Enantioresolution of Chiral Derivatives of Xanthones on (S,S)‐Whelk‐O1 and l‐Phenylglycine Stationary Phases and Chiral Recognition Mechanism by Docking Approach for (S,S)‐Whelk‐O1

The resolution of seven enantiomeric pairs of chiral derivatives of xanthones (CDXs) on (S,S)‐Whelk‐O1 and l ‐phenylglycine chiral stationary phases (CSPs) was systematically investigated using multimodal elution conditions (normal‐phase, polar‐organic, and reversed‐phase). The (S,S)‐Whelk‐O1 CSP, under polar‐organic conditions, demonstrated a very good power of resolution for the CDXs possessing an aromatic moiety linked to the stereogenic center with separation factor and resolution factor ranging from 1.91 to 7.55 and from 6.71 to 24.16, respectively. The chiral recognition mechanisms were also investigated for (S,S)‐Whelk‐O1 CSP by molecular docking technique. Data regarding the CSP–CDX molecular conformations and interactions were retrieved. These results were in accordance with the experimental chromatographic parameters regarding enantioselectivity and enantiomer elution order. The results of the present study fulfilled the initial objectives of enantioselective studies of CDXs and elucidation of intermolecular CSP–CDX interactions. Chirality 25:89–100, 2013. © 2012 Wiley Periodicals, Inc.     

Enantioseparation of Four Organophosphonate Derivatives on N‐(3,5‐Dinitrobenzoyl)‐ l‐leucine–n‐Propylamide Stationary Phase by Molecular Modeling

Four groups of organophosphonate derivatives enantiomers were separated on N‐(3,5‐dinitrobenzoyl)‐S‐leucine chiral stationary phase. The three‐dimensional structures of the complexes between the single enantiotopic chiral compounds and chiral stationary phase have been studied using molecular model and molecular dynamics simulation. Detailed results regarding the conformation, auto‐docking, and thermodynamic estimation are presented. The elution order of the enantiomer could be determined from the energy. The predicted chiral discrimination was obtained by computational results. Chirality 25:101–106, 2013. © 2012 Wiley Periodicals, Inc.     

A 3D Homochiral MOF [Cd2(d‐cam)3]•2Hdma•4dma for HPLC Chromatographic Enantioseparation

Up to now, some chiral metal‐organic frameworks (MOFs) have been reported for enantioseparation in liquid chromatography. Here we report a homochiral MOF, [Cd2(d‐cam)3]·2Hdma·4dma, used as a new chiral stationary phase for high‐performance liquid chromatographic enantioseparation. Nine racemates of alcohol, naphthol, ketone, and base compounds were used as analytes for evaluating the separation properties of the chiral MOF packed column. Moreover, some effects such as mobile phase composition, column temperature, and analytes mass for separations on this chiral column also were investigated. The relative standard deviations for the resolution values of run‐to‐run and column‐to‐column were less than 2.1% and 3.2%, respectively. The experimental results indicate that the homochiral MOF offered good recognition ability, which promotes the application of chiral MOFs use as stationary phase for enantioseparation. Chirality 28:340–346, 2016. © 2016 Wiley Periodicals, Inc.     

Resolution,determination of enantiomeric purity and chiral recognition mechanism of new xanthone derivatives on (S,S)‐whelk‐O1 stationary phase

The enantioresolution and determination of the enantiomeric purity of 32 new xanthone derivatives, synthesized in enantiomerically pure form, were investigated on (S ,S )‐Whelk‐O1 chiral stationary phase (CSP). Enantioselectivity and resolution (α and R_S) with values ranging from 1.41–6.25 and from 1.29–17.20, respectively, were achieved. The elution was in polar organic mode with acetonitrile/methanol (50:50 v/v ) as mobile phase and, generally, the (R )‐enantiomer was the first to elute. The enantiomeric excess (ee ) for all synthesized xanthone derivatives was higher than 99%. All the enantiomeric pairs were enantioseparated, even those without an aromatic moiety linked to the stereogenic center. Computational studies for molecular docking were carried out to perform a qualitative analysis of the enantioresolution and to explore the chiral recognition mechanisms. The in silico results were consistent with the chromatographic parameters and elution orders. The interactions between the CSP and the xanthone derivatives involved in the chromatographic enantioseparation were elucidated.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

