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.     

A novel stability-indicating HPLC method for the determination of enantiomeric purity of eluxadoline drug: Amylose tris(3,5-dichlorophenyl carbamate) stationary phase

A simple and sensitive stability-indicating chiral HPLC method has been developed and validated per International Conference on Harmonization guidelines for the determination of enantiomeric purity of eluxadoline (Exdl). The impact of different mobile phase compositions and chiral stationary phases on the separation of Exdl enantiomer along with process- and degradation-related impurities has been studied. Homogeneity of Exdl and stable results of Exdl enantiomer in all degraded samples reveal the fact that the proposed method was specific (stability indicating). Amylose tris(3,5-dichlorophenyl carbamate) stationary phase column Chiralpak IE-3 (150 × 4.6 mm, 3 μm) provided better resolution with polar organic solvents than cellulose derivative, crown ether, and zwitterion stationary phases and nonpolar solvents. The mobile phase consisted of acetonitrile, tetrahydrofuran, methanol, butylamine, and acetic acid in the ratio of 500:500:20:2:1.5 (v/v/v/v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min, column temperature of 35°C, injection volume of 10 μL, and UV detection of 240 nm. The United States Pharmacopeia (USP) resolution of the Exdl enantiomer was found to be more than 4.0 within a 65-min run time. Exdl enantiomer detector response linearity over the concentration range of 0.859–4.524 μg/mL was found to be R² = 0.9985. The limit of detection, limit of quantification, and average percentage recovery values were established as 0.283 μg/mL, 0.859 μg/mL, and 96.0, respectively.     

Preparative enantioseparation of loxoprofen precursor by recycling countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as a chiral selector

Recycling countercurrent chromatography was successfully applied to the resolution of 2‐(4‐bromomethylphenyl)propionic acid, a key synthetic intermediate for synthesis of nonsteroidal anti‐inflammatory drug loxoprofen, using hydroxypropyl‐β‐cyclodextrin as chiral selector. The two‐phase solvent system composed of n‐hexane/n‐butyl acetate/0.1 mol/L citrate buffer solution with pH 2.4 (8:2:10, v/v/v) was selected. Influence factors for the enantioseparation were optimized, including type of substituted β‐cyclodextrin, concentration of hydroxypropyl‐β‐cyclodextrin, separation temperature, and pH of aqueous phase. Under optimized separation conditions, 50 mg of 2‐(4‐bromomethylphenyl)propionic acid was enantioseparated using preparative recycling countercurrent chromatography. Technical details for recycling elution mode were discussed. The purities of both the S and R enantiomers were over 99.0% as determined by high‐performance liquid chromatography. The enantiomeric excess of the S and R enantiomers reached 98.0%. The recovery of the enantiomers from eluted fractions was 40.8–65.6%, yielding 16.4 mg of the S enantiomer and 10.2 mg of the R enantiomer. At the same time, we attempted to enantioseparate the anti‐inflammatory drug loxoprofen by countercurrent chromatography and high‐performance liquid chromatography using a chiral mobile phase additive. However, no successful enantioseparation was achieved so far.     

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.     

Stereoselective separation of (1S, 4S)‐sertraline from medicinal reaction mixtures by countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as stereoselective selector

Four stereoisomeric components were produced during the synthesis of the antidepressant drug (1S, 4S)‐sertraline hydrochloride due to the two chiral carbon centers in its chemical structure, including (1S, 4S), (1R, 4R), (1S, 4R), and (1R, 4S)‐isomer. Stereoselective separation of the target isomer (1S, 4S)‐sertraline from the medicinal reaction mixtures by countercurrent chromatography using hydroxypropyl‐β‐cyclodextrin as the stereoselective selector was investigated. A biphasic solvent system composed of n‐hexane/0.20 mol/L phosphate buffer solution with pH 7.6 containing 0.10 mol/L of hydroxypropyl‐β‐cyclodextrin (1:1, v/v) was selected for separation of cis‐sertraline and trans‐sertraline using reverse phase elution mode and (1S, 4S)‐sertraline was separated with (1R, 4R)‐sertraline using recycling elution mode. A fabricated in‐house analytical countercurrent chromatographic apparatus was used for optimization of the separation conditions. Stationary phase retention and peak resolution were investigated for separation of cis‐sertraline and trans‐sertraline by the analytical apparatus.     

