Direct Enantioseparation of Nitrogen‐Heterocyclic Pesticides on Cellulose‐Based Chiral Column by High‐Performance Liquid Chromatography

The enantiomeric separation of eight pesticides including bitertanol ( 1 ), diclobutrazol ( 2 ), fenbuconazole ( 3 ), triticonazole ( 4 ), imazalil ( 5 ), triapenthenol ( 6 ), ancymidol ( 7 ), and carfentrazone‐ethyl ( 8 ) was achieved, using normal‐phase high‐performance liquid chromatography on two cellulosed‐based chiral columns. The effects of isopropanol composition from 2% to 30% in the mobile phase and column temperature from 5 to 40 °C were investigated. Satisfactory resolutions were obtained for bitertanol ( 1 ), triticonazole ( 4 ), imazalil ( 5 ) with the (+)‐enantiomer eluted first and fenbuconazole ( 3 ) with the (—)‐enantiomer eluted first on Lux Cellulose‐2 and Lux Cellulose‐3. (+)‐Enantiomers of diclobutrazol ( 2 ) and triapenthenol ( 6 ) were first eluted on Lux Cellulose‐2. (—)‐Carfentrazone‐ethyl ( 8 ) were eluted first on Lux Cellulose‐2 and Lux Cellulose‐3 with incomplete separation. Reversed elution orders were obtained for ancymidol (7). (+)‐Ancymidol was first eluted on Lux Cellulose‐2 while on Lux Cellulose‐3 (—)‐ancymidol was first eluted. The results of the elution order at different column temperatures suggested that column temperature did not affect the optical signals of the enantiomers. These results will be helpful to prepare and analyze individual enantiomers of chiral pesticides. Chirality 27:32–38, 2015. © 2014 Wiley Periodicals, Inc.     

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.     

Enantioseparation and molecular modeling study of five β‐adrenergic blockers on Chiralpak IC column

A new high‐performance liquid chromatography (HPLC) method was developed for the enantiomeric resolution of five β‐adrenergic blockers on a Chiralpak IC column (250 mm × 4.6 mm, 5.0 μm particle size) in normal phase mode. The mobile phase used was n‐hexane‐ethanol‐diethylamine in different proportions at the flow rate of 1.0 mL/min with the column temperature of 25°C using a UV detector at 230 nm. The influences of base additives and alcohol modifiers were evaluated and optimized. The maximum resolution values for bevantolol, propranolol carteolol, esmolol, and metoprolol were 4.80, 2.77, 2.09, 2.30, and 1.11, respectively. To gain a better understanding of the interaction between chiral stationary phase and analyte enantiomers, the molecular docking of chiral stationary phase with five pairs of enantiomer was carried out using AutoDock molecular docking technique. By simulation studies, the mechanism of chiral recognition was determined. According to the results, hydrogen bond interactions and π‐π interactions were the chief interactions for the chiral recognition.     

Enantiomeric separation of type I and type II pyrethroid insecticides with different chiral stationary phases by reversed‐phase high‐performance liquid chromatography

The enantiomeric separation of type I (bifenthrin, BF) and type II (lambda‐cyhalothrin, LCT) pyrethroid insecticides on Lux Cellulose‐1, Lux Cellulose‐3, and Chiralpak IC chiral columns was investigated by reversed‐phase high‐performance liquid chromatography. Methanol/water or acetonitrile/water was used as mobile phase at a flow rate of 0.8 mL/min. The effects of chiral stationary phase, mobile phase composition, column temperature, and thermodynamic parameters on enantiomer separation were carefully studied. Bifenthrin got a partial separation on Lux Cellulose‐1 column and baseline separation on Lux Cellulose‐3 column, while LCT enantiomers could be completely separated on both Lux Cellulose‐1 and Lux Cellulose‐3 columns. Chiralpak IC provided no separation ability for both BF and LCT. Retention factor (k) and selectivity factor (α) decreased with the column temperature increasing from 10°C to 40°C for both BF and LCT enantiomers. Thermodynamic parameters including ?H and ?S were also calculated, and the maximum R_s were not always obtained at lowest temperature. Furthermore, the quantitative analysis methods for BF and LCT enantiomers in soil and water were also established. Such results provide a new approach for pyrethroid separation under reversed‐phase condition and contribute to environmental risk assessment of pyrethroids at enantiomer level.     