Resolution of (±)‐β‐methylphenylethylamine by a novel chiral stationary phase for Pirkle‐type column chromatography

In this study, a new Pirkle‐type chiral column stationary phase for resolution of β‐methylphenylethyl amine was described by using activated Sepharose 4B as a matrix, L ‐tyrosine as a spacer arm, and an aromatic amine derivative of L ‐glutamic acid as a ligand. The binding capacities of the stationary phase were determined at different pH values (pH = 6, 7, and 8) using buffer solutions as mobile phase, and enantiomeric excess (ee) was determined by HPLC equipped with chiral column. The ee was found to be 47%. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Superficially Porous Particle Based Hydroxypropyl‐β‐cyclodextrin Stationary Phase for High‐Efficiency Enantiomeric Separations

A superficially porous particle (SPP)‐based hydroxypropyl‐β‐cyclodextrin (HPBCD) chiral stationary phase (CSP) was produced and its chromatographic performance was compared to both 5 µm and 3 µm fully porous particle (FPP)‐based CSPs. The relative surface coverage of the HPBCD chiral selector on each particle was approximately equal, which resulted in equivalent enantiomeric selectivity (α) values on each phase when constant mobile phase conditions were used. Under such conditions, the SPP column resulted in greatly reduced analysis times and three times greater efficiencies compared to the FPP columns. When higher flow rates were used, efficiency gains per analysis times were five times greater for the SPP column compared to the FPP‐based columns. When the mobile phases were altered to give similar analysis times on each column, resolution values were doubled for the SPP column. Finally, the novel SPP based HPBCD column proved to be stable for 500 injections under high flow rate (4.5 mL/min) and high pressure (400 bar) conditions used for an ultrafast (~45 sec) enantiomeric separation. Chirality 27:788–794, 2015. © 2015 Wiley Periodicals, Inc.     

Chiral separation and modeling of baclofen,bupropion, and etodolac profens on amylose reversed phase chiral column

Chiral resolution of baclofen, bupropion, and etodolac profens was obtained with amylose derivatized chiral reversed stationary phase (carbamate groups). The eluent used for bupropion and etodolac was MeOH–water (20:80, v /v) and for baclofen was water–methanol (95:5, v /v). The eluent run rates, finding wavelength and temperature, were 1.0 mL/min, 220 nm and 27 ± 1 °C for all the eluents. The magnitude of the retardation factors for S‐ and R‐enantiomers of baclofen, bupropion, and etodolac were 1.37, 2.62, 2.25, 3.25, 1.8, and 3.0. The magnitudes of separation and resolution factors were 1.90, 1.44, and 1.67 and 2.77, 2.35, and 2.04. Limits of detection and quantitation were 1.0–2.0 and 5.1–10.0 μg/mL. Chiral recognition mechanisms were recognized by simulation and high‐performance liquid chromatography (HPLC) experiments. It was seen that hydrogen interactions, hydrophobic interactions, and π–π exchanges were the chief interactions for chiral recognition mechanisms. The described methods may be exploited for the chiral separation of baclofen, bupropion, and etodolac profens in any unknown sample.     

Enantiomeric separation of β‐blockers and tryptophan using heparin as stationary and pseudostationary phases in capillary electrophoresis

The separation methods of the enantiomers of two β‐blockers and tryptophan were studied using capillary electrochromatography with heparin covalently as well as non‐covalently, bonded onto the capillary inner wall as stationary phase and electrokinetic chromatography with heparin as pseudostationary phase. In the case of heparin, used as a stationary phase, the method was unable to resolve enantiomers in both cases β‐blockers and tryptophan. On the other hand, when heparin was used as a pseudostationary phase, the resolution of the enantiomers was obtained only with 3‐aminopropyltriethoxysilane which were immobilised onto the inner phase of the capillary. The results of this study let us infer that the electrostatic, hydrophobic, and steric interactions were involved in the separation mechanisms. The separation was achieved in less than 10 minutes under the optimized conditions: 30 mM phosphate buffer (pH 2.5) with the adding of 15 mg/mL of heparin at 15°C and 10 kV. The usefulness of heparin as a chiral selector both in electrokinetic chromatography using 3‐aminopropyltriethoxysilane attached to the capillary was demonstrated for the first time. The developed method was powerful, sensitive, and fast, and it could be considered an important alternative to conventional methods used for chiral separation.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