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.     

High‐performance liquid chromatographic enantioseparation of (RS)‐bupropion using isothiocyanate‐based chiral derivatizing reagents

A high‐performance liquid chromatographic (HPLC) method for enantioseparation of bupropion was developed using two isothiocyanate‐based chiral derivatizing reagents, (S)‐1‐(1‐naphthyl) ethyl isothiocyanate, (S)‐NEIT, and (R)‐α‐methyl benzyl isothiocyanate, (R)‐MBIT. The diastereomers synthesized with (S)‐NEIT were enantioseparated by reversed‐phase HPLC using gradient elution with mobile phase containing water and acetonitrile, whereas diastereomers synthesized with (R)‐MBIT were enantioseparated using triethyl amine phosphate buffer and methanol. Derivatization conditions were optimized and the method was validated for accuracy, precision and limit of detection. The limit of detection was found to be 0.040–0.043 µg/mL for each of the diastereomers prepared with (S)‐NEIT. Copyright © 2013 John Wiley & Sons, Ltd.     

HPLC法拆分(-)-2(S)-苄基-4-酮-4-(顺式-全氢化异吲哚-2-基)丁酸钙对映体

目的：建立刺激胰岛素分泌的新型降糖药物(-)-2 (S)-苄基-4-酮-4-(顺式-全氢化异吲哚-2-基)丁酸钙对映体的HPLC拆分方法。方法：采用Sumichiral OA-3300手性柱(250 × 4.6 mm I.D., 5 μm), 柱温35℃,以0.05 mol·L-1醋酸铵的甲醇溶液为流动相,检测波长为210 nm。结果：本品两对映体在22分钟内实现良好分离,分离度达3以上,S－异构体分别在0.028 ~ 5.6 μg mL-1和0.03 ~ 6.0 μg mL-1范围内线性关系良好,回归方程分别为：Y=1.32×103x-2.54 (r=0.9997)和Y=1.15×103x-1.78 (r=0.9998),最低检测限分别为0.15 ng和0.10 ng,方法精密度RSD低于1.0% (n=5)。结论：建立的对映体分离方法可用于本品光学异构体的质量控制。     

Determination of quinocide as impurity in primaquine tablets by capillary zone electrophoresis

A capillary zone electrophoretic method has been developed and validated for the determination of the impurity quinocide (QC) in the antimalarial drug primaquine (PQ). Different buffer additives such as native cyclodextrins and crown ethers were evaluated. Promising results were obtained when either β‐cyclodextrin (β‐CD) or 18‐crown‐6 ether (18C6) were used. Their separation conditions such as type of buffer and its pH, buffer additive concentration, applied voltage capillary temperature and injection time were optimized. The use of 18C6 offers slight advantages over β‐CD such as faster elution times and improved resolution. Nevertheless, migration times of less than 5 min and resolution factors (R_s) in the range of 2–4 were obtained when both additives were used. The method was validated with respect to selectivity, linearity, limits of detection and quantitation, analytical precision (intra‐ and inter‐day variability) and repeatability. Concentrations of 2.12 and 2.71% (w/w) of QC were found in pharmaceutical preparations of PQ from two different manufacturers. A possible mechanism for the successful separation of the isomers is also discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Development of a high‐performance liquid chromatography method for the simultaneous determination of chiral impurities and assay of (S)‐clopidogrel using a cellulose‐based chiral stationary phase in methanol/water mode

A simple reversed‐phase high‐performance liquid chromatography method for the chiral separation of the active pharmaceutical ingredient (S)‐clopidogrel has been developed on the cellulose‐based Chiralcel OJ‐RH chiral stationary phase. The S enantiomer was baseline resolved from its R impurity (impurity C) with a mobile phase consisting of methanol/water (100:15) without any interference coming from the other two potential chiral impurities A and B. The enantio‐ and chemoselective method was partially validated and compared with that reported in the United States Pharmacopoeia for the drug product. The versatility of the Chiralcel OJ‐RH allowed separating the enantiomers of the impurity B also under normal phase and setting up an efficient strategy to convert the racemic sample into the enantiomeric S form on a semipreparative scale.     

