Chiral Separation Method Research Articles

Residue and Dietary Risk Assessment of Chiral Cyflumetofen in Apple.

Ultra-performance convergence chromatography is an environmentally-friendly analytical method that uses dramatically reduced amounts of organic solvents. In addition, a robust and highly sensitive chiral separation method was developed for the novel chiral acaricide cyflumetofen by using ultra-performance convergence chromatography coupled with tandem mass spectrometry, which shows that stereoisomer recoveries determined for various apple parts ranged from 78.3% to 119.9%, with the relative standard deviations being lower than 14.0%. The half-lives of (−)-cyflumetofen and (+)-cyflumetofen obtained under 5-fold applied dosage equal to 22.13 and 22.23 days, respectively. For 1.5-fold applied dosage, the respective values were determined as 22.42 and 23.64 days, i.e., the degradation of (−)-cyflumetofen was insignificantly favored over that of its enantiomer. Importantly, cyflumetofen was unevenly distributed in apples, with its relative contents in apple peel, peduncle, and pomace equal to 50%, 22%, and 16%, respectively. The proposed method can be used to efficiently separate and quantify chiral pesticide with advantages of a shorter analysis time, greater sensitivity, and better environmental compatibility. Additionally, the consumption of apples with residue of cyflumetofen did not pose a health risk to the population if the cyflumetofen applied under satisfactory agricultural practices after the long-term dietary risk assessment.

High-fast enantioselective determination of prothioconazole in different matrices by supercritical fluid chromatography and vibrational circular dichroism spectroscopic study

Herein, we developed a rapid supercritical fluid chromatography (SFC) method for chiral separation and enantioselective determination of prothioconazole in soil and tomatoes. The potential effects of chiral stationary phases, co-solvents, column temperature, and back pressure on enantioseparation of prothioconazole have been studied in detail. Two prothioconazole enantiomers were best separated on cellulose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase with CO2-2-propanol (80:20, v/v) as the mobile phase, and the run time through SFC (about 4 min) was five-fold shorter than HPLC. Then, through comparing the experimental vibrational circular dichroism spectrum of the later-eluted component with the calculated pattern based on the (R)-configuration, it should be (R)-(-)-prothioconazole. Moreover, the modified QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction and cleanup procedures were applied in enantiomeric analysis of prothioconazole in two matrices. Good linearity (R2 ≥ 0.9992) and recoveries (91.84–101.66%, RSD ≤ 3.98%) for two enantiomers were achieved. This proposed method showed good accuracy and precision, and might be suitable for fast enantioselective determination and residual quantitative analysis of prothioconazole in food and environmental samples.

Enantiomeric impurity analysis using circular dichroism spectroscopy with United States Pharmacopeia liquid chromatographic methods

Over 300 chiral drug substances lack official United States Pharmacopeia (USP) methods for the enantiomeric purity determination. Because enantiomeric analysis typically requires specialized methods for each drug compound, developing protocols for each of these 300+ substances would be an expensive and laborious endeavor. Alternatively, if a detector capable of determining the enantiomeric composition without chiral separation could be used with certain drug compounds, this could be implemented relatively rapidly into official testing monographs. Circular dichroism (CD) detection following HPLC (HPLC-CD) has been proposed for this purpose but studies performed thus far have not prioritized its compatibility with validated regulatory methods. In this study, HPLC-CD was evaluated for enantiomeric purity determinations of 13 drug substances using HPLC methods consistent with assay protocols described in United States Pharmacopeia (USP) monographs. Of these selected substances, three (sitagliptin, timolol, and levalbuterol) showed no CD activity and one other (levofloxacin) could not be analyzed due to incompatibility of the mobile phase with the CD detector. For the remaining 9 substances, method validation was performed to determine the linearity, accuracy, precision and limits of quantitation of enantiomer impurities, which was compared to limits established by USP. It was found that enantiomeric impurities for four substances (pramipexole, levocetirizine, (S)-citalopram, and tolterodine) could be quantitatively determined at levels suitable to USP specifications. This analysis demonstrated that HPLC-CD does provide an effective enantiomeric characterization strategy for compatible chiral compounds, and can be implemented quickly and economically compared to traditional column-dependent chiral separation or derivatization methods.

Separation of enantiomers of new psychoactive substances by high-performance liquid chromatography.

