Teicoplanin Chiral Stationary Phase Research Articles

Fast, sensitive LC-MS resolution of -hydroxy acid biomarkers via SPP-teicoplanin and an alternative UV detection approach.

Enantioseparation of -hydroxy acids is essential since specific enantiomers of these compounds can be used as disease biomarkers for diagnosis and prognosis of cancer, brain diseases, kidney diseases, diabetes, etc., as well as in thefood industry to ensure quality. HPLC methods were developed for the enantioselective separation of 11 -hydroxy acids using asuperficially porous particle-based teicoplanin (TeicoShell) chiral stationary phase. The retention behaviors observed for the hydroxy acids were HILIC, reversed phase, and ion-exclusion. While both mass spectrometry and UV spectroscopy detection methods could be used, specific mobile phases containing ammonium formate and potassium dihydrogen phosphate, respectively, were necessary with each approach. The LC-MS mode was approximately two orders of magnitude more sensitive than UV detection. Mobile phase acidity and ionic strength significantly affected enantioresolution and enantioselectivity. Interestingly, higher ionic strength resulted in increased retention and enantioresolution. It was noticed that for formate-containing mobile phases, using acetonitrile as the organic modifier usually resulted in greater enantioresolution compared to methanol. However, sometimes using acetonitrile with high ammonium formate concentrations led to lengthy retention times which could be avoided by using methanol as the organic modifier. Additionally, the enantiomeric purities of single enantiomer standards were determined and it was shown that almost all standards contained some levels of enantiomeric impurities.

Understanding the Transition from High-Selective to High-Efficient Chiral Separations by Changing the Organic Modifier with Zwitterionic-Teicoplanin Chiral Stationary Phase.

The retention behavior of small molecules and N-protected amino acids on a zwitterionic teicoplanin chiral stationary phase (CSP), prepared on superficially porous particles (SPPs) of 2.0 μm particle diameter, has shown that efficiency and enantioselectivity, and so enantioresolution, dramatically change depending on the employed organic modifier. In particular, it was found that while methanol permits the boost of enantioselectivity and resolution of the amino acids, at the cost of efficiency, acetonitrile allows for the ability to reach extraordinary efficiency even at high flow rates (with reduced plate height <2 and up to 300,000 plates/m at the optimum flow rate). To understand these features, an approach based on the investigation of mass transfer through the CSP, the estimation of the binding constants of amino acids on the CSP, and the assessment of compositional properties of the interfacial region between bulk mobile phase and solid surface has been adopted.

Environmentally Sustainable Achiral and Chiral Chromatographic Analysis of Amino Acids in Food Supplements.

Two LC methods were developed for the achiral and chiral reversed-phase (RP) analysis of an amino acid (AA) pool in a food supplement, in compliance with the main paradigms of Green Chromatography. A direct achiral ion-pairing RP-HPLC method was optimized under gradient conditions with a water-ethanol (EtOH) eluent containing heptafluorobutyric acid (0.1%, v/v), to quantify the eight essential AAs (Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) contained in the food supplement. Thus, the usually employed acetonitrile was profitably substituted with the less toxic and more benign EtOH. The method was validated for Leu and Phe. The chiral LC method performed with a teicoplanin chiral stationary phase was developed with a water-EtOH (60:40, v/v) eluent with 0.1%, v/v acetic acid. The enantioselective analysis was carried out without any prior derivatization step. Both developed methods performed highly for all eight AAs and revealed that: (i) the content of six out of eight AAs was consistent with the manufacturer declaration; (ii) only L-AAs were present. Furthermore, it was demonstrated that a two-dimensional achiral-chiral configuration is possible in practice, making it even more environmentally sustainable. A molecular modelling investigation revealed interesting insights into the enantiorecognition mechanism of Lys.

High–throughput enantioseparation of Nα–fluorenylmethoxycarbonyl proteinogenic amino acids through fast chiral chromatography on zwitterionic-teicoplanin stationary phases

