Enantiomeric Resolution of Drug Compounds by Liquid Chromatography

Novel techniques have recently emerged to separate chiral drug compounds into pure enantiomers. The mechanism, experimental difficulties, and applicability of these methods can vary greatly, and the choices involved are not straightforward. The most significant new advances in the field of chiral separations have come from work done with liquid chromatographic systems and chiral stationary-phase columns. This review describes several commonly used approaches to chiral separation, diastereomeric derivatization, chiral mobile-phase additives, and three major types of chiral stationary phases. Although no single method can be judged superior for every drug application, it appears that chiral stationary phases have received the most attention recently and they are emphasized here.     

Indirect detection of anti-acetylcholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase

The inhibiting compounds were separated by micro-column liquid chromatography in the mobile phase containing the natural substrate acetylcholine. A home-made packed bed microbioreactor system containing immobilized enzyme acetylcholinesterase (ACHE) in human red blood cell membrane and choline oxidase (CHO) from alcaligenes was used for the post-column conversion of acetylcholine to hydrogen peroxide which was detected by an electrochemical detector. The inhibition effect of the solutes caused a decrease in the acetylcholinesterase activity, a decrease in the formation of hydrogen peroxide and also a decrease in the response corresponding to the concentration of the solutes. The rate of the enzyme regeneration was also recorded. The micro-system was compared with a conventional LC system comprising commercially prepared enzyme reactor. The stability of the enzymes is at least 3 weeks at ambient temperature. The limit of detection depends on biological activity of inhibition and for galanthamine was 1 pmol.     

高效液相色谱衍生化法测定脑脉康中冰片含量

目的：建立一种测定脑脉康中冰片含量的新方法。方法：柱前衍生化在乙腈中反应，以3，5－二硝苯苯甲酰氯为衍生化试剂（与冰片的摩尔比为20倍），4－二甲氨基吡啶为催化剂（与冰片的摩尔比为40倍），然后在ODS柱上，以甲醇：水（88：12）为流动相，检测波长225nm，高效液相色谱测定脑脉康中冰片含量。结果：冰片在1－10nmol范围内，色谱峰面程与进样量呈线性关系（R^2＝0．9996，P＜0．01），两批脑脉康中冰片的含量分别为7．9％和7．1％，RSD分别为1．0％和1．5％（n=5)，平均回收率为99．9％，RSD为1．8％（n=4).结论：高效液相色谱衍生化法用于测定脑脉康中的冰片含量方法简便，准确，可行。     

硫酸小诺霉素注射液的紫外分光光度测定

采用紫外分光光度法测定硫酸小诺霉素注射液的含量，以邻苯二醛为衍生化试剂，测定波长３３３ｎｍ。在２￣２０ｕ／ｍｌ范围内呈线性关系，ｒ＝０．９９９９。平均回收率为１００．０％，ＲＳＤ为０．８２％。本法简便、快速，结果准确。     

反相高效液相色谱-柱后衍生法分析检测鱿鱼中的生物胺

建立了水产品中多组分生物胺的反相高效液相色谱-柱后衍生-荧光检测方法。采用荧光试剂邻苯二甲醛（OPA）为柱后衍生化试剂,在Capcell Pak MG-C18色谱柱上,通过梯度洗脱使酪胺、腐胺、尸胺、组胺、胍丁胺、精胺和亚精胺等7种生物胺得到良好分离,在给定的浓度范围内,各组分生物胺呈现良好线性相关（R^2〉0.999）。在水产品中添加生物胺混合标准溶液,平均回收率为88.3%～110.1%,相对标准偏差RSD小于10%。结合水产品的感官鉴定、pH值和TVBN值测定等方法检测水产食品的新鲜程度,分析了鱿鱼在不同保藏温度、保藏时间下的生物胺种类及含量的变化。其中胍丁胺和尸胺在鱿鱼的保藏过程中发生最显著变化,可以作为其质量变化的参考指标。     

Chemical derivatization as a strategy to enhance detectability of agents used in cancer management

The bioanalysis of drugs used in the management of cancer is often complicated by the lack of selectivity and sensitivity. Chemical derivatization of these drugs prior to their chromatographic analysis represents a viable strategy to improve chromatographic resolution and to enhance detectability. This review provides examples of how this approach can meet these objectives. Derivatization of racemic cyclophosphamide with a chiral acylating agent, following hydroxyalkylation to introduce a reactive centre into the molecule, provides the basis for its stereospecific analysis. The analysis of dianhydrogalactitol is described, in which diethyldithiocarbamate is used as a nucleophilic derivatizing agent that improves chromatographic behaviour and analytical sensitivity. The final example that is described is the design and preparation of improved fluorogenic reagents (o-phthalaldehyde analogues) for the derivatization of peptides and application of these reagents to the trace analysis of leu-enkephalin in plasma.     

