A new stability indicating reverse phase high performance liquid chromatography method for the determination of enantiomeric purity of a DPP‐4 inhibitor drug linagliptin

A simple, sensitive, and stability indicating isocratic reverse phase high performance liquid chromatography method has been developed, optimized and validated for the separation and quantification of S‐enantiomer in linagliptin (R‐enantiomer) drug substance. Enantiomeric separation was achieved on a Cellulose tris(4‐chloro‐3‐methylphenylcarbamate) stationary phase. Mobile phase consists of aqueous diammonium hydrogen phosphate buffer and acetonitrile in the ratio of 35:65 v/v. Isocratic elution was performed at a flow rate of 1.0 mL/min, the column oven temperature was set at 40°C and detection was at 226 nm. The resolution between R and S enantiomers is found to be more than 4.0. The impact of mobile phase composition, pH of buffer and temperature on the resolution has been studied. The detector response is found to be linear over the concentration range of 0.17–1.7 μg/mL. LOD and LOQ levels of S‐enantiomer are found to be 0.057 and 0.172 μg/mL respectively. The recovery of S‐enantiomer is 99.8% w/w. The proposed method is validated for specificity, precision, linearity, accuracy and robustness.     

Direct separation of the enantiomers of ramosetron on a chlorinated cellulose‐based chiral stationary phase in hydrophilic interaction liquid chromatography mode

Ramosetron is an enantiopure active pharmaceutical ingredient marketed in Japan since 1996 and later in a few Southeast Asian countries predominantly as an antiemetic for patients receiving chemotherapy. In this study, a simple and rapid high‐performance liquid chromoatography method for the separation of ramosetron and its related enantiomeric impurity by using hydrophilic interaction liquid chromatography mode is presented. Chiral resolution was performed on an analytical column (100 mm × 4.6 mm id) packed with 3 μm particles of cellulose‐based Chiralpak IC‐3 chiral stationary phase. Using a mobile phase containing acetonitrile–water–diethylamine (100:10:0.1, v/v/v) and setting the column temperature at 35°C, the resolution value was 7.35. At a flow rate of 1 mL/min, the enantioseparation was completed within 5 min. The proposed method was partially validated and it has proven to be sensitive with limit of detection and limit of quantitation of the (S)‐enantiomer impurity of 44.5 and 133.6 ng/mL.     

Chiral separation and thermodynamic investigation of WCK 3023: A novel oxazolidinone antibacterial agent,application to pre‐clinical pharmacokinetic study

A chiral liquid chromatographic method was developed and validated for the quantification of R‐enantiomer impurity (RE) in WCK 3023 (S‐enantiomer), a new drug substance. The separation was achieved on Chiralpak IA (amylose‐based immobilized chiral stationary phase), using a mobile phase consisting of n‐hexane–ethanol–trifluoroacetic acid (70:30:0.2, v/v/v) at a flow rate of 1.0 mL/min. The method was extensively validated for the quantification of RE in WCK 3023 and proved to be robust. For RE the detector response was linear over the concentration range of 0.11–5 μg/mL. The limit of quantitation and limit of detection for RE were 0.11 and 0.04 μg/mL respectively. Average recovery of the RE was in the range of 98.11–99.55%. The developed method was specific, sensitive, precise and accurate for quantitative determination of RE in WCK 3023. The impact of thermodynamic parameters on the chiral separation was evaluated. The method was employed for controlling the enantiomeric impurity in the lots of WCK 3023 used for pre‐clinical studies. The method was successfully applied to evaluate the possible conversion of WCK 3023 to RE in rat serum samples during pre‐clinical pharmacokinetic studies.     