In this study, 31 racemates of Nα-FMOC (fluorenylmethoxycarbonyl) amino acids (AAs) with different chemico-physical characteristics (neutral nonpolar, neutral polar, acidic and basic) have been successfully resolved in fast enantioselective chromatography on recently-developed zwitterionic-teicoplanin chiral stationary phases (CSPs). The CSPs were prepared by covalently bonding the teicoplanin selector on fully-porous particles of narrow dispersion particle-size distribution (particle diameter 1.9 µm) and superficially-porous particles (2.0 µm). Both the zwitterionic-teicoplanin CSPs have proved to be ideal media for the separation of this important class of compounds. In particular, the zwitterionic CSP prepared on superficially-porous particles exhibited superior enantioselectivity and resolution, compared to that made of fully porous particles, in virtue of more favorable thermodynamics. The zwitterionic nature of these CSPs allowed avoiding the annoying effect of Donnan's exclusion of enantiomers from the stationary phase. This effect, on the opposite, was frequently observed on a commercial teicoplanin CSP (Teicoshell) employed for comparative purposes. Noticeably, on the zwitterionic-teicoplanin CSPs, by using either acetonitrile- or methanol-rich mobile phases (MPs), it was possible to favor speed over enantioresolution and vice versa. This work gives further replies to the request for rapid determination of enantiomeric excess of Nα-FMOC proteinogenic (and non–proteinogenic) AAs, typically used as preferred chiral synthons in the solid-phase synthesis of therapeutic peptides.

Development and Validation of Analytical Method for Clenbuterol Chiral Determination in Animal Feed by Direct Liquid Chromatography

A direct chiral liquid chromatography with UV detection method was developed and validated for enantiomeric determination of clenbuterol β2-agonist in feed samples for poultry. To obtain optimum chromatographic conditions for chiral separation, two commercially available columns packed with vancomycin and teicoplanin chiral stationary phase (150 × 2.1 mm i.d, 5 μm) were tested. Experimental design and response surface analysis were used to model analytical responses and to get mobile phase composition for acceptable enantioresolution and performance in low analysis time. Different sample treatments based on HCX SPE and on the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method were evaluated and compared to obtain the best extraction recoveries with negligible matrix effect. Special attention was paid to compatibility between sample preparation and chiral separation to get enantioresolution and low detection limits in the high-complex sample. QuEChERS-based extraction allowed obtaining cleaner feed extracts in low sample preparation time, providing a useful and reliable method capable to control the illegal use of clenbuterol enantiomers in feed for growth promotion in food-producing animals. Reproducible recoveries, from 64 to 101 % for clenbuterol enantiomers, with relative standard deviation values within the range 1–6 %, were achieved in feed samples of poultry spiked at low concentration levels of micrograms per gram.

Determination of salbutamol by direct chiral reversed‐phase HPLC using teicoplanin as stationary phase and its application to natural water analysis

A direct chiral LC-UV method was optimized for the determination of salbutamol (SAL) β2 -agonist in environmental water. Two commercially available columns were evaluated: teicoplanin Chirobiotic-T™ (150 × 2.1 mm i.d., 5 µm) and vancomycin Chirobiotic-V™ (150 × 2.1 mm i.d., 5 µm). Finally, teicoplanin chiral stationary phase was selected for SAL enantiomer resolution. In order to preserve its integrity and maintain the column performance for longer time, the use of additives such as triethylamine (TEA) in the mobile phase was avoided. Experimental design was applied to simultaneously evaluate the influence of several parameters involved in enantiomer separation and to establish the conditions for acceptable resolution and performance in short analysis time. Optimum mobile phase was methanol-20 mM ammonium acetate buffer at pH 4.5 (98:2, v/v). A solid-phase extraction procedure for sample pre-concentration and clean-up allowed the determination of chiral SAL residues in natural water samples spiked at low concentrations in the range 1.0-20 ng mL(-1) . Reproducible recoveries, between 77 and 98%, were obtained and matrix effect was negligible. Injection of sample solutions at low elution strength permitted the SAL enantioresolution in the natural water complex matrix with satisfactory sensitivity and precision.

Simultaneous Enantiomeric Determination of Acidic Herbicides in Apple Juice Samples by Liquid Chromatography on a Teicoplanin Chiral Stationary Phase

