Chiralpak AD-H Column Research Articles

Liquid chromatographic estimation of (S) - glycidyl butyrate in (R) - glycidyl butyrate

Aim:The present study was undertaken out of a commercial need for the synthesis of Linezolid with impurity limits within the specification.Materials and Methods:(R)-Glycidyl butyrate (RGB) is raw material for the synthesis of Linezolid drug substance. This RGB contains (S)-(+)-Glycidyl butyrate (SGB) and SGB appears in same concentration in the final Active Pharmaceutical Ingredient. So, a normal phase high-performance liquid chromatography (HPLC) method has been developed to determine the SGB level in the raw material. RGB and SGB were separated using an HPLC system equipped with quaternary gradient pumps on a Daicel chiralpak AD-H (250 × 4.6 mm) column with a mobile phase consisting 2.0 ml of ethanol in 1 000 ml of n-hexane. A 0.5 ml/minute flow rate and a 10 μl injection volume was used and the compounds were detected at 215 nm.Results:Method validation parameters demonstrated the same to be reliable, reproducible, and accurate one.Conclusion:Thus, the present study may be used for regular quality control of RGB to improve commercial feasibility for the synthesis of Linezolid.

Enantioselective determination of cetirizine in human plasma by normal-phase liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry

A highly sensitive and enantioselective method has been developed and validated for the determination of levocetirizine [(R)-cetirizine] in human plasma by normal-phase liquid chromatography coupled to tandem mass spectrometry with an atmospheric pressure chemical ionization (APCI) interface in the positive ion mode. Enantioselective separation was achieved on a CHIRALPAK AD-H column using an isocratic mobile phase consisting of a mixture of n-hexane, ethyl alcohol, diethylamine, and acetic acid (60:40:0.1:0.1, v/v/v/v). Levocetirizine-D 8 was used as an internal standard (IS). Levocetirizine and the IS were detected by multiple-reaction monitoring (MRM). Mass transitions of analyte and IS were m/z 389.2 → 201.1 and 397.2 → 201.1, respectively. Under optimized analytical conditions, a baseline separation of two enantiomers and IS was obtained in less than 11 min. Samples were prepared by a simple two-step extraction by protein precipitation using acetonitrile followed by liquid–liquid extraction with a n-hexane–dichloromethane mixture (50:50, v/v). The standard curve for levocetirizine was linear ( r 2 > 0.995) in the concentration range 0.5–300 ng/mL. Recovery was between 97.0 and 102.2% at low, medium, and high concentration. The limit of quantification (LOQ) was 0.5 ng/mL. Other method validation parameters, such as precision, accuracy, and stability, were very satisfactory. Finally, the proposed method was successfully applied to the study of enantioselective oral pharmacokinetics of levocetirizine in healthy Korean volunteers.

Chiral separation of potent corticotropin-releasing factor-1 receptor antagonists by supercritical fluid chromatography

Pyrazinones bearing an N-1-alkyl chain with a chiral center have been reported as potent antagonists of the corticotropin-releasing factor-1 receptor (CRF1R). Separation of individual enantiomers for preclinical testing was an important aspect of lead optimization. To evaluate the applicability and efficiency of supercritical fluid chromatography (SFC) for enantiomeric resolution of this class of compounds, enantiomeric pairs of eight pyrazinones with different structural characteristics were tested under an array of SFC conditions. The results showed that pyrazinones with a 1-cyclopropyl-2-methoxyethyl substituent were readily separated with a Chiralpak AD-H or Chiralcel OD-H column with ethanol as the modifier. On the other hand, analogs with a less polar alkyl substituent were not amenable to the general method and required further optimization of the chromatographic conditions. In addition, structural variations on the pyrazinone core and aromatic moiety had an impact on the chiral resolution of this class of compounds. This investigation led to the development of efficient chiral SFC methods for separating all eight pyrazinone enantiomeric pairs encompassing an array of structural variations.