New psychoactive substances are defined as compounds with consciousness-changing effects and have been developed simultaneously with classical drugs. They arise through structural modifications of illegal substances and are mainly produced to circumvent laws. Availability is simple, since new psychoactive substances can be purchased from the Internet. Among them many chemical drug compound classes are chiral and thus the two resulting enantiomers can differ in their effects. The aim of this study is to develop a suitable chiral high-performance liquid chromatography separation method for a broad spectrum of new psychoactive substances using cellulose tris(3,5-dichlorophenylcarbamate) as a chiral selector. Experiments were performed by high-performance liquid chromatography in normal-phase mode under isocratic conditions using ultraviolet detection. Direct separation was carried out on a high-performance liquid chromatography column (Lux® i-Cellulose-5, 3.5 μm, Phenomenex®), available since 2016. Excellent separation results were obtained for cathinones. After further optimization, even 47 instead of 39 out of 52 cathinones showed baseline separation. For amphetamine derivatives, satisfactory results were not achieved. Further, new psychoactive substances from other compound classes such as benzofuranes, thiophenes, phenidines, phenidates, morpholines, and ketamines were partially resolved, depending on the polarity and degree of substitution. All analytes, which were mainly purchased from the Internet, were proven to be traded as racemates.

Analysis of HETEs in human whole blood by chiral UHPLC-ECAPCI/HRMS

The biosynthesis of eicosanoids occurs enzymatically via lipoxygenases, cyclooxygenases, and cytochrome P450, or through nonenzymatic free radical reactions. The enzymatic routes are highly enantiospecific. Chiral separation and high-sensitivity detection methods are required to differentiate and quantify enantioselective HETEs in complex biological fluids. We report here a targeted chiral lipidomics analysis of human blood using ultra-HPLC-electron capture (EC) atmospheric pressure chemical ionization/high-resolution MS. Monitoring the high-resolution ions formed by the fragmentation of pentafluorobenzyl derivatives of oxidized lipids during the dissociative EC, followed by in-trap fragmentation, increased sensitivity by an order of magnitude when compared with the unit resolution MS. The 12(S)-HETE, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid [12(S)-HHT], and 15(S)-HETE were the major hydroxylated nonesterified chiral lipids in serum. Stimulation of whole blood with zymosan and lipopolysaccharide (LPS) resulted in stimulus- and time-dependent effects. An acute exposure to zymosan induced ∼80% of the chiral plasma lipids, including 12(S)-HHT, 5(S)-HETE, 15(R)-HETE, and 15(S)-HETE, while a maximum response to LPS was achieved after a long-term stimulation. The reported method allows for a rapid quantification with high sensitivity and specificity of enantiospecific responses to in vitro stimulation or coagulation of human blood.

Enantiomeric separation and quantification of R/S-amphetamine in urine by ultra-high performance supercritical fluid chromatography tandem mass spectrometry

To distinguish between legal and illegal consumption of amphetamine reliable analytical methods for chiral separation of the R- and S-enantiomers of amphetamine in biological specimens are required. In this regard, supercritical fluid chromatography (SFC) has several potential advantages over liquid chromatography, including rapid separation of enantiomers due to low viscosity and high diffusivity of supercritical carbon dioxide, the main component in the SFC mobile phase. A method for enantiomeric separation and quantification of R- and S-amphetamine in urine was developed and validated using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS). Sample preparation prior to UHPSFC-MS/MS analysis was a semi-automatic solid phase extraction method. The UHPSFC-MS/MS method used a Chiralpak AD-3 column with a mobile phase consisting of CO2 and 0.2% cyclohexylamine in 2-propanol. The injection volume was 2 μL and run-time was 6 min. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 136.1 > 119.0 and m/z 136.1 > 91.0). The calibration range was 50–10,000 ng/mL for each enantiomer. The between-assay relative standard deviations were in the range of 3.7–7.6%. Recovery was 92–93% and matrix effects ranged from 100 to 104% corrected with internal standard. After development and validation, the method has been successfully implemented in routine use at our laboratory for both separation and quantification of R/S-amphetamine, and has proved to be a reliable and useful tool for distinguishing intake of R- and S-amphetamine in authentic patient samples.