Enantioseparation of napropamide by supercritical fluid chromatography: Effects of the chromatographic conditions and separation mechanism

Supercritical fluid chromatography (SFC) is already used for enantioseparation in the pharmaceutical industry, but it is rarely used for the separation of chiral pesticides. Comparing with high performence liquid chromatography, SFC uses much more environmnetal friendly and economic mobile phase, supercritical CO₂. In our work, the enantioseparation of an amide herbicide, napropamide, using three different polysaccharide‐type chiral stationary phases (CSPs) in SFC was investigated. By studying the effect of different CSPs, organic modifiers, temperature, back‐pressure regulator pressures, and flow rates for the enantioseparation of napropamide, we established a rapid and green method for enantioseparation that takes less than 2 minutes: The column was CEL2, the mobile phase was CO₂ with 20% 2‐propanol, and the flow rate was 2.0 mL/min. We found that CEL2 demonstrated the strongest resolution capability. Acetonitrile was favored over alcoholic solvents when the CSP was amylose and 2‐propanol was the best choice when using cellulose. When the concentration of the modifiers or the flow rate was decreased, resolutions and analysis times increased concurrently. The temperature and back‐pressure regulator pressure exhibited only minor influences on the resolution and analysis time of the napropamide enantioseparations with these chiral columns. The molecular docking analysis provided a deeper insight into the interactions between the enantiomers and the CSPs at the atomic level and partly explained the reason for the different elution orders using the different chiral columns.     

Enantioseparation of chiral perfluorooctane sulfonate (PFOS) by supercritical fluid chromatography (SFC): Effects of the chromatographic conditions and separation mechanism

Perfluorooctane sulfonate (PFOS) is one of the most frequently detected perfluoroalkyl substances in environmental and human samples. Previous studies have shown that nonracemic PFOS in biological samples can be used as a marker of PFOS exposure sources. In recent years, supercritical fluid chromatography (SFC) has emerged as a powerful method to separate chiral compounds. In this study, a method of perfluoro‐1‐methylheptane sulfonate (1 m‐PFOS) enantioseparation by SFC was established. The optimal separation was obtained using a Chiralpak QN‐AX column with CO₂/2‐propanol (70/30, v/v) as the mobile phase with a flow rate of 1 mL/min, column temperature was 32°C, and BPR pressure was 1800 psi. The resolution (Rs) and retention time were 0.88 and 130 minutes, respectively. This method is more economic and greener than HPLC. Modifier pH and column temperature were determined to be significant factors of SFC chiral separation. Modifier pH is negatively correlated with the retention factors and Rs. Adsorption thermodynamics were used to explain the influence of temperature change, and it was concluded that the transfer of two enantiomers from the mobile phase to the stationary phase is enthalpy‐driven. Enantioseparation of 1 m‐PFOS by SFC follows the same rules of ion exchange as those for the chiral separation by HPLC.     

Study on the HPLC‐based separation of some ezetimibe stereoisomers and the underlying stereorecognition process

The enantioseparation of ezetimibe stereoisomers by high‐performance liquid chromatography on different chiral stationary phases, ie, 3 polysaccharide‐based chiral columns, was studied. It was observed that cellulose‐based Chiralpak IC column exhibited the best resolving ability. After the optimization of mobile phase compositions in both normal and reversed phase modes, satisfactory separation could be obtained on Chiralpak IC column, especially in normal phase mode. The use of prohibited solvents as nonstandard mobile phase gave rise to better resolution than that of standard mobile phases (n‐hexane/alcohol system). In addition, the presence of ethanol in nonstandard mobile phase has played an important role in enhancing chromatographic efficiency and resolution between ezetimibe stereoisomers. Various attempts were made to comprehensively compare the chiral recognition capabilities of immobilized versus coated polysaccharide‐based chiral columns, amylose‐based versus cellulose‐based chiral stationary phases, reversed versus normal phase modes, and standard versus nonstandard mobile phases. Moreover, possible solute‐mobile phase‐stationary phase interactions were derived to explain how stationary and mobile phases affected the separation. Then the method validation with respect to selectivity, linearity, precision, accuracy, and robustness was carried out, which was demonstrated to be suitable and accurate for the quantitative determination of (RRS)‐ezetimibe impurity in ezetimibe bulk drug.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Chiral HPLC Separation and Modeling of Four Stereomers of DL‐Leucine‐DL‐Tryptophan Dipeptide on Amylose Chiral Column