Separation and determination of polyurethane amine catalysts in polyether polyols by using UHPLC–Q‐TOF‐MS on a reversed‐phase/cation‐exchange mixed‐mode column

A simple, selective, and accurate ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was established and validated for the efficient separation and quantification of polyurethane amine catalysts in polyether polyols. Amine catalysts were primarily separated in polyether polyol‐based sample by solid‐phase extraction, and further baseline separated on a reversed‐phase/cation‐exchange mixed‐mode column (SiELC Primesep™ 200) using 0.1% trifluoroacetic acid/acetonitrile as a mobile phase in gradient elution mode at a flow rate of 0.2 mL/min. High‐resolution quadrupole time‐of‐flight mass spectrometry analysis in electrospray ionization positive mode allowed the identification as N,N′‐bis[3‐(dimethylamino)propyl]urea, N‐[2‐(2‐dimethylaminoethoxy)ethyl]‐N‐methyl‐1,3‐propanediamine, and N,N,N′,N′‐tetramethyldipropylenetriamine. The method was validated and presented good linearity for all the analytes in blank matrices within the concentration range of 0.20–5.0 or 0.1–2.0 μg/mL with the correlation coefficients (R²) ranging from 0.986 to 0.997. Method recovery ranged within 81–105% at all three levels (80, 100, and 120% of the original amount) with relative standard deviations of 1.0–6.2%. The limits of detection were in the range of 0.007–0.051 μg/mL. Good precision was obtained with relative standard deviation below 3.2 and 0.72% for peak area and retention time of three amines, respectively.     

Development and validation of a capillary electrophoresis method for determination of enantiomeric purity and related substances of esomeprazole in raw material and pellets

A capillary electrophoresis method using CDs for quality control of esomeprazole (ESO) in terms of enantiomeric purity and related substances in raw material and pellets was developed. ESO is the S‐enantiomer of omeprazole (OMZ). Several parameters were evaluated, including type and concentration of buffer and CD, concentration of additives and electrolyte pH. Resolution between the enantiomers of OMZ obtained for each parameter tested was calculated and the presence of the main related substance such as OMZ sulfone was carefully monitored. The optimized system consisted of 100 mM Tris‐phosphate buffer pH 2.5 with 20 mM 2‐hydroxypropyl‐β‐CD, 1 mM sodium dithionite, temperature at 15°C, voltage at 28 kV, and UV detection at 301 nm. Once optimized, the electrophoretic system was validated according to ICH guidelines. The limits of detection and quantification for R‐OMZ were 0.6 μg/mL (0.06% w/w of ESO) and 2.0 μg/mL (0.2% w/w of ESO), respectively. A mean concentration of R‐OMZ <0.2% limit established by the United States Pharmacopeia (USP) was found in the raw material and six‐pellet samples of ESO. No other impurities were found in the samples under these conditions. Therefore, the developed method was found to be appropriate not only for enantiomeric quality control of ESO but also for the analysis of ESO and the main related substance in raw material and pharmaceutical formulations as well as for stability indicating studies.     