直链淀粉手性固定相高效液相色谱法拆分比索洛尔对映体

采用直链淀粉手性固定相高效液相色谱法在正相条件下直接拆分了比索洛尔对映异构体。分别以异丙醇、乙醇为有机改性剂,考察了流动相的组成与配比、流速及柱温等因素对比索洛尔对映体分离的影响。确定了比索洛尔对映体的最佳拆分条件:流动相正己烷-乙醇-二乙胺(体积比为88∶12∶0.1),流速0.6 mL/min,检测波长270 nm,柱温20 ℃。该方法可快捷、简便地拆分比索洛尔对映体。     

Enantioselective separation of indole derivatives by liquid chromatography using immobilized cellulose (3,5-dimethylphenylcarbamate) chiral stationary phase

A new chiral stationary phase of 3,5-dimethylphenylcarbamates of cellulose chemically bonded to 3-aminopropyl silica gel was prepared, which may be used with a wide range of solvents including standard and non-standard ones. Several racemic indole derivatives have been resolved using standard and non-standard solvents on the immobilized chiral column (15 cm × 0.46 cm) at a flow rate of 1.0 mL/min or 0.5 mL/min with a UV detection at 230 nm. Separation of indole derivatives on immobilized and coated chiral stationary phases (CSP) in HPLC using a mixture of hexane/2-propanol as mobile phase was compared. The resolution factors for immobilized and coated chiral column were 0.57–2.02 and 0.61–4.03, respectively. It was found that both coated and immbolized chiral stationary phases were suitable for the separation of indole derivatives; however, the coated CSP possesses a higher resolving power than the immobilized one. The article is published in the original.     

Development and Validation of the Chiral HPLC Method for Daclatasvir in Gradient Elution Mode on Amylose-Based Immobilized Chiral Stationary Phase

A selective and specific high-performance liquid chromatography method for the determination of daclatasvir enantiomers has been developed and validated. Various immobilized polysaccharide-based chiral stationary phases were used to define a separation strategy utilizing normal-phase and polar organic chromatography modes. Excellent resolution between daclatasvir and its enantiomer was achieved on amylose tris (3-chlorophenylcarbamate) stationary phase, namely CHIRALPAK ID-3, using binary gradient containing acetonitrile:diethylamine and methanol:diethylamine as the mobile phase. The flow rate of the mobile phases was maintained at 1.0 mL min⁻¹ while the column oven temperature was maintained at 40 °C. The column effluent was monitored by UV detection at 315 nm. In comparison with isocratic method, the binary gradient method offered excellent peak shape and improved resolution between daclatasvir and its enantiomer while maintaining the specificity with diastereomers. The method was found to be precise, accurate, and linear (R ² > 0.999). Limit of detection and limit of quantitation of the enantiomer were found to be 0.083 µg mL⁻¹ as and 0.25 µg mL⁻¹, respectively. Recovery of the enantiomer was found to be in the range of 90 to 112 %.

     

直链淀粉-三[(S)-1-苯乙基氨基甲酸酯]手性固定相拆分布地奈德对映体及其制剂含量的测定

22R-布地奈德的药物活性比22S-布地奈德的强2~3倍,开发布地奈德对映体拆分和定量分析方法,可为其药物研发及质量控制提供重要依据。目前,主要以反相C₁₈固定相对布地奈德对映体进行拆分,而采用手性固定相对其进行拆分少有报道。通过考察固定相、流动相和柱温对布地奈德对映体拆分的影响,建立了基于直链淀粉-三[(S)-1-苯乙基氨基甲酸酯]手性固定相快速拆分和检测布地奈德对映体的高效液相色谱方法,其色谱条件如下:色谱柱为Chiralpak AS-RH色谱柱(150 mm×4.6 mm, 5.0 μm),流动相为乙腈-水(45∶55, v/v),柱温40 ℃,流速1.0 mL/min,二极管阵列检测器(DAD),检测波长246 nm,进样量10 μL。在该色谱条件下,布地奈德的两个对映体得到较好拆分,22R-布地奈德和22S-布地奈德的保留时间分别6.40 min和7.77 min,分离度为4.64; 22R-布地奈德和22S-布地奈德分别在各自范围内线性关系良好,相关系数(R²)均为0.9999,检出限分别为0.05 μg/mL和0.07 μg/mL,定量限分别为0.16 μg/mL和0.20 μg/mL; 4个添加水平的样品加标回收率为102.63%~104.17%,相对标准偏差(RSD)为0.08%~0.57%(n=6)。将该方法应用于1批次4个吸入用布地奈德混悬液实际样品进行检测,22R-布地奈德和22S-布地奈德的含量分别为283.15~284.63 μg/mL和259.86~261.51 μg/mL。该方法操作简便,分析时间短,重复性好,准确度高,可用于布地奈德对映体的拆分及其制剂的质量控制。     

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector,N‐methacryloyl‐l‐histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d ,l ‐phenylalanine, d ,l ‐tyrosine, and d ,l ‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l ‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l ‐enantiomers of aromatic amino acids migrated before d ‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.     