(±)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis

Praeruptorin A is a coumarin compound naturally occurring in the roots of Peucedanum praeruptorum Dunn., a commonly used traditional Chinese medicine for the treatment of certain respiratory diseases and hypertension. Although previous studies indicated the relaxant effects of (±)-praeruptorin A on tracheal and arterial preparations, little is known about the functional characteristics of the enantiomers. In the present study, the two enantiomers were successfully isolated and identified by using a preparative Daicel Chiralpak AD-H column, and their relaxant effects on aorta rings were observed and compared. (+)-Praeruptorin A showed more potent relaxation than (−)-praeruptorin A against KCl- and phenylephrine-induced contraction of rat isolated aortic rings with intact endothelium. Removal of the endothelium remarkably reduced the relaxant effect of (+)-praeruptorin A but not that of (−)-praeruptorin A. Pretreatment of aortic rings with N ω-nitro- l-arginine methyl ester ( l-NAME, an inhibitor of nitric oxide synthase) or methylene blue (MB, a soluble guanylyl cyclase inhibitor) resulted in similar changes of the relaxant effects of the two enantiomers to endothelium removal. Molecular docking studies also demonstrated that (+)-praeruptorin A was in more agreement to nitric oxide synthase pharmacophores than (−)-praeruptorin A. On the other hand, the two enantiomers of praeruptorin A could slightly attenuate the contraction of rat aortic rings induced by internal Ca 2+ release from sarcoplasmic reticulum (SR). These findings indicated that (+)-praeruptorin A and (−)-praeruptorin A exerted distinct relaxant effects on isolated rat aorta rings, which might be mainly attributed to nitric oxide synthesis catalyzed by endothelial nitric oxide synthase.

High-Performance Liquid Chromatographic Method for The Separation of Enantiomeric Gatifloxacin

A high-performance liquid chromatographic method has been developed in normal-phase conditions for the separation of enantiomeric gatifloxacin, (+/-) 1-cyclopropyl-6-fluoro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-quinoline-3-carboxylic acid, an antibiotic in bulk drug. The method involved the use of an amylose-based Chiralpak AD-H (150 mm x 4.6 mm, 5 microm) column using a mobile phase system containing n-hexane-ethanol-diethylamine (85:15:0.1% v/v). The conditions affording the best resolution were found by selection and variation of the mobile-phase compositions and the differences in separation capability of the method is noted. Relative standard deviation of retention times and peak areas were better than 0.2% and 0.4%, respectively, for precision. Gatifloxacin sample solution and mobile phase are found to be stable for at least 48 h.

A Validated Chiral LC Method for the Enantiomeric Separation of Nateglinide and the Quantitative Determination of Its l-Enantiomer

A simple and accurate chiral liquid chromatographic method was developed for the enantiomeric purity determination of d-nateglinide and quantitative determination of l-nateglinide in bulk drug samples. Good resolution (Rs > 6.0) between d-enantiomer and l-enantiomer of nateglinide were achieved with Chiralpak AD-H (250 × 4.6 mm, 5 μm particle size) column using hexane and ethanol (90:10 v/v) as mobile phase at 25 °C temperature. Flow rate was kept as 1.0 mL min−1 and elution was monitored at 210 nm. The effects of the mobile phase composition, the flow rate and the temperature on the chromatographic separation were investigated. Developed method is capable to detect (LOD) and quantitate (LOQ) l-nateglinide to the levels of 0.3 and 1.0 μg mL−1 respectively, for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of l-nateglinide at LOQ concentration was 5.2. The percentage recoveries of l-nateglinide from d-nateglinide ranged from 97.9 to 99.7. The test solution and mobile phase was found to be stable up to 24 h after preparation. The developed method was validated with respect to LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.

Enantiomeric Separation of Docetaxel Starting Material by Chiral LC Using Amylose Based Stationary Phase

The present work narrates an LC method for determination of enantiomeric purity of (4S,5R)-3,5-oxazolidinedicarboxylic acid 2,2-dimethyl-4-phenyl-3-(1,1-dimethylethyl) ester, which is a key starting material of docetaxel, an antineoplastic agent and belongs to the taxoid family. Resolution between enantiomers was achieved ≥4.0 within 30 min on Chiralpak AD-H (250 mm × 4.6 mm) column using n-hexane, 2-propanol, ethanol and trifluoroacetic acid (97.5, 1.5, 1.0, 0.15 v/v) as mobile phase at a flow rate of 1.0 mL min−1 at 25 °C. The detection was made at 215 nm using UV detection. The effects of 2-propanol, ethanol and temperature on enantioselectivity and resolution of enantiomers were evaluated. The method was validated for (4R,5S)-enantiomeric impurity in terms of accuracy, precision and linearity in the range of 0.5–5 μg mL−1 and the correlation coefficient was >0.999. The recoveries were 93.2–104.4%. The limits of detection (LOD) and quantification (LOQ) of unwanted (4R,5S)-enantiomer was 0.1 and 0.5 μg mL−1. The method was found to be suitable for rapid quantification of (4R,5S)-enantiomer of docetaxel starting material.