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).     

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.     

Strategy Approach for Direct Enantioseparation of Hyoscyamine Sulfate and Zopiclone on a Chiral αl‐Acid Glycoprotein Column and Determination of Their Eutomers: Thermodynamic Study of Complexation

Rapid and simple isocratic high‐performance liquid chromatographic methods with UV detection were developed and validated for the direct resolution of racemic mixtures of hyoscyamine sulfate and zopiclone. The method involved the use of α_l‐acid glycoprotein (AGP) as chiral stationary phase. The stereochemical separation factor (?) and the stereochemical resolution factor (R_s) obtained were 1.29 and 1.60 for hyoscyamine sulfate and 1.47 and 2.45 for zopiclone, respectively. The method was used for determination of chiral switching (eutomer) isomers: S‐hyoscyamine sulfate and eszopiclone. Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on the chiral AGP column. The influence of mobile phase, concentration and type of uncharged organic modifier, ionic strength, and column temperature on enantioselectivity were studied. Calibration curves were linear in the ranges of 1–10 µg mL^‐1 and 0.5–5 µg mL^‐1 for S‐hyoscyamine sulfate and eszopiclone, respectively. The method is specific and sensitive, with lower limits of detection and quantifications of 0.156, 0.515 and 0.106, 0.349 for S‐hyoscyamine sulfate and eszopiclone, respectively. The method was used to identify quantitatively the enantiomers profile of the racemic mixtures of the studied drugs in their pharmaceutical preparations. Thermodynamic studies were performed to calculate the enthalpic ΔH and entropic ΔS terms. The results showed that enantiomer separation of the studied drugs were an enthalpic process. Chirality 28:49–57, 2016. © 2015 Wiley Periodicals, Inc.     

Resolution and determination of enantiomeric purity of the enantiomers of felodipine using chiral-AGP® as stationary phase

A direct chiral chromatographic reversed phase method for the determination of the enantiomers of felodipine is described. The influence of charged and uncharged modifiers as well as the effect of the mobile phase pH on the enantiomeric resolution is discussed. A high mobile phase pH and the addition of 2-propanol as organic modifier gave the highest separation factor (α = 1.3). The high mobile phase pH (pH = 7.6) is outside the recommended pH limit of silica based columns but was necessary to achieve baseline resolution of (R)- and (S)-felodipine. Improvement of column efficiency by increasing column temperature was utilized for optimization of the enantiomeric resolution (Rs = 1.7). The enantiomers of felodipine and three related compounds were separated within 15 min. The enantiomeric purity of (R)- and (S)-felodipine in injections and (R)-felodipine in bulk substance was higher than 99.5% and no racemization was observed after storage at accelerated conditions. A poor Chiral-AGP® column used for a long period was restored using a simple wash step together with repacking the top of the chromatographic column. © 1995 Wiley-Liss, Inc.     

Chiral separation of phospine‐containing α‐amino acid derivatives using two complementary cellulosic stationary phases in supercritical fluid chromatography

Enantiomeric separations of six amino‐acid derivatives have been studied using packed‐column supercritical fluid chromatography with two polysaccharide‐based enantioselective stationary phases: cellulose tris(3,5‐dimethylphenylcarbamate) and cellulose tris(3‐chloro‐4‐methylphenylcarbamate) (Lux Cellulose‐1 and ‐2). The effect of analyte structure on retention and separation was studied. Varied mobile phase compositions were investigated: alcohol modifier percentage was increased from 3 to 40% but smaller amounts were most effective in separating these compounds. Besides, ethanol was preferred to methanol or isopropanol as it proved to be a good compromise to achieve sufficient resolution in a reasonable analysis time. Moreover, a carbon dioxide‐ethanol mixture allows performing analyses in safe and green conditions. The effect of temperature at constant mobile phase composition was explored between 10 and 40°C. In most cases, increasing the temperature improved the chiral separation, up to an optimum temperature. The results are discussed in line with the structure variation of the racemic derivatives analyzed and the two columns are compared. The two columns were shown to provide complementary selectivities for the investigated solutes: whereas Lux 1 provided separation for five of the six racemates, Lux 2 could resolve the last racemic mixture. Finally, optimized conditions of separation are defined. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Chiral 3D Open‐Framework Material Ni(D‐cam)(H2O)2 Used as GC Stationary Phase

