Enantioselective gas chromatographic assay with electron-capture detection for dl-ritalinic acid in plasma

Enantioselective gas chromatographic assays for the quantitation of methylphenidate and its major metabolite ritalinic acid in plasma are described. The procedures involved the extraction of methylphenidate enantiomers from alkanised plasma. The plasma was then washed to ensure complete removal of methylphenidate before saturation with sodium carbonate to promote the extraction of ritalinic acid enantiomers with ethyl acetate-isopropanol (60:40) solvent mixture. Subsequently, ritalinic acid enantiomers were converted back into methylphenidate enantiomers by Fisher-Speier esterification. N-Heptafluorobutyryl-L-prolyl chloride, a chiral acylating reagent, was used to convert the enantiomers of methylphenidate into their corresponding diastereomeric amide derivatives, which were separated cleanly on an achiral capillary column (OV-225) and quantitated with electron-capture detection. The assays were sensitive, reliable and reproducible.     

Determination of (13)C natural abundance of amino acid enantiomers in soil: methodological considerations and first results

The application of a combined gas chromatography-combustion/isotope ratio mass spectrometry (GC-C/IRMS) method for stable carbon isotope analysis of amino acid enantiomers in soil samples is presented. Triplicate delta(13)C analyses of pentafluoropropionyl (PFP) isopropyl ester derivatives of 27 amino acid enantiomers revealed that discrimination of (13)C during derivatization is different for different amino acid enantiomers and different amounts. Injection of increasing amounts of amino acid derivatives showed that the isotopic signal varied up to 10 per thousand for D-aspartic acid. Correction for the delta(13)C signal of underivatized amino acid enantiomers is possible for all investigated amino acid enantiomers using logarithmic functions. Operating the GC-C/IRMS system in the split-mode (split ratio 1:12) is possible but resulted in a higher isotopic discrimination. The detection limit approached 3 ng for some amino acid enantiomers in the splitless mode, while the lower limit of routine determination exceeded 10 ng injection amount. The upper limit at which accurate stable isotope values were obtained was 200 ng injection amount. Compound-specific delta(13)C analysis of alanine, valine, aspartic and glutamic acid showed that the D-forms were enriched in (13)C relative to the L-forms, suggesting that microbes significantly contributed to the formation of the D-enantiomers in soil.     

C18柱串联冠醚手性柱高效分离3种芳香族氨基酸对映体

将C18柱与手性冠醚柱串联,建立了一种反相高效液相色谱法用于3种芳香族氨基酸对映体同时拆分的方法.考察了反相色谱流动相的组成、pH值、柱温、流速对对映体拆分的影响.实验结果表明,当流动相为HClO4-乙睛溶液(86:14,V/V,pH 2.0)、柱温20℃、流速0.4 mL/min时,3种氨基酸对映体可获得基线分离.进一步对比了C18柱、冠醚手性柱和串联顺序不同的4种分离模式,结果表明,C18柱不能拆分氨基酸对映体,仅能分离不同种类氨基酸;冠醚手性柱可分离氨基酸映体,但不同种类氨基酸色谱峰出现重叠;串联模式能实现3种氨基酸对映体的基线分离,实现双柱优势互补,而串联顺序对分离影响不大,仅影响色谱峰的峰形.     

人血清白蛋白与氨基酸对映异构体的差异性识别研究

以人血清白蛋白为识别主体,利用表面等离子体共振(SPR)技术实时研究了其与5种氨基酸对映异构体相互作用过程中的动态行为。实验结果显示,每种氨基酸分子的L-和D-型异构体在与人血清白蛋白作用过程中均存在明显的动力学差异。动力学数据分析表明,虽然各种氨基酸的L-和D-型异构体与人血清白蛋白作用的结合速率常数、解离速率常数无统一规律,但在解离平衡常数或结合平衡常数上有一致性。每种氨基酸的L-型异构体与人血清白蛋白的亲和力均大于D-型,这表明人血清白蛋白在与氨基酸对映异构体作用的过程中基于手性识别作用而产生了明显的差异性结合。     

The solute molecule—Rigid or partially flexible? calculation of benzodiazepineR F values as an example

Summary The liquid-chromatographic separation of the enantiomers of amino acid esters as benzophenone Schiff-base derivatives on polysaccharide-derived chiral stationary phases (CSPs) is described. The performance of Chiralcel OF was superior to that of the other CSPs for resolution of benzophenone imine derivatives of amino acid ethyl and methyl esters. The enantiomers of most of the amino acid esters examined as their benzophenone imine derivatives were resolved to baseline on Chiralcel OF. The L-(−) enantiomers of all the analytes were preferentially retained on Chiralcel OF. The resolution of several imine derivatives of amino acid esters was investigated, as was the effect of eluent composition on the resolution of amino acid ethyl esters as their benzophenone imine derivatives.     