Direct Chiral Resolution and its Application to the Determination of the Pesticide Tetramethrin in Soil by High-Performance Liquid Chromatography Using Polysaccharide-Type Chiral Stationary Phase

A high-performance liquid chromatography method is presented for the enantioseparation and quantitation of tetramethrin. The separation is achieved on amylose 3,5-dimethylphenyl-carbamate CSP (Chiralpak AD-H column) using a mobile phase consisting of a mixture of n-hexane-ethanol-2-propanol (99:0.9:0.1, v/v/v). Baseline chiral separation for the four isomers of tetramethrin is obtained within 20 min. Each of the resolutions of the two pairs of enantiomers is more than 2.0. The absolute configurations of each isomer are determined. The accuracy, precision, linearity, and limits of detection and quantitation of the method are investigated. For the determination of isomers in soil, the soil sample is extracted with acetone in an ultrasonic bath. The percentage recoveries from soil are in the range of 73.5% to 87.9%.

Supercritical fluid chromatography comparison of the poly( trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases

The poly( trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1′-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.

Separation of maxi-K channel opening 3-substitued-4-arylquinolinone atropisomers by enantioselective supercritical fluid chromatography

Many 3-substitued-4-arylquinolinones containing an ortho substituent on the aryl ring were known as a class of compounds with maxi-K opening activity. These quinolinones, which contained a stereogenic axis in their structures due to their bulky ortho substituents on the two aryl rings, exhibited atropisomerism. The rotationally hindered atropisomers could have differential biological and pharmacological activity, and it was highly desirable to separate them and test the individual atropisomers in biological assays. To explore the potential of supercritical fluid chromatography (SFC) to separate the atropisomers of this class of compounds, six 3-substitued-4-arylquinolinones with various hydrophilic and hydrophobic substituents in various positions were screened using three alcoholic modifiers (methanol, ethanol and 2-propanol) with four polysaccharide-based chiral stationary phases (Chiralpak AD-H and AS-H, Chiralcel OD-H and OJ-H). Our results showed that all six compounds studied were successfully resolved under multiple SFC conditions regardless of their structural differences and polarity. The majority of the separations were completed within 10 min. The Chiralpak AD-H column appeared to be superior to the other three chiral columns, and methanol and ethanol showed higher successful rate than 2-propanol in separating atropisomers of this class of compounds. These SFC methods were efficient and easily scalable for preparative separation. Thus, SFC was found to be the methodology of choice for resolving the atropisomers of this class of compounds.

A Validated LC Method for the Determination of Chiral Purity of (1S)-6,11-Dioxo-1,2,3,4,6,11-Hexahydropyridazino[1,2-b]Phthalazine-1-Carboxylic Acid: A Key Intermediate of Cilazapril

A simple and accurate normal phase liquid chromatographic method was developed for the determination of chiral purity of (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, S-enantiomer used as key intermediate in the manufacturing of cilazapril bulk drug. Chromatographic separation between (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, and its opposite enantiomer (1R)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, R-enantiomer was achieved using a Chiralpak AD-H column using a mobile phase containing hexane, isopropyl alcohol and tri-fluoro acetic acid (80:20:0.1 v/v/v). The resolution between the two enantiomers was found to be more than 3.2. The limit of detection (LOD) and limit of quantitation (LOQ) of the R-enantiomer was 0.15 and 0.5 μg mL−1, respectively, for 10 μL injection volume. The percentage recoveries of the R-enantiomer ranged from 96.5 to 105.3 in the bulk samples of (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid. The test solution and mobile phase was observed to be stable up to 24 h after the preparation. The developed method was validated as per International Conference on Harmonization guidelines in terms of LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.

Development and implementation of a stereoselective normal-phase liquid chromatography–tandem mass spectrometry method for the determination of intrinsic metabolic clearance in human liver microsomes