Rapid quantification of amlodipine enantiomers in human plasma by LC‐MS/MS: application to a clinical pharmacokinetic study

A rapid, simple, specific and sensitive LC‐MS/MS method has been developed and validated for the enantiomeric quantification of amlodipine (AML) isomers [R‐amlodipine (R‐AML) and S‐amlodipine (S‐AML)] with 200 μL of human plasma using R‐AML‐d₄ and S‐AML‐d₄ as corresponding internal standards as per regulatory guidelines. A simple liquid–liquid extraction process was used to extract these analytes from human plasma. The total run time was 3.5 min and the elution of R‐AML, S‐AML, R‐AML‐d₄ and S‐AML‐d₄ occurred at 1.62, 2.51, 1.63 and 2.53 min, respectively. This was achieved with a mobile phase consisting of 0.2% ammonia–acetonitrile (20:80, v/v) at a flow rate of 1 mL/min on a Chiralcel OJ RH column. A linear response function was established for the range of concentrations 0.1–10 ng/mL (r >0.998) for each enantiomer. The intra‐ and inter‐day precision values for both enantiomers met the acceptance criteria. Both enantiomers were stable in a set of stability studies, viz. bench‐top, auto‐sampler, freeze–thaw cycles and long‐term. The current assay was successfully applied to a pharmacokinetic study to quantitate AML enantiomers following oral administration of 10 mg AML tablet to humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of an ion‐exchange chromatography method for heparin and its impurities in heparin products

An anion‐exchange liquid chromatography method for the determination of heparin and its impurities (dermatan sulfate and oversulfated chondroitin sulfate) was developed using chemometric‐assisted optimization, including multivariate experimental design and response surface methodology. The separation of heparin, dermatan sulfate, and oversulfated chondroitin sulfate (R_s above 2.0) was achieved on a Dionex RF IC IonPac AS22 column with a gradient elution of 10–70% of 2.5 M sodium chloride and 20 mM Tris phosphate buffer (pH 2.1) at a flow rate of 0.6 mL/min and UV detection at 215 nm. Method validation shows good linearity (r > 0.99), acceptable precision (%relative standard deviations <11.4%) and trueness (%recovery of 92.3–103.9%) for all analytes. The limits of detection for dermatan sulfate and oversulfated chondroitin sulfate are equivalent to 0.11% w/w (10.5 μg/mL) and 0.07% w/w (7.2 μg/mL), while the limits of quantification are 0.32% w/w (31.5 μg/mL) and 0.22% w/w (22.0 μg/mL) relative to heparin, respectively. The method is specific for heparin, dermatan sulfate, and oversulfated chondroitin sulfate without interference from mobile phase and sample matrices and could be used for accurate quantitation the drug and its impurities in a single run. Applications of the method reveal contents of heparin between 90.3 and 97.8%. Dermatan sulfate and oversulfated chondroitin sulfate were not detected in any of the real‐life samples.     

Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl‐γ‐cyclodextrin‐modified micellar electrokinetic chromatography

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl‐γ‐cyclodextrin (HP‐γ‐CD)‐modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate–15 mM borate buffer (pH 9.0) containing 30 mM HP‐γ‐CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)‐ and (R)‐enantiomers of regioisomeric 8‐, 11‐, 12‐, and 15‐HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8‐ and 12‐HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8‐HETE was found to be a mixture of 98% (R)‐enantiomer and 2% (S)‐enantiomer, while the 12‐HETE was a mixture of 98% (S)‐enantiomer and 2% (R)‐enantiomer. The present study demonstrates that the HP‐γ‐CD‐modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs.     

Direct separation and quantitative determination of glimepiride isomers by high performance liquid chromatography

A simple and sensitive high‐performance liquid chromatographic procedure for the determination of the trans isomer of glimepiride is reported. Chromatography accomplished direct separation of the cis and trans isomers of glimepiride on a Dikmonsil C18 (250×4.6 mm, 5 μm) column with a mobile phase consisting of methanol‐acetonitrile‐NH₄Ac buffer solution (1.5 mol L^–1, pH = 4.5) (1.1 : 1.3 : 1.0, v/v) at a flow rate 0.5 mL min^–1. The resolution (R_S) was 1.73 with a retention time of 24.885 and 23.018 min for the cis and the trans isomer, respectively. A standard linear calibration curve was established for the trans isomer of glimepiride over the range of 4.95–198.00 μg mL^–1 with a correlation coefficient of 0.99997. This method has been successfully used to analyze four different kinds of glimepiride product.     