Gas-chromatographic separation of enantiomers of 2-chlorobutane and 2-bromobutane on a cyanuric acid-modified Carboblack C adsorbent

The enantiomers of 2-chlorobutane and 2-bromobutane were separated by gas chromatography on a Carboblack C adsorbent modified by 10% cyanuric acid. Upon thorough mechanical stirring of a solution, cyanuric acid was shown to form chiral supramolecular structures on the surface of adsorbent particles analogously to the Kondepudi formation of chiral crystals of achiral molecules. The enantiomers of 2-bromobutane and 2-chlorobutane were found to undergo, respectively, complete and partial separation on the cyanuric acid-modified Carboblack C adsorbent. The separation of enantiomers is due to a high enantioselectivity of the modified adsorbent. The analysis of thermodynamic functions of adsorption showed that the differences in specific retention volumes of enantiomers are caused by the fact that one of enantiomers is adsorbed predominantly within the cavity of the supramolecular structure of cyanuric acid and another one is adsorbed on its surface. Separation on the proposed chiral stationary phase is characterized by the relative standard deviation of retention volumes no more than 7%.     

毛细管电色谱-电喷雾-飞行时间/质谱联用分离分析混合氨基酸对映体

建立了一种简单高效的毛细管电色谱-电喷雾-飞行时间/质谱(CEC-ESI-TOF/MS)联用技术分离混合氨基酸对映体的方法。 以D,L-精氨酸、D,L-缬氨酸和D,L-谷氨酸为研究对象,通过优化CEC分离条件和MS检测条件,3种混合氨基酸对映体的6个组分在15 min内实现了分离,分离度分别为3.03、1.59和1.37。 该方法为混合对映体的分离分析提供了参考方法和基础数据。     

Separation of the enantiomers of pyrethroic acids and their esters by high-performance liquid chromatography on a polysaccharide-derived chiral stationary phase

Summary The liquid-chromatographic separation of the enantiomers of pyrethroic acids and their esters has been investigated on a polysaccharide-derived chiral stationary phase (CSP), Chiralpak AS. Good separation of the enantiomers of underivatized pyrethroic acids was achieved on the column, and the enantiomers of pyrethroic acid methyl and ethyl ester derivatives were also resolved.     

Direct TLC Resolution of (±)-Ketamine and (±)-Lisinopril by Use of (+)-Tartaric Acid or (−)-Mandelic Acid as Impregnating Reagents or Mobile Phase Additives. Isolation of the Enantiomers

Direct resolution of the enantiomers of the racemic drugs ketamine and lisinopril has been achieved by TLC. Enantiomerically pure tartaric acid and mandelic acid were used as chiral impregnating reagents and as mobile phase additives. When (?)-mandelic acid was used as chiral impregnating reagent use of ethyl acetate–methanol–water 3:1:1 (v/v) as mobile phase enabled successful resolution of the enantiomers of both compounds. For lisinopril, the mobile phase acetonitrile–methanol–water–dichloromethane 7:1:1:0.5 (v/v) was successful when (+)-tartaric acid was used as impregnating agent. When (+)-tartaric acid was used as mobile phase additive the mobile phase acetonitrile–methanol–(+)-tartaric acid (0.5% in water, pH 5)–glacial acetic acid 7:1:1.1:0.7 (v/v) enabled successful resolution of the enantiomers of lisinopril. The effects on resolution of temperature, pH, and the amount of chiral selector were also studied. The separated enantiomers were isolated and identified. Spots were detected with iodine vapour. LODs were 0.25 and 0.27 μg for each enantiomer of ketamine with (+)-tartaric acid and (?)-mandelic acid, respectively, whereas for lisinopril LODs were 0.14 and 0.16 μg for each enantiomer with (+)-tartaric acid (both conditions) and (?)-mandelic acid, respectively.     

Chromatographic determination of amino acid enantiomers in beers and raw materials used for their manufacture

Using gas chromatography (GC) on a chiral stationary phase, accompanied by high-performance liquid chromatography, beers and raw materials used for manufacturing (hops, barley grains, malts) were investigated for the pattern and quantities of amino acid enantiomers. Although L-amino acids were most abundant, certain D-amino acids were detected in all beers and most of the raw materials. Highest amounts of D-amino acids were detected in special beers such as Berliner Weisse that underwent bottle-conditioning with lactic cultures, and Belgian fruit beers produced by spontaneous fermentation. It is demonstrated that GC on chiral stationary phases is highly suitable for the quantitative determination of amino acid enantiomers in beers and raw materials used for their manufacture. Quantities, relative amounts and pattern of amino acid enantiomers can serve in particular as chiral markers for the authenticity of special beers.     