Chiral separation of helical chromenes with chloromethyl phenylcarbamate polysaccharide‐based stationary phases

Two chloromethyl phenylcarbamate‐based chiral stationary phases, one containing an amylose‐type chiral selector (Lux Amylose 2, from Phenomenex) and the other a cellulose‐type one (Lux Cellulose‐4, from Phenomenex), were successfully used for the chiral resolution of three helical chromenes featuring a helicene‐like structure. The compound bearing a phenyl substituent on the helicene‐like structure was enantioresolved at 25°C with Lux Cellulose‐4 and a n‐hexane/1‐propanol 99:1 v/v eluent. With a n‐hexane/2‐propanol 99.8:0.2 v/v mobile phase, the same column (operated at 35°C) provided the separation of the four isomers of the compound having a hexyl residue on the helicene‐like motif and an additional asymmetric carbon. Lux Amylose‐2 was necessary for the enantioseparation of the compound having the sole hexyl residue on the helical scaffold. For the last compound a n‐hexane/2‐propanol 99.8:0.2 v/v eluent was used, and the column temperature was fixed at 5°C. The enantiomer elution order was appraised by using electronic circular dichroism and theoretical calculations. Notably, different thermodynamics of retention and enantioseparation were observed for molecules with pronounced structural similarity, that is, the enantiomer pairs of the compound containing the additional asymmetric carbon atom. Indeed, both entropically and enthalpically controlled adsorption and separation processes were observed.     

Separation and quantitation of oxprenolol in urine and pharmaceutical formulations by HPLC using a Chiralpak IC and UV detection

Abstract  

A stereoselective HPLC method has been developed for the simultaneous determination of oxprenolol enantiomers in urine and pharmaceutical products. Enantiomeric resolution of oxprenolol was achieved on cellulose tris(3,5-dichlorophenylcarbamate) immobilized onto a 5 μm spherical porous silica chiral stationary phase (CSP) known as Chiralpak IC with UV detection at 273 nm. The mobile phase consisted of n-hexane:isopropanol:triethylamine 70:30:0.1 (v/v/v) at a flow rate of 1.0 cm³/min. The method was validated for its linearity, accuracy, precision, and robustness. The calibration curves were linear over the range of 0.5–75 μg/cm³, with a detection limit of 0.1 μg/cm³ for each enantiomer. An average recovery of 99.0% and a mean relative standard deviation of 2.6% at 40.0 μg/cm³ for S-(−)- and R-(+)-enantiomers were obtained. The overall recoveries of oxprenolol enantiomers from pharmaceutical formulations were in the range 97.5–99.0%, with RSDs ranging from 0.6 to 0.8%. The mean extraction efficiency of oxprenolol from urine was in the range of 86.0–93.0% at 0.5–5 μg/cm³ for each enantiomer. The assay method proved to be suitable as a chiral quality control for oxprenolol formulations using HPLC and for therapeutic drug monitoring.     

Green micellar HPLC analysis of three angiotensin‐converting enzyme inhibitors in their mixtures with hydrochlorothiazide and modeling of their retention behavior by fitting to Foley's model

We present an environmentally friendly method for the analysis of three angiotensin‐converting enzyme inhibitors and hydrochlorothiazide simultaneously using a green micellar eluent for the first time. The chromatographic separation of enalapril maleate, lisinopril dihydrate, benazepril hydrochloride, and hydrochlorothiazide was implemented on an octadecyl silica column with a solution containing sodium dodecyl sulfate (0.12 M), 1‐propyl alcohol (10% v/v), triethylamine (0.3% v/v), and H₃PO₄ (0.02 M) at pH 3.6 as the mobile phase and UV detection at 210 nm. Validity of the method was confirmed and it exhibited good linearity within the ranges of 5.0–50.0 μg/mL for hydrochlorothiazide and 10.0–60.0 μg/mL for the three angiotensin‐converting enzyme inhibitors with a limit of detection of 0.39 to 1.15 μg/mL for all the studied drugs. The developed micellar high‐performance liquid chromatography method enables the quantification of the targeted angiotensin‐converting enzyme inhibitors in combined tablets with hydrochlorothiazide by isocratic elution. There is no need for special precautions to prevent broadening and splitting of their chromatographic peaks. The method fulfills the society rights for safe and green analytical methods. The retention behavior of the four studied drugs was fitted to Foley's model and their association equilibria to the micelles (K _AM) and to the surface‐modified stationary phase (K _AS) were calculated.     