A Validated LC Method for Determination of the Enantiomeric Purity of Montelukast Sodium in Bulk Drug Samples and Pharmaceutical Dosage Forms

A new and accurate chiral liquid chromatographic method has been developed for determination of the enantiomeric purity of montelukast sodium (R enantiomer) in bulk drugs and dosage forms. Normal phase chromatographic separation was performed on an immobilized amylose-based chiral stationary phase with n-hexane–ethanol–1,4-dioxane–trifluoroacetic acid–diethylamine 65:25:10:0.3:0.05 (v/v) as mobile phase at a flow rate of 1.0 mL min^?1. The elution time was approximately 15 min. The resolution (R _S) between the enantiomers was >3. The mobile phase additives trifluoroacetic acid and diethylamine played a key role in achieving chromatographic resolution between the enantiomers and also in enhancing chromatographic efficiency. Limits of detection and quantification for the S enantiomer were 0.07 and 0.2 μg, respectively, for a test concentration of montelukast sodium of 1,000 μg mL^?1 and 10 μL injection volume. The linearity of the method for the S enantiomer was excellent (R ² > 0.999) over the range from the LOQ to 0.3%. Recovery of the S enantiomer from bulk drug samples and dosage forms ranged from 97.0 to 103.0%, indicative of the high accuracy of the method. Robustness studies were also conducted. The sample solution stability of montelukast sodium was determined and the compound was found to be stable for a study period of 48 h.     

A Validated LC Method for Separation and Quantification of Voriconazole and Its Enantiomer

A simple liquid chromatographic method was developed for the separation and quantification of voriconazole and its enantiomer in drug substance. The separation was achieved on Chiral cel-OD (250 mm × 4.6 mm × 10 μm) using mobile phase consisting of n-hexane and ethanol in the ratio 9:1 (v/v) with a flow rate of 1.0 mL min⁻¹, at 27 °C column temperature and detection at 254 nm with an injection volume of 20 μL. Ethanol was used as diluent. The method is capable of detecting the (2S, 3R) enantiomer down to 0.0075% and can quantify down to 0.021% with respect to sample concentration. The method is rapid and the resolution achieved was about 3.0. This method can be employed for the quantification of (2S, 3R) enantiomer in voriconazole drug substance.     

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.     

(RS)‐Propranolol: enantioseparation by HPLC using newly synthesized (S)‐levofloxacin‐based reagent,absolute configuration of diastereomers and recovery of native enantiomers by detagging

Diastereomers of (RS)‐propranolol were synthesized using (S)‐levofloxacin‐based new chiral derivatizing reagents (CDRs). Levofloxacin was chosen as the pure (S)‐enantiomer for its high molar absorptivity (ε_o ～ 24000) and availability at a low price. Its ‐COOH group had N‐hydroxysuccinimide and N‐hydroxybenzotriazole, which acted as good leaving groups during nucleophilic substitution by the amino group of the racemic (RS)‐propranolol; the CDRs were characterized by UV, IR, ¹H‐NMR, high resolution mass spectrometry (HRMS) and carbon, hydrogen, nitrogen, and sulphur fundamental elemental components analyser (CHNS). Diastereomers were separated quantitatively using open column chromatography; absolute configuration of the diastereomers was established and the reagent moiety was detagged under microwave‐assisted acidic conditions. (S)‐ and (R)‐propranolol as pure enantiomers and (S)‐levofloxacin were separated, isolated and characterized. Optimized lowest‐energy structures of the diastereomers were developed using Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6‐31G* basis set (based on density functional theory) for explanation of elution order and configuration. In addition, RP HPLC conditions for separation of diastereomers were optimized with respect to pH, concentration of buffer, flow rate of mobile phase and nature of organic modifier. HPLC separation method was validated as per International Conference on Harmonization guidelines. With the systematic application of various analytical techniques, absolute configuration of the diastereomers (and the native enantiomers) of (RS)‐propranolol was established. Copyright © 2016 John Wiley & Sons, Ltd.