Metal‐organic frameworks (MOFs) have been explored for analytical applications because of their outstanding properties such as high surface areas, flexibility and specific structure features, especially for chromatography application in recent years. In this work, a chiral MOF Ni(D‐cam)(H₂O)₂ with unusual integration of molecular chirality, absolute helicity, and 3‐D intrinsic chiral net was chosen as stationary phase to prepare Ni(D‐cam)(H₂O)₂‐coated open tubular columns for high‐resolution gas chromatographic (GC) separation. Two fused‐silica open tubular columns with different inner diameters and lengths, including column A (30 m × 250 µm i.d.) and column B (2 m × 75 µm i.d.), were prepared via a dynamic coating method. The chromatographic properties of the two columns were investigated using n‐dodecane as the analyte at 120 °C. The number of theoretical plates (plates/m) of the two metal–organic framework (MOF) columns was 1300 and 2750, respectively. The racemates, isomer and linear alkanes mixture were used as analytes for evaluating the separation properties of Ni(D‐cam)(H₂O)₂‐coated open tubular columns. The results showed that the columns offered good separations of isomer and linear alkanes mixture, especially racemates. Chirality 26:27–32, 2013. © 2013 Wiley Periodicals, Inc.     

A validated HPLC method for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) with fluorescence detection in raw material and pill form: application to an in vitro dissolution test and a content uniformity test

A simple, sensitive and rapid HPLC method with fluorescence detection for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) in the raw material and pill form was developed. Liquid chromatography was performed on a C₁₈ column (250 × 4.6 mm i.d., 5 µm particle size), the mobile phase consisted of methanol and 0.05 M sodium dihydrogen phosphate buffer (80 : 20, v/v), and the apparent pH of the mobile phase was adjusted to 3. The fluorescence detector was operated at excitation/emission wavelengths of 275/400 nm. The proposed method allows the determination of DDB within concentration range 0.1–1.5 µg/mL with a limit of detection of 0.032 µg/mL, a limit of quantification of 0.097 µg/mL and a correlation coefficient of 0.9997. The proposed method has been successfully applied for the analysis of DDB in its pills with a percentage recovery of 98.45 ± 0.32. The method was fully validated according to ICH guidelines. Moreover, the high sensitivity of the method permits its use in an in vitro dissolution test for DDB under simulated intestinal conditions. In addition, the proposed method was extended to a content uniformity test according to USP guidelines. Copyright © 2014 John Wiley & Sons, Ltd.     

Enantiomeric Separation of Bicyclo[2.2.2]octane‐Based 2‐Amino‐3‐Carboxylic Acids on Macrocyclic Glycopeptide Chiral Stationary Phases

Direct high‐performance liquid chromatographic (HPLC) separation of four bicyclo[2.2.2]octane based 2‐amino‐3‐carboxylic acid enantiomers were developed on chiral stationary phases (CSPs) containing different macrocyclic glycopeptide antibiotic selectors. The analyses were performed under reversed‐phase, polar organic and polar ionic mode on macrocyclic‐glycopeptide‐based Chirobiotic T, T2, TAG, and R columns. The effects of the mobile phase composition including the acid and base modifier, the structure of the analytes, and the temperature on the separations were investigated. Experiments were achieved at constant mobile phase compositions on different stationary phases in the temperature range 5–40°C. Thermodynamic parameters were calculated from plots of ln k or ln α versus 1/T. It was recognized that the enantioseparations in reversed‐phase and polar organic mode were enthalpically driven, but under polar‐ionic conditions entropically driven enantioseparation was observed as well. Baseline separation and determination of elution sequence were achieved in all cases. Chirality 26:200–208, 2014. © 2014 Wiley Periodicals, Inc.     