HPLC enantioseparation of racemic bupropion,baclofen and etodolac: modification of conventional ligand exchange approach by pre‐column formation of chiral ligand exchange complexes

Separation of racemic mixture of (RS)‐bupropion, (RS)‐baclofen and (RS)‐etodolac, commonly marketed racemic drugs, has been achieved by modifying the conventional ligand exchange approach. The Cu(II) complexes were first prepared with a few l ‐amino acids, namely, l ‐proline, l ‐histidine, l ‐phenylalanine and l ‐tryptophan, and to these was introduced a mixture of the enantiomer pair of (RS)‐bupropion, or (RS)‐baclofen or (RS)‐etodolac. As a result, formation of a pair of diastereomeric complexes occurred by ‘chiral ligand exchange’ via the competition between the chelating l ‐amino acid and each of the two enantiomers from a given pair. The diastereomeric mixture formed in the pre‐column process was loaded onto HPLC column. Thus, both the phases during chromatographic separation process were achiral (i.e. neither the stationary phase had any chiral structural feature of its own nor did the mobile phase have any chiral additive). Separation of diastereomers was successful using a C₁₈ column and a binary mixture of MeCN and TEAP buffer of pH 4.0 (60:40, v/v) as mobile phase at a flow rate of 1 mL/min and UV detection at 230 nm for (RS)‐Bup, 220 nm for (RS)‐Bac and 223 nm for (RS)‐Etd. Baseline separation of the two enantiomers was obtained with a resolution of 6.63 in <15 min. Copyright © 2016 John Wiley & Sons, Ltd.     

基于直链淀粉衍生物手性固定相的高效液相色谱-串联质谱法拆分4种β-受体阻滞剂对映体及其分离机制的探讨

建立了以直链淀粉衍生物为手性固定相的高效液相色谱-串联质谱(HPLC-MS/MS)直接拆分普萘洛尔、美托洛尔、阿罗洛尔和卡维地洛4种β-受体阻滞剂对映体的方法。考察了手性固定相的种类、流动相改性剂和添加剂的体积分数、柱温和流速等对4种药物对映体分离的影响。结果表明:在Chiralpak AD-H手性色谱柱上,在正己烷-乙醇-二乙胺(20∶80∶0.03,v/v/v)为流动相、流速0.550 mL/min、柱温40℃的条件下,普萘洛尔、美托洛尔、阿罗洛尔和卡维地洛对映体均达到基线分离,分离度分别为1.37、1.80、2.09和4.70。通过热力学研究及对映体结构分析对拆分机理进行了探讨,发现4种药物对映体的手性拆分均为焓驱动过程,而固定相的手性空腔对不同药物的拆分影响较大。研究结果为β-受体阻滞剂的深入研究提供了参考方法。     

Efficient preparative separation of 6‐(4‐aminophenyl)‐5‐methyl‐4, 5‐dihydro‐3(2H)‐pyridazinone enantiomers on polysaccharide‐based stationary phases in polar organic solvent chromatography and supercritical fluid chromatography

6‐(4‐Aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone is a key synthetic intermediate for cardiotonic agent levosimendan. Very few studies address the use of chiral stationary phases in chromatography for the enantioseparation of this intermediate. This study presents two efficient preparative methods for the isolation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone in polar organic solvent chromatography and supercritical fluid chromatography using polysaccharide‐based chiral stationary phases and volatile organic mobile phases without additives in isocratic mode. Under optimum conditions, Chiralcel OJ column showed the best performance (α = 1.71, Rs = 5.47) in polar organic solvent chromatography, while Chiralpak AS column exhibited remarkable separations (α = 1.81 and Rs = 6.51) in supercritical fluid chromatography with an opposite enantiomer elution order. Considering the sample solubility, runtime and solvent cost, the preparations were carried out on Chiralcel OJ column and Chiralpak AS column (250 × 20 mm i.d.; 10 µm) in polar organic mode and supercritical fluid chromatography mode with methanol and CO₂/methanol as mobile phases, respectively. By utilizing the advantages of chromatographic techniques and polysaccharide‐based chiral stationary phases, this work provides two methods for the fast and economic preparation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone, which are suitable for the pharmaceutical industry.     