A novel and chiral liquid chromatography-ultraviolet method has been developed for simultaneous determination of mecoprop, fenoprop and diclofop acid herbicides. Optimization of mobile phase composition for enantiomeric separation on a teicoplanin stationary phase was done by experimental design and response surface analyses. When chiral separation of herbicide mixtures was optimized, multiple response analysis was necessary to determine combination of experimental factors, methanol percentage and triethylamine concentration in the aqueous buffer, which simultaneously optimize experimental responses. Under optimum conditions, acceptable resolution between the three enantiomeric pairs and between enantiomers of each studied herbicide was achieve in low analysis time. Applicability of the chromatographic method for simultaneous chiral herbicide determination was evaluated in commercial apple juice samples. Previously, a clean-up and preconcentration step on a polymeric solid phase extraction (SPE) cartridge (OASIS® MCX) was done. In order to get enantioresolution in the complex matrix, compatibility between the sample preparation procedure and the chiral liquid chromatography analysis was necessary. Thus, a focusing step was required after herbicide elution from the SPE sorbent to obtain an injection solution with low organic solvent composition. Detection limits were around maximum residue levels permitted by European Regulations in fresh fruit, allowing herbicide determination at enantiomer spiked levels from 0.075 to 0.750 μg g−1, with recoveries in the range 65–99 % and relative standard deviations of ≤4 %. Due to co-extracted endogenous matter, which partially overlapped with mecoprop enantiomers, mathematical deconvolution was the best choice as integration tool minimizing errors in peak area estimation.

Evaluation and Comparison of a 3,5‐Dimethylphenyl Isocyanate Teicoplanin with Phenyl Isocyanate Teicoplanin Chiral Stationary Phase Using RP‐HPLC

AbstractHPLC enantiomeric separations of 8 α‐amino acids were achieved using two self‐made chiral stationary phases (CSP)–phenyl isocyanate teicoplanin (Phe‐TE) and 3,5‐dimethylphenyl isocyanate teicoplanin (DMP‐TE), using reversed phase mobile phases. The Phe‐TE or the DMP‐TE CSP was prepared from the TE using derivative agents, phenyl isocyanate or 3,5‐dimethylphenyl isocyanate, respectively. The chromatographic results were given as the retention, selectivity, resolution factor and the enantioselective free energy difference corresponding to the separation of the two enantiomers. The effect of pH, organic modifier type and amount were discussed, and the stereoselectivities for two TE‐based CSPs were compared. The chiral selectivity factor for six α‐amino acids on DMP‐TE is somewhat bigger than that on Phe‐TE CSP under reversed phase (RP) mode. Comparison of the enantiomeric separations using self‐made Phe‐TE and DMP‐TE was conducted to gain a better understanding of the chiral recognition mechanism of the macrocyclic glycopeptide CSP.

Enantioseparation of Novel Amino Analogs of Indole Phytoalexins on Macrocyclic Glycopeptide-Based Chiral Stationary Phase

Direct chiral separation of the enantiomers of spirobrassinin, 1-methoxyspirobrassinin and ten novel cis- and trans-diastereoisomers of 2-amino analogs of indole phytoalexin 1-methoxyspirobrassinol methyl ether on macrocyclic glycopeptide-based chiral stationary phase (CSP) with teicoplanin (Chirobiotic T) was studied. Normal phase eluents containing n-hexane with modifiers ethanol and 2-propanol were used. The effects of mobile phase composition, structure of the analytes and temperature were investigated. Chiral resolution on teicoplanin CSP was achieved only in the case of trans-diastereoisomers. The van’t Hoff plots were found to show linear behavior in all cases. It was found that studied normal phase enantioseparations were enthalpy driven. The elution order of the enantiomers was determined in some cases.

Evaluation of dalbavancin as chiral selector for HPLC and comparison with teicoplanin‐based chiral stationary phases

Dalbavancin is a new compound of the macrocyclic glycopeptide family. It was covalently linked to 5 microm silica particles using two different binding chemistries. Approximately 250 racemates including (a) heterocyclic compounds, (b) chiral acids, (c) chiral amines, (d) chiral alcohols, (e) chiral sulfoxides and sulfilimines, (f) amino acids and amino acid derivatives, and (g) other chiral compounds were tested on the two new chiral stationary phases (CSPs) using three different mobile phases. As dalbavancin is structurally related to teicoplanin, the same set of chiral compounds was screened on two commercially available teicoplanin CSPs for comparison. The dalbavancin CSPs were able to separate some enantiomers that were not separated by the teicoplanin CSPs and also showed improved separations for many racemates. However, there were other compounds only separated or better separated on teicoplanin CSPs. Therefore, the dalbavancin CSPs are complementary to the teicoplanin CSPs.

