates in this family of drugs. Racemic drugs structurally related to print molecule, were completely resolved into two spots with the MIP plates. In general the retention of (+)-R-isomers was greater than that of (-)-S-isomers, indicating lower stereoselectivity of the MIPs to the dextrorotatory isomers. The method offers a rapid, sensitive and reliable method for quality control for these drugs.