Photografted methacrylate‐based monolithic columns coated with cellulose tris(3,5‐dimethylphenylcarbamate) for chiral separation in CEC

A chiral capillary monolithic column for enantiomer separation in capillary electrochromatography was prepared by coating cellulose tris(3,5‐dimethylphenylcarbamate) on porous glycidyl methacrylate‐co‐ethylene dimethacrylate monolith in capillary format grafted with chains of [2(methacryloyloxy)ethyl] trimethylammonium chloride. The surface modification of the monolith by the photografting of [2(methacryloyloxy)ethyl] trimethylammonium chloride monomer as well as the coating conditions of cellulose tris(3,5‐dimethylphenylcarbamate) onto the grafted monolithic scaffold were optimized to obtain a stable and reproducible chiral stationary phase for capillary electrochromatography. The effect of organic modifier (acetonitrile) in aqueous mobile phase for the enantiomer separation by capillary electrochromatography was also investigated. Several pairs of enantiomers including acidic, neutral, and basic analytes were tested and most of them were partially or completely resolved under aqueous mobile phases. The prepared monolithic chiral stationary phases exhibited a good stability, repeatability, and column‐to‐column reproducibility, with relative standard deviations below 11% in the studied electrochromatographic parameters.     

Rapid and economic chiral-HPLC method of nebivolol enantiomers resolution in dosage formulation

A fast, economic, reproducible, accurate, effective, rugged and selective chiral-HPLC method was developed and validated for the enantiomeric resolution of nebivolol enantiomers [(+)-RRRS and (-)-SSSR)] in dosage formulation. The method was rapid as chiral separation occurred within only 12 min. The mobile phase used was n-heptane-ethanol-DEA (85:15:0.1, v/v) at 3.0 mL/min flow-rate with 225 nm detection. The column used was an amylase-based 3-AmyCoat (150 × 46 mm) [tris-(3,5-dimethylphenyl carbamate)]. The capacity factors of (+)-RRRS and (-)-SSSR enantiomers were 7.85 and 10.90 while the separation and resolution factors were 1.39 and 1.83, respectively. The limits of detection and quantitation for (+)-RRRS enantiomer were 4.5 and 10.00 μg/mL, while these values for (-)-SSSR enantiomer were 4.1 and 8.2 μg/mL, respectively. The linearity was observed in the concentrations range of 0.10-1.0 mg/mL for both enantiomers. The π-π interactions, hydrogen bonds, dipole-dipole interactions and steric effects control the chiral resolution of nebivolol enantiomers on the reported chiral column. The reported method can be used for the quality control of nebivolol in pharmaceutical preparations with good economy. In addition, this method can also be used for the analysis of (+)-RRRS and (-)-SSSR) enantiomers in biological and environmental samples.     

How mobile phase composition and column temperature affect enantiomer elution order of liquid crystals on amylose tris(3-chloro-5-methylphenylcarbamate) as chiral selector

A comprehensive study into the effects of mobile phase composition and column temperature on enantiomer elution order was conducted with a set of chiral rod-like liquid crystalline materials. The analytes were structurally similar and comprised variances such as length of terminal alkyl chain, presence of chlorine, number of phenyl rings, and type of chiral center. Experiments were carried out in polar organic and reversed-phase modes using amylose tris(3-chloro-5-methylphenylcarbamate) immobilized on silica gel as the chiral stationary phase. For all liquid crystals, reversal of elution order of enantiomers was observed based on type of used cosolvent and/or its content in the mobile phase; for some of the liquid crystals a temperature-induced reversal was also observed. Both linear and nonlinear dependencies of natural logarithm of enantioselectivity on temperature were found. Tested mobile phases comprised pure organic solvents and binary and tertiary mixtures of acetonitrile with organic solvents and/or water. Effect of acidic/basic mobile phase additives was also tested. Effect of structure of chiral selector is briefly discussed.     