The stereoselective determination of stereoisomers in biological samples provides vital information on stereospecific metabolism and pharmacokinetic profiles of the drugs. Despite the unique advantage and the great success of normal-phase (NP) HPLC for the separations of drug stereoisomers using polysaccharide-type chiral stationary phases (CSPs), the technique is rarely applied to quantitative HPLC–MS–MS bioanalysis. This is, at least in part, due to the incompatibility between the usual mobile phase ( n-hexane or n-heptane) in normal-phase HPLC and the MS ionization sources which poses a potential detonation hazard. An environmentally friendly and nonflammable alternative solvent, ethoxynonafluorobutane (ENFB), was reported previously to potentially provide an ideal solution for combining the powers of stereoselective NP chromatographic separation and MS–MS detection. In this study, a stereoselective NP-HPLC–MS–MS method was developed using ENFB to quantify a pair of Bristol Myers Squibb (BMS) proprietary drug stereoisomers and their ketone metabolite for an in vitro study, which demonstrated, for the first time, the practical applicability and utility of ENFB for bioanalysis in pharmaceutical industry. The effects of different organic modifiers and temperature, as well as the comparison between ENFB and the usual solvent, heptane, for the separation, are discussed. The resolution of the stereoisomers was achieved using 63% of 3:1 mixture of ethanol and methanol with 37% ENFB on a Chiralpak AD-H column at 50 °C. High sensitivity was obtained using the MS–MS detection in the positive ion atmospheric pressure chemical ionization (APCI) mode. The lower limit of quantitation (LLOQ) for the first stereoisomer and the ketone metabolite was 5 ng/mL, and was 10 ng/mL for the second isomer in the human liver microsome–potassium phosphate buffer matrix. The linear dynamic range of 5–1000 ng/mL for both isomers and 10–1000 ng/mL for the metabolite were demonstrated with R 2 ≥ 0.997. The precision of the analysis was <5% R.S.D. at or above the nominal concentration of 80 ng/mL, and <20% R.S.D. at 8 ng/mL. The mean bias was less than 15%. Extraction recovery and acceptable matrix interference were demonstrated using one isomer and the ketone, and better than 75% recovery and less than 25% ion suppression or interference were found. The method was successfully implemented for an in vitro intrinsic metabolic clearance study.

Separation of the Two Enantiomers of T-3811ME by Normal-Phase HPLC Using Modified Amylose as Chiral Stationary Phase

A normal-phase enantioselective high-performance liquid chromatographic method is developed for the separation of the undesired enantiomer from T-3811ME and quantitaion of the undesired enantiomer at low levels using a Chiralpak AD-H column (4.6 x 150 mm) packed with modified amylose stationary phase. The 2% water-modified 2-propanol is used for the method development activities, including exploration of various organic modifiers, optimization of additive acid concentration, column screening, and column temperature optimization. The final optimized method separated the undesired enantiomer from T-3811ME and is proven to be robust, sensitive, linear, accurate, and precise.

Verification of the need for optical purity measurement of chiral pesticide standards as agricultural reference materials

In this study, optical purity measurement was performed on eight kinds of commercially available pesticide, namely, Dimethenamid-P, Dichlorprop-P, Fluazifop-P butyl, Indoxacarb, Metalaxyl-M, Mecoprop-P, Quizalofop-P ethyl, and Uniconazole-P, in both enantiomer and racemate forms. Chiral separation of each pesticide was achieved by using HPLC with a photodiode-array (PDA) detector and a circular dichroism (CD) detector, which were connected in series. The chiral column used for the enantiomeric separation was a normal phase Chiralpak AD-H column. The mobile phase was n-hexane, with various alcohols added as polar modifiers. The study included investigation of the effects on enantiomeric separation of the percentage of alcohol used, the presence or absence of acid in the mobile phase, and the column temperature, and the optimum detection wavelengths of both PDA and CD detectors. Enantiomeric excess (ee) was calculated as an expression of optical purity. As a result, the ee of some pesticides investigated was approximately (over 95%) in accord with the data provided by manufacturers. However, the ee of Indoxacarb, Uniconazole-P, Quizalofop-P ethyl, and Fluazifop-P butyl was in the range 34.1–94.5%. These results suggest that there is a need to conduct optical purity tests, in addition to a chemical purity test, for optically active pesticides.

Simultaneous chromatographic separation of enantiomers, anomers and structural isomers of some biologically relevant monosaccharides

A one-step chiral high-performance liquid chromatography (HPLC) method was developed to separate anomers and enantiomers of some carbohydrates—glucose, fructose, arabinose, ribose, fucose, mannose, lyxose and xylose, using a Chiralpak AD-H column. The method allows the carbohydrate identification and determination of the absolute configuration ( d or l) and simultaneously also determine the configuration of the anomeric center (α or β) of the monosaccharide. The method was applied to matrices involved in food chain and human health, as part of a general strategy for the structure determination of their constituents, due to the extreme importance of sugars since they are involved in food authenticity and nutritional characteristics and the biological role depends on the enantiomer and the anomeric form of a given monosaccharide.

A Validated chiral LC Method for the Enantiomeric Separation of Galantamine

A rapid isocratic chiral HPLC method has been developed for the separation of R-galantamine from S-galantamine. Good resolution viz. Rs > 3 between R- and S- forms of galantamine was achieved with chiralpak AD-H (250 × 4.6 mm) column using n-hexane, 20% propionic acid in isopropanol and diethyl amine in the ratio of 80:20:0.2 (v/v) as mobile phase at ambient temperature. Flow rate was kept as 0.8 mL min−1 and the elution was monitored at 289 nm. This method was further used to determine the amount of R-galantamine in pure and pharmaceutical formulations of S-galantamine. This method is capable to detect and quantitate R-galantamine to the levels of 0.21 and 0.84 μg mL−1, respectively. The method was validated as per International Conference on Harmonization (ICH) guidelines in terms of limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy, specificity, robustness and ruggedness.