Analysis of methamphetamine and amphetamine chiral enantiomers in hair by high performance liquid chromatography-tandem mass spectrometry

A chiral separation method was developed for the analysis of methamphetamine (METH) and amphetamine (AM) in hair by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The samples were extracted using methanol by ultrasonication in a high-temperature water bath. The chromatographic separation was performed on a SUPELCO Astec CHIROIOTIC® V2 column with the mobile phase of methanol-20 mmol/L ammonium acetate aqueous solution containing 0.1% (v/v) formic acid (99:1, v/v). All the compounds showed linear relationship in the range of 15-300 ng/mg (r>0.99). The limits of detection (LODs) of METH and AM were 0.1 ng/mg and 0.15 ng/mg, respectively. The limits of quantification (LOQs) of METH and AM were 0.4 ng/mg and 0.5 ng/mg, respectively. The inter-day precisions were all ≤ 6.8%, and the intra-day precisions were all ≤ 11.4%. Fifty drug abuse samples were determined by this method. Up to 70% of the cases were determined as S-(+)-METH and S-(+)-AM, while 18% of the cases were S-(+)-METH, S-(+)-AM, R-(-)-METH and R-(-)-AM. The proposed method is rapid, simple, and provides technical support and a scientific basis for the chiral analysis in actual hair samples for forensic toxicant identification.

Enantiomeric separation of D, L-isoleucine in citric acid-zinc (Ⅱ) medium by capillary electrophoresis

The enantiomeric separation of unmodified D,L-isoleucine was achieved in citric acid-zinc(Ⅱ) medium by capillary electrophoresis (CE) with contactless conductivity detector (C4D). In the conventional chiral separation methods of amino acid, a chiral complex used as the chiral selector was added into the eluent in order to yield a chiral environment. However, in this study a non-chiral solution, i. e. 2.8 mmol/L NaOH+0.8 mmol/L citric acid+2.0 mmol/L zinc acetate was used as the running buffer, and the citric acid-zinc(Ⅱ) acted the role of a chiral selector. Under the optimum experimental conditions:uncoated fused-silica capillary (45 cm×50 μm, Leff=40 cm), separation voltage of +13 kV, electrokinetic injection of 11 kV×8 s, the enantiomers of D,L-isoleucine were baseline separated within 8 min with the resolutions (Rs) of 2.0. The calibration curve of each enantiomer showed good linearity in the range from 1.0 mg/L to 20 mg/L, with the limits of detection of 0.40 mg/L. The intra-and inter-day precisions were examined. The RSDs of peak area and migration time were found to be below 5.0% and 2.5% (n=6), respectively, indicating good repeatability (intra-day) and reproducibility (inter-day) of the method. Interference experiment was also tested. As a result, other common amino acids did not interfere with the detection. The proposed method provided a potential new way to further investigate the enantioseparation of unmodified or native amino acids.

Enantioselective separation of RS-mandelic acid using β-cyclodextrin modified Fe3O4@SiO2/Au microspheres.

Chiral magnetic microspheres show great potential in direct enantioseparation. Herein novel chiral magnetic microspheres were prepared by self-assembling β-cyclodextrin on magnetic Fe3O4@SiO2/Au composite microspheres. The prepared materials were characterized by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), vibrating sample magnetometry (VSM) and Fourier transform infrared spectroscopy (FTIR). The results showed that the functional microspheres have a three-ply structure and high saturation magnetization. β-Cyclodextrin-immobilized magnetic Fe3O4@SiO2/Au microspheres (Fe3O4@SiO2/Au/β-CD) were applied in the direct chiral separation of RS-mandelic acid. The analysis results of HPLC revealed that the Fe3O4@SiO2/Au/β-CD magnetic microspheres exhibited stronger enantioselective adsorption for R-mandelic acid than S-mandelic acid. The chiral selector functionalized magnetic microspheres are therefore expected to be an efficient and economical chiral separation method for use in further research.

Chiral separation of quinolones by liquid chromatography and capillary electrophoresis.

The quinolones are derivatives of oxoquinolines and mostly known for their antibacterial and antiviral activities. Many quinolones are chiral compounds having asymmetric centers and important due to their enantioselective biological activities. In order to study the biological activities of quinolone enantiomers, to control the manufacturing of homochiral drugs and to prepare necessary quantities of pure enantiomers for preclinical or clinical trials, respective chiral separation methods are urgently needed. In this context, the present review discusses chromatographic and electrophoretic methods for the enantioseparation of chiral quinolones and provides some useful information on their physical and pharmaceutical properties. The drawbacks of currently used techniques are revealed and ways to overcome them are outlined. Moreover, recommendations for an optimal choice of a separation protocol are given.