Screening Approach for Chiral Separation of β‐Aminoketones by HPLC on Various Polysaccharide‐Based Chiral Stationary Phases

Nine β‐aminoketones were synthesized via Mannich reaction when benzaldehyde was condensed with some primary amines and acetophenone. The purified compounds were identified by using spectroscopic methods. The enantiomeric separation of these derivatives was carried out by high‐performance liquid chromatography (HPLC) using several coated and immobilized polysaccharide stationary phases, namely, Chiralcel^® OD‐H, Chiralcel^® OD, Chiralcel^® OJ, Chiralpak^® AD, Chiralpak^® IA, and Chiralpak^® IB using different mobile phases composed of n‐hexane and alcohol mixed in various ratios or pure ethanol or isopropanol. The retention behavior and selectivity of these chiral stationary phases were examined in isocratic normal phase mode. The results indicate that cellulose derivatives have higher enantioselectivity than amylose derivatives for the separation of racemic β‐amino ketones. Chirality 27:332–338, 2015. © 2015 Wiley Periodicals, Inc.     

Unusual Temperature‐Induced Retention Behavior of Constrained β‐Amino Acid Enantiomers on the Zwitterionic Chiral Stationary Phases ZWIX(+) and ZWIX(–)

The effects of temperature on the chiral recognition of cyclic β‐amino acid enantiomers on zwitterionic [Chiralpak ZWIX(+) and ZWIX(–)] chiral stationary phases were investigated. Experiments were performed at different mobile phase compositions and under 10°C column temperature increments in the temperature range 10–50°C. Apparent thermodynamic parameters and T_iso values were calculated from plots of ln k and ln α versus 1/T, respectively. Unusual temperature behavior was observed, especially on the ZWIX(–) column, where the application of MeOH/MeCN (50/50 v/v) containing 25 mM triethylamine and 50 mM formic acid as mobile phase led to nonlinear van't Hoff plots and increasing retention time with increasing temperature. On both columns, both enthalpically and entropically driven separations were observed. Chirality 26:385–393, 2014. © 2014 Wiley Periodicals, Inc.     

Stopped‐Flow Enantioselective HPLC‐CD Analysis and TD‐DFT Stereochemical Characterization of Methyl Trans‐3‐(3,4‐Dimethoxyphenyl)Glycidate

Caffeic acid‐derived polyethers are a class of natural products isolated from the root extracts of comfrey and bugloss, which are endowed with intriguing pharmacological properties as anticancer agents. The synthesis of new polyether derivatives is achieved through ring‐opening polymerization of chiral 2,3‐disubstituted oxiranes, whose absolute configurations define the overall stereochemistry of the produced polymer. The absolute stereochemistry of one of these building blocks, methyl trans‐3‐(3,4‐dimethoxy‐phenyl)glycidate ( 3 ), was therefore characterized by the combination of enantioselective high‐performance liquid chromatography (HPLC), electronic circular dichroism (ECD) spectroscopy, and time‐dependent density functional theory (TD‐DFT) calculations. Initial efforts aiming at the isolation of enantiomers by means of a standard preparative HPLC protocol followed by offline ECD analysis failed due to unexpected degradation of the samples after collection. The stopped‐flow HPLC‐CD approach, by which the ECD spectra of enantiomers are measured online with the HPLC system, was applied to overcome this issue and allowed a fast, reliable, and chemical‐saving analysis, while avoiding the risks of sample degradation during the collection and processing of enantiomeric fractions. Subsequent TD‐DFT calculations identified ( (2S,3R)-3 as the first eluted enantiomeric fraction on the Lux Cellulose‐2 column, therefore achieving a full stereochemical characterization of the chiral oxirane under investigation. Chirality 27:914–918, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

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.     