Analysis of neomycins A, B and C by high-performance liquid chromatography with post-column reaction detection

The analysis of the antibiotics neomycins A, B and C was investigated. The separation of the components was studied using reversed-phase and reversed-phase ion-pair chromatography. The optimum separation was obtained utilizing a Lichrosorb RP-2 column with a mobile phase consisting of 75 mg/l sodium dodecyl sulphate, 0.5M Na2SO4 and 0.015 M sodium acetate buffer at pH 7.0. Using this mobile phase, baseline separation was obtained for all three compounds in approximately 20 min. Detection was via post-column derivatization of the analytes with ortho-phthalaldehyde in the presence of mercaptoethanol to form fluorescent iso-indole products. This system is applied to the analysis of a number of formulated products containing neomycin.     

Separation and determination of the two components of glycerol formal by high-performance liquid chromatography

The two isomeric components of glycerol formal, 1,3-dioxan-5-ol and 1,3-dioxolane-4-methanol, are marginally separated (R_s = 1.0) by polar-bonded-phase high-performance liquid chromatography (HPLC) on a cyanopropyl column with acetone—hexane as the eluent. Esterification of these components with 3,5-dinitrobenzoyl chloride produces derivatives which are, however, completely resolved (R_s > 2) by normal-phase HPLC on silica; derivatization has the added advantage of introducing an ultraviolet-absorbing chromophore into each component. Preparative scale chromatography is used to isolate each of the derivatives, which are characterized by their UV, NMR and mass spectral properties. These esters are used as reference standards for an analytical method based on derivatization and normal-phase chromatography. In this way a sample of glycerol formal is calibrated for use as a standard in the direct determination of the two components by polar-bonded-phase HPLC.     

Analytical Chiral Separation of the Stereoisomers of a Novel Carbonic Anhydrase Inhibitor and Its Deethylated Metabolite,and the Assignment of Absolute Configuration of the Human Metabolite and Chiral Degradation Products

Several approaches to the separation of four stereoisomers, 1–4, of a novel, topically active, carbonic anhydrase inhibitor, 1, with two chiral centers in the molecule and four isomers, 5–8, of its chiral metabolite, 5, were evaluated. These methods include nonchiral derivatization followed by separation on chiral stationary phases (CSPs) and chiral derivatization and separation on nonchiral columns and on CSPs. Baseline separation of stereoisomers 1–4 was achieved in less than 15 min after chiral derivatization with (S)-(+)-l-(l-naphthyl)ethyl isocyanate (NEIC) and chiral chromatography on a (R)-N-(3,5-dinitrobenzoyl)phenyl glycine (DNBPG) column under normal phase (NP) conditions. Similarly, isomers 5-8 were baseline separated in less than 20 min after derivatization with NEIC and chromatography on nonchiral (nitrophenyl) and chiral [(S)-(3,5-dinitrobenzoyl)leucine; DNBL] columns in series under the same NP chromatographic conditions. Only partial separation of the diastereomeric derivatives was observed on a variety of nonchiral columns. In addition, all other direct and indirect chiral separation approaches gave only partial separation of at least two stereoisomers within the group of 1–4 or 5–8. The details of chiral separations using various methods and separation () and capacity factors (k) of the derivatized isomers 1–8 on a series of chiral and nonchiral columns are presented. Using these methods, the absolute configuration of the human metabolite of 1 was established as S ₁ S ₂ (5), and the heat (HD) and light (LD) degradation products of 1 as R ₁ S ₂ (3) and S₁ S ₂ (5), respectively.     

Chiral separation of β-blockers after derivatization with (−)-menthyl chloroformate by reversed-phase high performance liquid chromatography

Optimum conditions of chiral derivatization reaction of beta-blockers acebutolol, arotinolol, beta-xolol, bisoprolol, celiprolol, metoprolol and pindolol) with (-)-menthyl chloroformate were investigated for the resolution by HPLC. With more than 30 times molar excess of (-)-menthyl chloroformate chiral derivatization reactions were completed within one hour at room temperature except arotinolol and celiprolol. Diastereomeric derivatives of beta-blockers were well resolved on the ODS column using acetonitrile-methanol-water as a mobile phase.