Preparation of filter paper with chiral separation function by oxidation and Schiff-base reaction

Paper chromatography (PC) is a highly flexible, fast, and efficient separation method. In this study, a chromatographic paper with a chiral separation function was investigated and a paper chromatographic method for chiral separation was developed. Single-factor experiments and orthogonal tests were used to determine the optimal oxidation conditions for filter paper. The optimal conditions were that sodium periodate 4% (mass percentage), pH 2 buffer solution, reaction temperature 45℃, and reaction time 4 h. Under the optimal conditions, the aldehyde group content of a dialdehyde-based filter paper was 57.93% (amount of substance percentage). A paper-based chiral separation material was synthesized by a microwave-assisted Schiff-base reaction of the oxidized filter paper with L-glutamic acid. The developing solvent for separating racemic tartaric acid using this chiral filter paper consisted of 100 mL of n-butanol, 50 mL of 50%(v/v) acetic acid, and 0.1000 g of bromophenol blue. The rate of flow (Rf) values of L-tartaric acid and D-tartaric acid were 0.52 and 0.40, respectively. This method does not require large-scale equipment to perform the chiral separation and it is therefore suitable for general teaching, research, and industrial applications.

Enantioselective analysis of fluoxetine in pharmaceutical formulations by capillary zone electrophoresis

Fluoxetine is an antidepressant, a selective serotonin reuptake inhibitor (SSRI) used primarily in the treatment of major depression, panic disorder and obsessive compulsive disorder. Chiral separation of racemic fluoxetine is necessary due to its enantioselective metabolism. In order to develop a suitable method for chiral separation of fluoxetine, cyclodextrin (CD) modified capillary electrophoresis (CE) was employed. A large number of native and derivatized, neutral and ionized CD derivatives were screened to find the optimal chiral selector. As a result of this process, heptakis(2,3,6-tri-O-methyl)-β-CD (TRIMEB) was selected for enantiomeric discrimination. A factorial analysis study was performed by orthogonal experimental design in which several factors are varied at the same time to optimize the separation method. The optimized method (50mMphosphate buffer, pH=5.0, 10mMTRIMEB, 15°C, + 20kV, 50mbar/1s, detection at 230nm) was successful for baseline separation of fluoxetine enantiomers within 5min. Our method was validated according to ICH guidelines and proved to be sensitive, linear, accurate and precise for the chiral separation of fluoxetine.

Chiral separation of ketoprofen on an achiral NH2 column by HPLC using vancomycin as chiral mobile phase additive

A high-performance liquid chromatographic method for chiral separation of ketoprofen racemate was developed. (R)- and (S)-ketoprofen enantiomers were separated on a LiChrosorb NH2 column (250 mm × 4.6 mm, i.d 5 µm) at 20 °C, using 2-propanol/potassium dihydrogen phosphate buffer (pH 6.0, 0.05 M) (50:50 v/v). Containing vancomycin as the mobile phase, at a flow rate of 0.8 ml min−1 and detection wavelength of UV, the detector was set at 310 nm. Under these conditions, ketoprofen enantiomers could be separated with a selectivity factor (α) of 2.172 and a resolution (Rs) of 4.78 using extremely low concentrations of the vancomycin chiral additive.

Chiral separation and determination of enantiomeric purity of the pharmaceutical formulation of cefadroxil using coated and immobilized amylose-derived and cellulose-derived chiral stationary phases

Background and objectives In the present article, we describe the development of a simple, direct, and isocratic high-performance liquid-chromatographic method for chiral separation and the determination of the enantiomeric purity of cefadroxil. Cefadroxil has three chiral centers; the existence of eight different stereoisomers is possible. Only one of these isomers is currently under development as an antibiotic agent and, consequently, the other seven isomers are considered as unwanted chiral impurities.Materials and methods An analytical chiral separation was carried out to check its enantiomeric purity. The separation was carried out by exploiting the high efficiency of several coated/immobilized cellulose and amylose chiral stationary phases under normal-phase and polar-organic modes. The effects of type and concentration of the alcoholic modifiers, 2-propanol and ethanol, on the separation of stereoisomers were studied for optimum resolution.Results and conclusion Complete baseline separation of stereoisomers with good resolution was achieved within 40 min under normal-phase mode on Chiralpak IB column using hexane–2-propanol (60 : 40 v/v) as the mobile phase, without any organic additives, at a flow rate of 0.4 ml/min at 25°C and with the ultraviolet detection set at 268 nm. This process was found to be suitable for rapid enantiomeric purity analysis and a quality control of cefadroxil in pharmaceutical formulations without interference of the excipients. The chiral recognition mechanisms of separation were also discussed.