Binding of Dipeptides and Amino Acids to Teicoplanin Chiral Stationary Phase: Apparent Homogeneity of Some Heterogeneous Systems

The binding between Teicoplanin glycopeptide antibiotic and some dipeptides and amino acids has been studied by nonlinear liquid chromatography. A Teicoplanin-based chiral stationary phase, specifically designed to achieve maximum selectivity and loading by reducing non-specific interactions, has been prepared and packed into a microbore column. The adsorption isotherms of the enantiomers of proline, alanine, and alanine-alanine (Ala-Ala) have been measured through frontal analysis. The experimental binding data have been interpreted in the context of the ordinary homogeneous Michaelis-Menten model and by considering an heterogeneous model that accounts for a broad adsorption energy distribution (AED). AED has been achieved by the analysis of adsorption isotherms. Besides confirming the importance of the terminal D-Ala-D-Ala moiety in the molecular recognition between the dipeptide and the macrocyclic antibiotic Teicoplanin (it was found that Teicoplanin behaves as a molecular filter toward the enantiomers of Ala-Ala), this study shows that a heterogeneous adsorption model is needed for the correct interpretation of binding data.

Evaluation and Comparison of a 3,5‐Dimethylphenyl Isocyanate Teicoplanin and Phenyl Isocyanate Teicoplanin Chiral Stationary Phases

AbstractTwo chiral stationary phases (CSP), phenyl isocyanate teicoplanin CSP (Ph‐TE) and 3,5‐dimethylphenyl isocyanate teicoplanin CSP (DMP‐TE) were prepared from the macrocyclic glycopeptide teicoplanin CSP (TE) using derivative agents, phenyl isocyanate or 3,5‐dimethylphenyl isocyanate, respectively. The evaluation and comparison of these two self‐made CSP were conducted using HPLC enantiomeric separations in two different mobile phase modes, i.e., the reversed phased (RP) mode and the polar organic (PO) mode. The chromatographic results were given as the retention, selectivity, and resolution factors. Twelve racemic analytes, including eight amino acids and four non‐amino acid compounds, amino‐(4‐hydroxyphenyl) acetic acid, 4‐(1‐hydroxyethyl)‐2‐methoxy phenol, lamivudine and oxazepam reached enantioseparation using the RPLC mobile phase on these two new CSP. The results show that the retention factors and stereoselectivity factors on DMP‐TE are somewhat bigger than those on Ph‐TE for most analytes in the RPLC mobile phase. Six amino alcohols reached enantiomeric separation on these two CSP using the PO mobile phase. The retention of these amino alcohols on DMP‐TE is longer than that on Ph‐TE but the selectivities have little difference between these two CSP. Comparison of the enantiomeric separations using self‐made Ph‐TE and DMP‐TE were conducted in order to gain a better understanding of the chiral recognition mechanism of the macrocyclic glycopeptide CSP.

Enantioselective HPLC Analysis and In-vitro Kinetic Study Decomposition of Potential β-Blocker Drug in Guinea Pig Serum

A method using off-line solid-phase extraction and liquid chromatography has been developed for the analysis of enantiomers of derivatives of aryloxyaminopropanol in guinea pig blood serum. The paper presents the results of HPLC enantioseparation of the derivative of aryloxyaminopropanol obtained by using of four chiral stationary phases [macrocyclic antibiotic (vancomycin, teicoplanin, teicoplanin aglycone, and methylated teicoplanin aglycone)] and the mixture of methanol/acetonitrile/acetic acid/triethylamine (45:55:0.3:0.2, v/v/v/v) as mobile phase for all tested stationary phases. The presence of saccharide moieties in the chiral stationary phase (vancomycin and teicoplanin chiral stationary phases) plays an important role, together with charge interactions and steric interactions, in the separation of enantiomers of derivatives of aryloxyaminopropanol. The in-vitro degradation studies of individual (R)-enantiomer and (S)- enantiomer in blood serum demonstrated a difference in the final concentration of the enantiomers after the incubation at 37°C. It was found that the rate constant of the (R)-enantiomer is lower than the (S)-enantiomer.

Effects of a strongly adsorbed additive on process performance in chiral preparative chromatography

The shapes of elution profiles are often significantly influenced by the presence of strongly adsorbed additives in the mobile phase. This aspect needs to be considered in quantitative optimization of preparative chromatography. The theoretical study carried out here is based on available thermodynamic information for the enantiomers of three β-blockers, alprenolol, propranolol, and atenolol, on a teicoplanin chiral stationary phase (Chirobiotic T) using methanol/acetonitrile as the mobile phase and acetic acid/triethylamine as the additive. The properties of this strong additive made it possible to tune the binary elution profiles in any combination of the following apparent band shapes: anti-Langmuir/anti-Langmuir, anti-Langmuir/Langmuir and Langmuir/Langmuir. Optimization of the productivity and yield, when performing repetitive batch injections, was investigated using the equilibrium dispersive model. We show that it is important to consider the invisible additive perturbation peak when defining the cycle time and therefore a model-based optimization needs to take this into account. Furthermore, both productivity and yield could be improved for the two unusual shape combinations in comparison to the traditional Langmuir/Langmuir case.