Hydrophobic ionic liquid modified thermoresponsive molecularly imprinted monolith for the selective recognition and separation of tanshinones

A hydrophobic ionic liquid modified thermoresponsive molecularly imprinted monolith was synthesized using N‐isopropylacrylamide as a thermoresponsive monomer and a long‐chain hydrophobic ionic liquid as an auxiliary modification monomer. The ionic‐liquid‐modified thermoresponsive molecularly imprinted polymer was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. When the column temperature was 50°C, the synthesized monolithic column was successfully applied to the selective separation of homologue tanshinones within 7 min and eluted only by water (mobile phase) (theoretical plates more than 1.00 × 10⁵ per meter). The negative Gibbs free energy (≤–2.37) values showed that the transfer of the tanshinones from the mobile phase to the stationary phase on this monolithic column was a thermodynamically spontaneous process. Good linearity of the five tanshinones by thermoresponsive monolith was obtained in the range of 0.100–25.0 μg/mL. The limit of detection (S/N = 3) and limit of quantitation (S/N = 10) were less than 0.0390 and 0.0630 μg/mL, respectively, with a relative standard deviation of <4.8%. In this proposed thermoresponsive chromatography method, the separation of homologue analytes can be achieved by changing the column temperature, and the use of water as the mobile phase would decrease the economic cost and organic pollution.     

Preparation and evaluation of monodispersed,submicron, non‐porous silica particles functionalized with β‐CD derivatives for chiral‐pressurized capillary electrochromatography

Submicron, non‐porous, chiral silica stationary phase has been prepared by the immobilization of functionalized β‐CD derivatives to isocyanate‐modified silica via chemical reaction and applied to the pressurized capillary electrochromatography (pCEC) enantio‐separation of various chiral compounds. The submicron, non‐porous, cyclodextrin‐based chiral stationary phases (sub_μm‐CSP2) exhibited excellent chiral recognition of a wide range of analytes including clenbuterol hydrochloride, mexiletine hydrochloride, chlorpheniramine maleate, esmolol hydrochloride, and metoprolol tartrate. The synthesized submicron particles were regularly spherical and uniformly non‐porous with an average diameter of around 800 nm and a mean pore size of less than 2 nm. The synthesized chiral stationary phase was packed into 10 cm × 100 μm id capillary columns. The sub_μm‐CSP2 column used in the pCEC system showed better separation of the racemates and at a higher rate compared to those used in the capillary liquid chromatography mode (cLC) system. The sub_μm‐CSP2 possessed high mechanical strength, high stereoselectivity, and long lifespan, demonstrating rapid enantio‐separation and good resolution of samples. The column provided an efficiency of up to 170 000 plates/m for n‐propylbenzene.     

Chiral separation and quantitation of dorzolamide hydrochloride enantiomers by high‐performance liquid chromatography

A direct HPLC method for chiral separation of dorzolamide hydrochloride (4S,6S) and its enantiomer (4R,6R) was developed. Dorzolamide (4S,6S) and its antipode were separated on a chiral‐α₁‐acid glycoprotein column (150×4.0 mm, 5 μm). The influences of pH, temperature, flow rate, buffer concentration, and organic modifiers of the mobile phase on the retention and enantioselectivity were evaluated. The mobile phase consisted of an ammonium acetate buffer of pH 7.0. The method was validated for linearity, repeatability, accuracy, LOD, and LOQ. Calibration curves were constructed in the range of 0.5–10 μg/mL for dorzolamide (4S,6S) and 0.2–5 μg/mL for its enantiomer (4R,6R). Repeatability (n=6) showed less than 2% RSD. LOD and LOQ of the two enantiomers were found to be 0.2 and 0.5 for dorzolamide (4S,6S), 0.05 and 0.2 for its enantiomer (4R,6R), respectively. The proposed method was applied to the determination of dorzolamide enantiomer (4R,6R) in a raw material and two different eye drop samples.