Stereospecific Metabolism of the Tobacco-Specific Nitrosamine, NNAL.

Among the most potent carcinogens in tobacco are the tobacco-specific nitrosamines (TSNAs), with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) being the most potent as well as one of the most abundant. NNK is extensively metabolized to the equally carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Of the two NNAL enantiomers, (S)-NNAL not only appears to be preferentially glucuronidated and excreted in humans but also exhibits higher stereoselective tissue retention in mice and humans and has been shown to be more carcinogenic in mice than its (R) counterpart. Due to the differential carcinogenic potential of the NNAL enantiomers, it is increasingly important to know which UGT enzyme targets the specific NNAL enantiomers for glucuronidation. To examine this, a chiral separation method was developed to isolate enantiomerically pure (S)- and (R)-NNAL. Comparison of NNAL glucuronides (NNAL-Glucs) formed in reactions of UGT2B7-, UGT2B17-, UGT1A9-, and UGT2B10-overexpressing cell microsomes with pure NNAL enantiomers showed large differences in kinetics for (S)- versus (R)-NNAL, indicating varying levels of enantiomeric preference for each enzyme. UGT2B17 preferentially formed (R)-NNAL-O-Gluc, and UGT2B7 preferentially formed (S)-NNAL-O-Gluc. When human liver microsomes (HLM) were independently incubated with each NNAL enantiomer, the ratio of (R)-NNAL-O-Gluc to (S)-NNAL-O-Gluc formation in HLM from subjects exhibiting the homozygous deletion UGT2B17 (*2/*2) genotype was significantly lower (p = 0.012) than that with HLM from wild-type (*1/*1) subjects. There was a significant trend (p = 0.015) toward a decreased (R)-NNAL-O-Gluc/(S)-NNAL-O-Gluc ratio as the copy number of the UGT2B17*2 deletion allele increased. These data demonstrate that variations in the expression or activity of specific UGTs may affect the clearance of specific NNAL enantiomers known to induce tobacco-related cancers.

Rapid chiral separation of racemic cetirizine in human plasma using subcritical fluid chromatography-tandem mass spectrometry

A method for fast chiral separation of cetirizine and quantitation of levocetirizine in human plasma using subcritical fluid chromatography with tandem mass spectrometry was developed and validated. The chromatographic separation was performed using a Chiralpak IE column (2.1mm×150mm, 5μm) with an isocratic elution of CO2/organic modifier (55/45, v/v) at a flow rate of 0.85mL/min. The organic modifier was composed of water/methanol (5/95, v/v). The makeup flow was optimized at water/methanol (10/90, v/v) and 0.2mL/min. The most influential parameters on the separation of cetirizine affecting resolution, retention time and sensitivity were selected by fractional factorial design. The 3 selected factors were optimized by response surface methodology. Tandem mass spectrometry was used at electrospray ionization, positive ion mode, and multiple-reaction monitoring mode. Isotope-labeled cetirizine-d4 was used as the internal standard. The sample preparation of human plasma was conducted by solid phase extraction of hydrophilic-lipophilic balance (HLB) type. The developed method was validated for selectivity, linearity, precision, accuracy, recovery, limit of quantitation (LOQ), and limit of detection (LOD). The real human plasma samples were analyzed and the pharmacokinetic results were compared with results of previous research. The developed method was found to be reliable based on the similarity between the results of the current and previous methods. The chiral separation for cetirizine and economic feasibility were compared with those of previous studies using normal phase-HPLC or reversed phase-HPLC. The established analytical method could be successfully applied to pharmacokinetic study with reduction in the analysis time and costs.

Enantioseparation of Racemic Flurbiprofen by Aqueous Two-Phase Extraction With Binary Chiral Selectors of L-dioctyl Tartrate and L-tryptophan.