Determination of the 2,3-pentadienedioic acid enantiomer interconversion energy barrier 1. Classical kinetic approach

A classical kinetic method was used to determine the energy barrier for the inter-conversion of 2,3-pentadienedioic acid enantiomers. Each individual enantiomer was isolated by collecting the appropriate peaks from the HPLC enantiomeric separation, of racemic 2,3-pentadienedioic acid. The isolated enantiomers were racemized at 22 degrees C using various interconversion times. The ratio of enantiomers in each reaction solution was determined by HPLC at 22 degrees C. The corresponding peak areas of the enantiomers and the interconversion times obtained from the HPLC chromatograms were used to calculate both the interconversion rate constants describing (+)--> (-) and (-) --> (+) interconversions as well as the energy barriers. It was confirmed that the interconversion of 2,3-pentadienedioic acid enantiomers is a firstorder kinetic reaction. Both semiempirical and ab initio methods were used to explore the mechanism of the interconversion of 2,3-pentadienedioic acid enantiomers, and to calculate the interconversion energy barrier. Comparison of the interconversion energy barriers found by the ab initio method (deltaG# = 110.7 kJ/mol) and by classical kinetics in the mobile phase solution at 22 degrees C (delta Gapp = 93.9+/-0.2 kJ/mol) shows a difference which may be attributed to the different conditions assumed in the theoretical calculation (i.e., a gaseous state) and the actual experimental conditions (i. e., liquid solution) and a possible catalytic effect of the solution composition.     

扁桃酸及其衍生物对映体的手性识别研究

以两种手性氨基醇作为主体,运用1H NMR分别考察其对客体7个扁桃酸衍生物对映异构体的手性识别能力。结果表明,主客体物质的量之比为1∶1时,主体诱导客体对叔丁基扁桃酸消旋体最高产生37.6 Hz的化学位移差值。     

Preparation of the both enantiomers of threo-2-amino-3-methylhexanoic acid by enzymatic resolution and their conversion to optically active forms of threo-4-methylheptan-3-ol,a pheromone component of the smaller european elm bark beetle

The both enantiomers of threo-2-amino-3-methylhexanoic acid were prepared by resolving its racemic N-acetate with Aspergillus acylase. The amino acid enantiomers were converted to optically active forms of threo-4-methylheptan-3-ol, a pheromone component of Scolytus multistriatus.     

Separation and determination of the enantiomers of lactic acid and 2‐hydroxyglutaric acid by chiral derivatization combined with gas chromatography and mass spectrometry

Lactic acid and 2‐hydroxyglutaric acid are chiral metabolites that have two distinct d‐ and l ‐enantiomers with distinct biochemical properties. Perturbations of a single enantiomeric form have been found to be closely related to certain diseases. Therefore, the ability to differentiate the d and l enantiomers is important for these disease studies. Herein, we describe a method for the separation and determination of lactic acid and 2‐hydroxyglutaric acid enantiomers by chiral derivatization (with l‐ menthol and acetyl chloride) combined with gas chromatography and mass spectrometry. The two pairs of above‐mentioned enantiomers exhibited linear calibration curves with a correlation coefficient (R²) exceeding 0.99. The measured data were accurate in the acceptable recovery range of 88.17–102.30% with inter‐ and intraday precisions (relative standard deviations) in the range of 4.23–17.26%. The limits of detection for d‐ lactic acid, l‐ lactic acid, d‐ 2‐hydroxyglutaric acid, and l‐ 2‐hydroxyglutaric acid were 0.13, 0.11, 1.12, and 1.16 μM, respectively. This method was successfully applied to analyze mouse plasma. The d‐ lactic acid levels in type 2 diabetes mellitus mouse plasma were observed to be significantly higher (P < 0.05, t‐test) than those of normal mice, suggesting that d‐ lactic acid may serve as an indicator for type 2 diabetes mellitus.     

A crosslinked chiral stationary phase for the separation of enantiomers by gas and supercritical fluid chromatography

Crosslinking experiments for the chiral stationary phase OV-225-L-Val-t-butylamide within both fused silica and glass capillary columns have been carried out. Amino acid enantiomers were separated on crosslinked columns by both GC and SFC methods. In SFC, the a values of amino acid enantiomers are independent of the density of the mobile phase, and they are hig her than those obtained by GC for the tested enantiomers with the same column due to the lower column temperature used in SFC.     

Sequential analysis of malic acid and both enantiomers of lactic acid in wine using a high-performance liquid chromatographic column-switching procedure.