Chiral high‐performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL‐leucine‐tryptophan DL‐dipeptide on AmyCoat‐RP column are described. The mobile phase applied was ammonium acetate (10 mM)‐methanol‐acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL‐, DD‐, DL‐, and LD‐ stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0–2.3 and 5.6–14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π–π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL‐leucine‐DL‐tryptophan in unknown matrices. Chirality 28:642–648, 2016. © 2016 Wiley Periodicals, Inc.     

Enantioseparation of chiral pharmaceuticals by vancomycin‐bonded stationary phase and analysis of chiral recognition mechanism

The drug chirality is attracting increasing attention because of different biological activities, metabolic pathways, and toxicities of chiral enantiomers. The chiral separation has been a great challenge. Optimized high‐performance liquid chromatography (HPLC) methods based on vancomycin chiral stationary phase (CSP) were developed for the enantioseparation of propranolol, atenolol, metoprolol, venlafaxine, fluoxetine, and amlodipine. The retention and enantioseparation properties of these analytes were investigated in the variety of mobile phase additives, flow rate, and column temperature. As a result, the optimal chromatographic condition was achieved using methanol as a main mobile phase with triethylamine (TEA) and glacial acetic acid (HOAc) added as modifiers in a volume ratio of 0.01% at a flow rate of 0.3 mL/minute and at a column temperature of 5°C. The thermodynamic parameters (eg, ΔH, ΔΔH, and ΔΔS) from linear van 't Hoff plots revealed that the retention of investigated pharmaceuticals on vancomycin CSP was an exothermic process. The nonlinear behavior of lnk′ against 1/T for propranolol, atenolol, and metoprolol suggested the presence of multiple binding mechanisms for these analytes on CSP with variation of temperature. The simulated interaction processes between vancomycin and pharmaceutical enantiomers using molecular docking technique and binding energy calculations indicated that the calculated magnitudes of steady combination energy (ΔG) coincided with experimental elution order for most of these enantiomers.     

Enantioseparation of racecadotril using polysaccharide‐type chiral stationary phases in polar organic mode

Enantioseparation of the antidiarrheal drug, racecadotril, was investigated by liquid chromatography using polysaccharide‐type chiral stationary phases in polar organic mode. The enantiodiscrimininating properties of 4 different chiral columns (Chiralpak AD, Chiralcel OD, Chiralpak AS, Chiralcel OJ) with 5 different solvents (methanol, ethanol, 1‐propanol, 2‐propanol, and acetonitrile) at 5 different temperatures (5–40 °C) were investigated. Apart from Chiralpak AS column the other 3 columns showed significant enantioseparation capabilities. Among the tested mobile phases, alcohol type solvents were superior over acetonitrile, and significant differences in enantioselective performance of the selector were observed depending on the type of alcohol employed. Van't Hoff analysis was used for calculation of thermodynamic parameters which revealed that enantioseparation is mainly enthalpy controlled; however, enthropic control was also observed. Enantiopure standard was used to determine the enantiomer elution order, revealing chiral selector—and mobile‐phase dependent reversal of enantiomer elution order. Using the optimized method (Chiralcel OJ stationary phase, thermostated at 10 °C, 100% methanol, flow rate: 0.6 mL/min) baseline separation of racecadotril enantiomers (resolution = 3.00 ± 0.02) was achieved, with the R‐enantiomer eluting first. The method was validated according to the ICH guidelines, and its application was tested on capsule and granules containing the racemic mixture of the drug.     