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two-phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two-phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L-dioctyl tartrate and L-tryptophan, which were screened from amino acids, β-cyclodextrin derivatives, and L-tartrate esters. Factors such as the amounts of L-dioctyl tartrate and L-tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L-dioctyl tartrate, 80 mg; L-tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L-dioctyl tartrate and L-tryptophan, which enantioselectively recognized R- and S-enantiomers in top and bottom phases, respectively. Compared to conventional liquid-liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers.

Development of Electrophoretic Methods for Simultaneous Determination of Enantiomeric Ratio and Composition of Diastereomeric Salt Mixtures

Obtaining enantiomeric pure compounds is—among other techniques—possible in a resolvation experiment via diastereomeric salt formation, excellently exemplified by a modified Pope–Peachy method performed in supercritical carbon dioxide as solvent. The salt precipitation is followed by supercritical fluid extraction (SFE) to separate the diastereomeric salts and the unreacted enantiomers. To evaluate the extraction efficiency, conversion and enantioselectivity achieved, it is essential to determine the enantiomer excess and the residual resolving agent content in extracts and raffinates. Carefully chosen experimental parameters enable the simultaneous determination of certain anions and cations in capillary electrophoresis in a single run, which has not been reported for diastereomeric mixtures so far. In this paper, a partially validated chiral selective cyclodextrin enabled capillary electrophoresis method is presented for the characterization of cis-permethrinic acid samples resolved with (R)-1-phenylethylamine prepared by the SFE-based resolvation technique. To evaluate the efficiency of the resolvation, a cyclodextrin enabled chiral separation method was developed applying permethylated-β-cyclodextrin as chiral selector. The theoretical possibility of the widespread application of the developed method (with minor adjustments) is justified for other selectands and selectors. The developed methods can be thereby applied for the fast and reliable control of resolvation experiments.

Validated chiral high performance liquid chromatography separation method and simulation studies of dipeptides on amylose chiral column

Chiral resolution of dl-alanine-dl-tyrosine and dl-leucine-dl-phenylalanine dipeptides was achieved on AmyCoat-RP column. The mobile phase used for dl-alanine-dl-tyrosine was acetonitrile-ammonium acetate (10mM, pH 6.0) [50:50, v/v]. It was acetonitrile–methanol–ammonium acetate (10mM; pH adjusted to 4.5 with glacial acetic acid) [50:20:30, v/v] for dl-leucine-dl-phenylalanine. The flow rate of the mobile phases was 0.8mL/min with UV detection at 275nm. The values of retention factors for ll-, dd-, dl- and ld-stereomers of dl-alanine-dl-tyrosine were 1.71, 2.86, 5.43 and 9.42, respectively. The values of separation and resolution factors were 1.67, 1.90 and 1.73 and 2.88, 6.43 and 7.90, respectively. Similarly, these values for dl-leucine-dl-phenylalanine stereomers were 1.50, 2.88, 3.50 and 4.07 (retention factors), 1.92, 1.22 and 1.62 (separation factors) and 2.67, 1.55 and 2.30 (resolution factors). The limits of detections and quantitation were ranged from 2.03 to 6.40 and 6.79 to 21.30μg/mL, respectively. The modeling studies were in agreement with the elution orders. The mechanism of chiral recognition was established by modeling and chromatographic studies. It was observed that hydrogen bondings and π–π interactions are the major forces for chiral separation.

Scope of partial least-squares regression applied to the enantiomeric composition determination of ketoprofen from strongly overlapped chromatographic profiles.

Valuable quantitative information could be obtained from strongly overlapped chromatographic profiles of two enantiomers by using proper chemometric methods. Complete separation profiles where the peaks are fully resolved are difficult to achieve in chiral separation methods, and this becomes a particularly severe problem in case that the analyst needs to measure the chiral purity, i.e., when one of the enantiomers is present in the sample in very low concentrations. In this report, we explore the scope of a multivariate chemometric technique based on unfolded partial least-squares regression, as a mathematical tool to solve this quite frequent difficulty. This technique was applied to obtain quantitative results from partially overlapped chromatographic profiles of R- and S-ketoprofen, with different values of enantioresolution factors (from 0.81 down to less than 0.2 resolution units), and also at several different S:R enantiomeric ratios. Enantiomeric purity below 1% was determined with excellent precision even from almost completely overlapped signals. All these assays were tested on the most demanding condition, i.e., when the minor peak elutes immediately after the main peak. The results were validated using univariate calibration of completely resolved profiles and the method applied to the determination of enantiomeric purity of commercial pharmaceuticals.