Enantiomeric impurities in chiral catalysts,auxiliaries, and synthons used in enantioselective syntheses. Part 5

The enantiomeric excess of chiral starting materials is one of the important factors determining the enantiopurity of products in asymmetric synthesis. Fifty‐one commercially available chiral reagents used as building blocks, catalysts, and auxiliaries in various enantioselective syntheses were assayed for their enantiomeric purity. The test results were classified within five impurities level (ie, <0.01%, 0.01%‐0.1%, 0.1%‐1%, 1%‐10%, >10%). Previously from 1998 to 2013, several reports have been published on the enantiomeric composition of more than 300 chiral reagents. This series of papers is necessitated by the fact that new reagents are forthcoming and that the enantiomeric purity of the same reagent can vary from batch to batch and/or from supplier to supplier. This report presents chiral liquid chromatography (LC) and gas chromatography (GC) methods to separate enantiomers of chiral compounds and evaluate their enantiomeric purities. The accurate and efficient LC analysis was done using newly introduced superficially porous particle (SPP 2.7 μm) based chiral stationary phases (TeicoShell, VancoShell, LarihcShell‐P, and NicoShell).     

High‐Performance Liquid Chromatographic Enantioseparation of Cyclic β‐Amino Acids on Zwitterionic Chiral Stationary Phases Based on Cinchona Alkaloids

Stereoselective high‐performance liquid chromatographic separations of eight sterically constrained cyclic β‐amino acid enantiomer pairs were carried out using the newly developed Cinchona alkaloid‐based zwitterionic chiral stationary phases Chiralpak ZWIX(+) and ZWIX(?). The effects of the mobile phase composition, the nature and concentrations of the acid and base additives, the counterions and temperature on the separations were investigated. The changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calculated from the linear van't Hoff plots derived from the ln α vs. 1/T curves in the studied temperature range (10–50°C). The values of the thermodynamic parameters depended on the nature of the selectors and the structures of the analytes. Unusual temperature behavior was observed on the ZWIX(?) column: decreased retention times were accompanied by increased separation factors with increasing temperature. On the ZWIX(+) column only enthalpically, whereas on the ZWIX(?) column both enthalpically and entropically driven separations were observed. The elution sequence was determined in all cases and was observed to be the opposite on ZWIX(+) and on ZWIX(?). Chirality 27:563–570, 2015. © 2015 Wiley Periodicals, Inc.     

Enantiomeric Separations of Pyriproxyfen and its Six Chiral Metabolites by High‐Performance Liquid Chromatography

Pyriproxyfen is a chiral insecticide, and over 10 metabolites have been identified in the environment. In this work the separations of the enantiomers of pyriproxyfen and its six chiral metabolites were studied by high‐performance liquid chromatography (HPLC). Both normal phase and reverse phase were applied using the chiral columns Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralcel OD, Chiralcel OD‐RH, Chiralpak AY‐H, Chiralpak AD‐H, Chiracel OJ‐H, (R,R)‐Whelk‐O 1, and Lux Cellulose‐3. The effects of the chromatographic parameters such as mobile phase composition and temperature on the separations were investigated and the enantiomers were identified with an optical rotation detector. The enantiomers of these targets could obtain complete separations (resolution factor Rs > 1.5) on Chiralpak IA, Chiralpak IB, Chiralcel OD, Chiralpak AY‐H, or Chiracel OJ‐H under normal conditions. Chiralcel OJ‐H showed the best chiral separation results with n‐hexane as mobile phase and isopropanol (IPA) as modifier. The simultaneous enantiomeric separation of pyriproxyfen and four chiral metabolites was achieved on Chiralcel OJ‐H under optimized condition: n‐hexane/isopropanol = 80/20, 15°C, flow rate of 0.8 ml/min, and UV detection at 230 nm. The enantiomers of pyriproxyfen and the metabolites A , C , and D obtained complete separations on Chiralpak IA, Chiralpak IC, and Lux Cellulose‐3 under reverse phase using acetonitrile/water as the mobile phase. The retention factors (k) and selectivity factors (α) decreased with increasing temperature, and the separations were better under low temperature in most cases. The work is of significance for the investigation of the environmental behaviors of pyriproxyfen on an enantiomeric level. Chirality 28:245–252, 2016. © 2016 Wiley Periodicals, Inc.