A liquid chromatographic column-switching method for the sequential determination of malic acid and both enantiomers of lactic acid in wine is described. The procedure involves the heart cutting of lactic acid enantiomers from a reversed-phase high-performance liquid chromatography chromatogram, retaining them, and back-flushing them through a chiral ligand-exchange column in which they are separated. The method is used to determine the concentration of lactic acid enantiomers in commercial wines. The results are in satisfactory agreement with those of other methods. The malic acid contents of various wines are also determined. The total analysis time for one experiment is approximately 10 min.     

Derivatives for separation of amino acid enantiomers

Summary An optimum gas chromatographic separation of all protein amino acids in one run on capillaries coated with Chirasil-Val is difficult to achieve. Overlap of enantiomers of different amino acids may occur because the relative retention times depend upon the overall polarity of the stationary phase, the film thickness and the actual temperature programm. Employment of different derivatives formed by esterification with isopropanol, n-propanol, isobutanol and n-butanol and by acylation with trifluoroacetic, pentafluoropropionic and heptafluorobutyric anhydrides yields patterns of relative elution of all amino acid enantiomers. Thus, even critical pairs of amino acid enantiomers can be separated or shifted in their relative retention times. All amino acid enantiomers can be separated and quantitatively estimated.     

手性非水毛细管电泳法测定沙美特罗替卡松粉吸入剂中昔萘酸沙美特罗对映体

昔萘酸沙美特罗是目前治疗哮喘夜间发作和哮喘维持治疗的理想药物之一,它在临床上以外消旋体形式给药。昔萘酸沙美特罗的两个对映体在药理活性和毒理作用等方面差异较大,建立昔萘酸沙美特罗对映体的手性分离分析方法对提高手性药物质量、保证临床用药安全有效具有重要意义。该文以L(+)-酒石酸-硼酸络合酸为手性选择剂,建立了测定沙美特罗替卡松粉吸入剂中昔萘酸沙美特罗对映体含量的非水毛细管电泳法。实验考察了L(+)-酒石酸浓度、硼酸浓度和表观pH(apparent pH, pH^* )对手性分离效果的影响。优化的缓冲溶液为:含120.0 mmol/L L(+)-酒石酸和120.0 mmol/L硼酸的甲醇溶液,pH^* 为0.93;其他实验条件为:未涂层弹性熔融石英毛细管(内径50.0 μm,总长度64.5 cm,有效长度55.5 cm),重力进样17.5 cm×10.0 s,检测波长225 nm,室温,工作电压20.0 kV。在优化的实验条件下,昔萘酸沙美特罗的两个对映体在18.0 min内获得了2.18的分离度;在27.5~800.0 mg/L质量浓度范围内,与峰面积呈现良好的线性关系,相关系数(r)大于0.9990;检出限和定量限分别为7.5和25.0 mg/L;加标回收率为98.1%~101.9%,相对标准偏差为1.2%~1.9%。随机购买市面上出售的沙美特罗替卡松粉吸入剂,对其昔萘酸沙美特罗对映体的含量进行了分析检测。结果显示,昔萘酸沙美特罗对映体1和对映体2的标示量百分含量均为98.7%, RSD分别为2.5%和2.7%。该方法操作简便易行,结果准确可靠,消耗低,可用于市售沙美特罗替卡松粉吸入剂中昔萘酸沙美特罗对映体的含量测定。     

Direct TLC Resolution of (±)-Ketamine and (±)-Lisinopril by Use of (+)-Tartaric Acid or (−)-Mandelic Acid as Impregnating Reagents or Mobile Phase Additives. Isolation of the Enantiomers

Direct resolution of the enantiomers of the racemic drugs ketamine and lisinopril has been achieved by TLC. Enantiomerically pure tartaric acid and mandelic acid were used as chiral impregnating reagents and as mobile phase additives. When (−)-mandelic acid was used as chiral impregnating reagent use of ethyl acetate–methanol–water 3:1:1 (v/v) as mobile phase enabled successful resolution of the enantiomers of both compounds. For lisinopril, the mobile phase acetonitrile–methanol–water–dichloromethane 7:1:1:0.5 (v/v) was successful when (+)-tartaric acid was used as impregnating agent. When (+)-tartaric acid was used as mobile phase additive the mobile phase acetonitrile–methanol–(+)-tartaric acid (0.5% in water, pH 5)–glacial acetic acid 7:1:1.1:0.7 (v/v) enabled successful resolution of the enantiomers of lisinopril. The effects on resolution of temperature, pH, and the amount of chiral selector were also studied. The separated enantiomers were isolated and identified. Spots were detected with iodine vapour. LODs were 0.25 and 0.27 μg for each enantiomer of ketamine with (+)-tartaric acid and (−)-mandelic acid, respectively, whereas for lisinopril LODs were 0.14 and 0.16 μg for each enantiomer with (+)-tartaric acid (both conditions) and (−)-mandelic acid, respectively.