Chiral recognition mechanisms with macrocyclic glycopeptide selectors

Macrocyclic glycopeptide selectors are naturally occurring antibiotics produced by microorganisms. They were found to be excellent chiral selectors for a wide range of enantiomers, including amino acids. Four selectors are commercialized as chiral stationary phases (CSP) for chromatography. They are ristocetin, teicoplanin, vancomycin, and the teicoplanin aglycone (TAG). The key docking interaction for amino acid recognition was established to be a charge-charge interaction between the anionic carboxylate group of the amino acid and a cationic amine group of the macrocyclic peptidic selector basket. The carbohydrate units are responsible for secondary interactions. However, they hinder somewhat the charge-charge docking interaction. The TAG selector is more effective for amino acid enantioseparations than the other CSPs. The "sugar" units are however useful allowing for chiral recognitions of other analytes, e.g., beta-blockers, not possible with the aglycone. Thermodynamic studies established that normal phase and reversed phase enantioseparations were enthalpy-driven. With polar waterless mobile phases used in the polar ionic mode, some separations were enthalpy-driven and others were entropy-driven. The linear solvation energy method was tentatively used to gain knowledge about the chiral recognition mechanism. It appeared to be a viable approach with neutral molecules but it failed with ionizable solutes. With molecular solutes and the teicoplanin CSP, the study showed a significant role of the surface charge-induced dipole interaction and steric effects. The remarkable complementary enantioselectivity effect observed with the four CSPs is discussed.

Comparative Enantioseparation of Seven Amino Alcohols on Teicoplanin‐Based Chiral Stationary Phases

The macrocyclic antibiotics represent a relatively new class of chiral selectors in separation science and teicoplanin‐based chiral stationary phases (CSP) have been used successfully in a number of applications in high‐performance liquid chromatography. In the present studies, we self‐prepared two bonded CSPs–teicoplanin (TE) and teicoplanin phenyl isocyanate (TE‐Phe). Seven amino alcohols, propranolol, bisoprolol fumarate, atenolol, salbutamol, isoproterenol, metoprolol, and labetalol were enantioseparated on both self‐made CSPs using methanol as mobile phase and acetic acid (HOAc) and triethylamine (TEA) as mobile phase additives. On both CSPs, the different enantioseparation behavior of analytes with different structure was compared. The influence of the concentration of mobile phase additives (HOAc and TEA) on the enantioseparation was investigated. In all conditions, the retention factors (k′) of seven analytes on TE‐Phe CSP were larger than that on TE CSP. However, the separation factors (α) and resolutions (Rs) on TE‐Phe CSP were smaller than that on TE CSP. The results indicated that the derivatized TE‐Phe CSP is not efficient as original teicoplanin CSP. Our observations also suggested that, for teicoplanin‐based CSPs, π‐π interactions and dipole‐dipole between solutes and CSPs mainly contribute to the retention of solutes on CSPs while hydrogen bonding and steric interactions play important roles in the chiral recognition for teicoplanin‐based CSPs.

Could linear solvation energy relationships give insights into chiral recognition mechanisms?: 2. Characterization of macrocyclic glycopeptide stationary phases

Five parameter linear solvation energy relationships (LSER) are known to have little or no shape recognition ability. However, it is proposed to use LSER studies to get insights into chiral recognition mechanisms. Since the two enantiomers have exactly the same five A–V solute descriptors being still separated by chiral stationary phases (CSPs), it can be considered that they form two different transient diastereoisomers with the CSP. It is then possible to perform LSER studies on the enantioselectivity factors taken as the two enantiomer retention factor ratios. In a first step, the five a–v system parameters of four CSPs of the macrocyclic glycopeptide types were determined using a set of test solutes with known A–V descriptors, both in the reversed phase and the normal phase modes. In a second step, the A–V descriptors of 18 enantiomeric pairs were tentatively established using five achiral columns with known a–v parameters. This was successful for the five molecular enantiomers only. It was found that the predicted retention factor for the molecular enantiomers separated on a given CSP corresponded either to retention factor of the first experimentally eluted enantiomer or to the second one or to none of them. Using the enantioselectivity factors it was possible to obtain the Δa–Δv parameters corresponding to the difference in CSP properties seen by the two enantiomers. For the five molecular enantiomeric pairs in the reversed phase mode with a teicoplanin CSP, it was found that there was an elevated contribution by the e coefficient that we interpret as a possible interaction between surface charges on the teicoplanin CSP and solute induced dipoles. Steric effects, seen on the v parameter, are second in magnitude followed by H-bond and polar interactions. Only one solute could be studied in the normal phase mode showing a different mechanism with polar and steric major interactions.