Evaluation of the chiral recognition properties and the column performances of three chiral stationary phases based on cellulose for the enantioseparation of six dihydropyridines by high‐performance liquid chromatography

Separations of six dihydropyridine enantiomers on three commercially available cellulose‐based chiral stationary phases (Chiralcel OD‐RH, Chiralpak IB, and Chiralpak IC) were evaluated with high‐performance liquid chromatography (HPLC). The best enantioseparation of the six chiral drugs was obtained with a Chiralpak IC (250 × 4.6 mm i.d., 5 μm) column. Then the influence of the mobile phase including an alcohol‐modifying agent and alkaline additive on the enantioseparation were investigated and optimized. The optimal mobile phase conditions and maximum resolution for every analyte were as follows respectively: n‐hexane/isopropanol (85:15, v /v) for nimodipine (R  = 5.80) and cinildilpine (R  = 5.65); n‐hexane/isopropanol (92:8, v /v) for nicardipine (R  = 1.76) and nisoldipine (R  = 1.92); and n‐hexane/isopropanol/ethanol (97:2:1, v /v/v) for felodipine (R  = 1.84) and lercanidipine (R  = 1.47). Relative separation mechanisms are discussed based on the separation results, and indicate that the achiral parts in the analytes' structure showed an important influence on the separation of the chiral column.     

Enantiomeric resolution,thermodynamic parameters,and modeling of clausenamidone and neoclausenamidone on polysaccharide‐based chiral stationary phases

The aim of the paper is to describe a new synthesis route to obtain synthetic optically active clausenamidone and neoclausenamidone and then use high‐performance liquid chromatography (HPLC) to determine the optical purities of these isomers. In the process, we investigated the different chromatographic conditions so as to provide the best separation method. At the same time, a thermodynamic study and molecular simulations were also carried out to validate the experimental results; a brief probe into the separation mechanism was also performed. Two chiral stationary phases (CSPs) were compared with separate the enantiomers. Elution was conducted in the organic mode with n‐hexane and iso‐propanol (IPA) (80/20 v/v) as the mobile phases; the enantiomeric excess (ee) values of the synthetic R‐clausenamidone and S‐clausenamidone and R‐neoclausenamidone and S‐ neoclausenamidone were higher than 99.9%, and the enantiomeric ratio (er) values of these isomers were 100:0. Enantioselectivity and resolution (α and Rs, respectively) levels with values ranging from 1.03 to 1.99 and from 1.54 to 17.51, respectively, were achieved. The limits of detection and quantitation were 3.6 to 12.0 and 12.0 to 40.0 ug/mL, respectively. In addition, the thermodynamics study showed that the result of the mechanism of chiral separation was enthalpically controlled at a temperature ranging from 288.15 to 308.15 K. Furthermore, docking modeling showed that the hydrogen bonds and π‐π interactions were the major forces for chiral separation. The present chiral HPLC method will be used for the enantiomeric resolution of the clausenamidone derivatives.     

Simultaneous determination of guaifenesin enantiomers and ambroxol HCl using 50‐mm chiral column for a negligible environmental impact

Chiral stationary phases are conveniently used for enantiomeric separation of drugs by liquid chromatography. Consumption of large volumes of hazardous solvents is considered as a common challenge for the sustainability of this technique. To this end, a columnar chromatography has been adopted using 50‐mm‐length stationary phases. The study comprised five Phenomenex Lux cellulose‐ and amylose‐based columns for the separation of guaifenesin (GUA) enantiomers. In addition, an experimental design was used to optimize the gradient profile for the separation of racemic GUA and ambroxol HCl (AMB) binary mixture. The chromatographic method was achieved using Lux Cellulose‐1 (50 × 4.6 mm) as a chiral stationary phase and ethanol/water as a mobile phase with linear gradient elution of 20% to 70% ethanol in 6 minutes at a flow rate of 1.0 mL min^?1 and UV detection at 270 nm. Linearity ranges were found to be 50 to 1000 μg mL^?1 and 15 to 450 μg mL^?1 for each GUA enantiomer and AMB, respectively. Environmental, health and safety tool was used to assess and compare greenness of the proposed and reported methods. Short column indeed reduces the environmental impact by decreasing waste by about 60% and utilizing only 1‐mL ethanol in the mobile phase. The proposed method is a safer alternative for the simultaneous determination of drugs in their combined pharmaceutical formulation. The method has been validated and compared favorably with a reported one.