Comparison of Atmospheric Pressure Photoionization and Atmospheric Pressure Chemical Ionization for Normal-Phase LC/MS Chiral Analysis of Pharmaceuticals

In this work, we compared APPI and APCI for normal-phase LC/MS chiral analysis of five pharmaceuticals. Performance was compared both by FIA and by on-column analysis using a ChiralPak AD-H column under optimized conditions. By comparison, APPI generated more reproducible signals and was less susceptible to ion suppression than APCI. APPI generated higher peak area and lower baseline noise, and therefore much higher S/N ratios. APPI sensitivity (i.e., S/N ratio) was approximately 2-130 times higher than APCI by FIA and was approximately 2.6-530 times higher than APCI by on-column analysis depending on specific compounds. The better APPI sensitivity as compared to APCI was more dramatic by on-column analysis than by FIA. APCI sensitivity was degraded by ion suppression caused by LC column bleeding components and by elevated APCI baseline noise relative to APPI. On-column APPI LODs (at S/N = 3) were 83, 16, 17, 95, and 7 pg for enantiomer #1, and 104, 23, 19, 122, and 17 pg for enantiomer #2 for benzoin, naringenin, mianserin, mephenesin, and diperodon, respectively, on a Waters ZQ. APPI offers no concern of explosion hazard relative to APCI corona needle discharge or ESI high voltage discharge when flammable solvents (e.g., hexane) are used as mobile phases. Whether APPI dopants are required depends on the IP(s) of mobile-phase solvent(s) and solvent complexes, and photon energies of VUV lamps. Dopant was not necessary for hexane-based mobile phases due to their self-doping effects. Dopants did enhance Kr lamp APPI sensitivity when MeOH was used as the mobile phase. However, dopants became unnecessary for the MeOH mobile phase when the Ar lamp was used.

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 ( r 2 > 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.

Chiral separation of β‐blocker drug nadolol by HPLC – A kinetic and equilibrium study

A Chiralpak AD-H column packed with amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel was used to study the enantioseparation of nadolol by HPLC. The bed voidage, axial dispersion coefficient, overall mass transfer coefficients as well as equilibrium constants for the chromatographic enantiomeric separation were evaluated by moment analysis on the basis of the solid film linear driving force model. The equilibrium constants were found to be 3.81, 5.24, 9.45 and 19.41 for the stereoisomers (SRS)-, (SRR)-, (RSS)- and (RSR)-nadolol, respectively. Their overall mass transfer coefficients were found to be 1841.8, 1254.8, 799.4 and 401.7 min(-1) respectively. Temperature effect on the enantiomeric separation and thermodynamic properties including enthalpy and entropy change of binding to the amylose tris(3,5-dimethylphenylcarbamate) stationary phase were also investigated. The moment analysis and the parameters obtained were used to simulate nadolol elution profiles. The simulated results matched the experimental profiles well, which confirmed the validity of model parameters obtained in this study.

Chiral separation of selected proline derivatives using a polysaccharide type stationary phase by high-performance liquid chromatography

Proline derivatives, such as Boc-proline, Boc-2-methylproline, Boc-2-methylproline benzyl ester and Boc-2-methyl-4-hydroxy-proline benzyl ester, have been widely used as a building block leading to a variety of pharmaceutical compounds. Therefore, there is a wide interest in the chiral separation of these compounds. High-performance liquid chromatography (HPLC) methods were developed using a Chiralpak AD-H column to separate enantiomers of these proline derivatives. The effect of mobile phase composition and column temperature was studied. For the proline derivatives studied in this work, good resolution was achieved using a mobile phase composition of hexane, ethanol and 0.1% TFA. For prolines containing carboxyl or hydroxy group, resolution was changed dramatically corresponding to changes as little as 1% of ethanol in the mobile phase, suggesting that the dominant chiral recognition is from hydrogen bonding interactions. On the other hand, for prolines containing a benzyl ester instead of hydroxy group next to the chiral center, resolution was not affected as significantly with the changes of ethanol content in the mobile phase, indicating a different leading chiral recognition mechanism, such as inclusion, steric effect, or possible π–π interaction. Linearity, precision and limit of detection were also measured for Boc-2-methylproline and Boc-2-methylproline benzyl ester.