Separation and Thermodynamic Studies of Chiral Sulfoxides on Teicoplanin‐Based Stationary Phase

Three different teicoplanin chiral stationary phases (CSPs) (native teicoplanin, teicoplanin aglycon, and methylated teicoplanin aglycon) were used to study the enantioseparation and temperature behaviour of a set of chiral sulfoxides. The chiral analytes studied were 2‐, 3‐, 4‐ toluyl methyl sulfoxides and phenyl methyl sulfoxides with different 2‐, 3‐, 4‐halogen substituents on the aromatic ring. The effect of temperature on the LC separation of racemic aromatic sulfoxides was studied between 10 and 50°C in a methanol mobile phase. The van't Hoff plots were constructed and thermodynamic data were determined from the slope and the intercept of linear van't Hoff plots. The van't Hoff plots (ln k versus 1/T and ln α versus 1/T) were linear for all enantiomers. Given the linearity of the van't Hoff plots, thermodynamic parameters, such as the change in enthalpies Δ(ΔH 2,1) and the entropies Δ(ΔS 2,1) for the sulfoxide enantiomers could be calculated. In addition, the elution order did not reverse in the temperature range of this study and the mechanism of enantioseparation did not vary.

Enantioseparation of Seven Amino Alcohols on Teicoplanin Chiral Column

Under the polar organic mode, seven amino alcohols, propranolol, metoprolol, bisoprololfumarate, atenolol, salbutamol, isoprenaline, metoprolol and labetalol, were enantioseparated on teicoplanin chiral stationary phase (CSP), using methanol as mobile phase and acetic acid (HOAc) and triethylamine (TEA) as mobile phase additives. When the proportion of HOAc and TEA was 1:1, the retention factors and the resolutions of seven amino alcohols decreased with the increase of HOAc/TEA concentration, and the separation factors being kept constant. The excessive acid increased the polarity of mobile phase, and therefore the eluting ability was increased. Without HOAc additive, the retention factors of the amino alcohols decreased with the increase of the amount of TEA, with the separation factors being kept constant except labetalol. The polarity of mobile phase increased with the increasing of TEA concentration; therefore, the hydrogen bonding between solutes and CSPs decreased. All the results showed that the proportion of HOAc and TEA did not change the essentiality of chiral separation.

Development of an HPLC Method for the Quantitation of Bisoprolol Enantiomers in Pharmaceutical Products using a Teicoplanin Chiral Stationary Phase and Fluorescence Detection

A selective high performance liquid chromatographic (HPLC) method was developed for the separation and quantification of bisoprolol enantiomers in pharmaceutical products. The method is highly specific where another co-formulated drug, hydrochlorothiazide, did not interfere. Baseline resolution was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP), known as Chirobiotic T, with fluorescence detection at excitation/emission wavelengths 275/305 nm. The polar ionic mobile phase (PIM) consisting of methanol‐glacial acetic acid‐triethylamine, (100:0.020:0.025), (v/v/v) has been used at a flow rate of 1.5 mL/min. All analytes with S‐(−)‐atenolol as the internal standard were conducted at room temperature. The stability of bisoprolol enantiomers under different degrees of temperature was also studied. The results showed that the drug is stable for at least 7 days at 70°C. The method was validated for its linearity, accuracy, precision, and robustness. An experimental design was used during validation to evaluate method robustness. The calibration curves were linear over the range of 5‐250 ng/mL for each enantiomer, with a correlation coefficient of 0.999 for both enantiomers. The overall recoveries of S‐(−)‐ and R‐(+)‐bisoprolol from pharmaceutical products ranged from 97.6 to 100.5% with %RSD ranging from 0.7 to 2.6%. The limit of quantification (LOQ) and limit of detection (LOD) for each enantiomer were 5 and 2 ng/mL, respectively. The method proved to be of chiral quality control for bisoprolol formulations by HPLC.