Derivatization reactions in the gas-liquid chromatographic analysis of drugs in biological fluids

Alkylation, acylation, silylation and other derivatization reactions applied to the gas chromatographic analysis of drugs in biological matrices are reviewed. Reaction conditions are discussed in relation to reaction mechanisms. Detector-oriented labelling of drugs, and derivatization with chiral reagents for the separation of enantiomers are surveyed. Data on the sample clean-up, derivatization and GLC analysis of more than 300 drugs and related compounds are listed.     

Some strategies for improving specificity and sensitivity in the analysis of anti-cancer drugs

Some approaches are discussed for introducing specificity and sensitivity into analytical methods for anti-tumour agents which include a liquid chromatographic step. Various modes of HPLC have been exploited to monitor these drugs specifically and at therapeutically low levels. The use of column switching technology and chemical derivatization techniques to enhance both specificity and sensitivity are discussed. Multiple columns (linked through switching valves) containing packings exhibiting different affinities for the analytes cisplatin and riboxamide provide (a) a high degree of selectivity with convenient analysis times, (b) the opportunity for preconcentration of analytes, (c) improved longevity of analytical columns, (d) a solution to the ‘general elution problem’, and (e) allow direct application of biological fluid to the HPLC system. The use of chemical derivatization techniques (pre- and post-column) to achieve improved sensitivity and altered chromatographic and chemical properties of these and other anti-tumour agents (galactitol, tamoxifen, emetine) is also described. The high chemical reactivity of many anti-tumour agents often requires their rapid derivatization after a biological sample is drawn to prevent chemical degradation in the sample vial. The use of chemical and photochemical derivatization techniques combined with spectrophotometric, fluorometric and voltammetric detectors illustrates the power and utility of derivatization technology in trace drug analysis.     

The application of chemical derivatization to clinical drug analysis

The sensitivity and selectivity achievable in the analysis of drug substances from biological matrices is often limited by the physical and chemical properties of the analyte. These limitations are further exacerbated by the inherent reactivity of most drugs in biological systems (i.e., their propensity for undergoing biotransformation). One very powerful approach that has been taken to improve the quality of the analytical methodology is to alter the physico-chemical properties of the drug through chemical modification (derivatization) during some stage of the analytical sequence. This approach has been successfully applied to situations and has resulted in improved chemical stability, analytical selectivity and sensitivity. In most cases, drug analysis from biological fluids involves a chromatographic step; the derivatization reaction can be carried out either prior or subsequent to chromatography. In this paper, examples of the advantages (and limitations) offered by the introduction of a chemical derivatization step in clinical drug analysis will be presented. Specifically, focus will be placed on analysis of chemically-reactive antineoplastic agents and peptides/proteins. The latter represent an emerging class of drugs which present significant analytical challenges. The use of o-phthalaldehyde analogues offering improved derivative stability and increased sensitivity will be described.     

The application of chemical derivatization to clinical drug analysis

1. The sensitivity and selectivity achievable in the analysis of drug substances from biological matrices is often limited by the physical and chemical properties of the analyte. These limitations are further exacerbated by the inherent reactivity of most drugs in biological systems (i.e., their propensity for undergoing biotransformation).

2. One very powerful approach that has been taken to improve the quality of the analytical methodology is to alter the physico-chemical properties of the drug through chemical modification (derivatization) during some stage of the analytical sequence. This approach has been successfully applied to situations and has resulted in improved chemical stability, analytical selectivity and sensitivity. In most cases, drug analysis from biological fluids involves a chromatographic step; the derivatization reaction can be carried out either prior or subsequent to chromatography.

3. In this paper, examples of the advantages (and limitations) offered by the introduction of a chemical derivatization step in clinical drug analysis will be presented. Specifically, focus will be placed on analysis of chemically-reactive antineoplastic agents and peptides/proteins. The latter represent an emerging class of drugs which present significant analytical challenges. The use of o-phthalaldehyde analogues offering improved derivative stability and increased sensitivity will be described.     

Recent progress in derivatization methods for LC and CE analysis

The derivatization procedure with a suitable fluorescence or chemiluminescence reagent is performed for the purpose of increasing the detection sensitivity and selectivity, in high-performance liquid chromatography (HPLC) and/or capillary electrophoresis (CE). In this article, recent derivatization methods and their applications to biosamples are described. In HPLC, femto mol order of mass detection limits are obtained by derivatization. Regarding the fluorescence reagents, the use of water-soluble reagents has been effective to avoid an undesired adsorption in the process of determination of peptides. In CE, the advantages of having extremely low mass detection limits (ranging from atto to yocto mol level) and requiring only a very short analysis time (less than a few minutes) are made possible by using laser-induced fluorescence or near infra-red detections.     

A rapid screening procedure for acidic and neutral drugs in blood by high resolution gas chromatography

A rapid high resolution gas chromatographic method for screening acidic and neutral drugs in blood is described. The procedure involves a single extraction with ethyl acetate. Using flame ionization detection, without derivatization and with on-column methylation, more than 60 drugs of toxicologic importance are detected.     

Gas chromatographic analysis of cetiedil, a candidate antisickling agent, in human plasma with nitrogen-sensitive detection

In this report a sensitive gas chromatographic assay for cetiedil, a candidate antisickling agent, in human plasma is described. After a triple extraction procedure, cetiedil was analyzed without derivatization with a nitrogen-phosphorus detector (with papaverine used as the internal standard.) Cetiedil was measured in plasma samples taken from human volunteers administered the drug intravenously.     

4-(6-Methoxy-2-naphthyl)-2-butyl chloroformate enantiomers: New reagents for the enantiospecific analysis of amino compounds in biogenic matrices

(+)- and (−)-4-(6-methoxy-2-naphthyl)-2-butyl chloroformate (NAB-C) were prepared from the prochiral nonsteroidal anti-inflammatory agent nabumetone with the aim of developing easily detectable chloroformate reagents for the enantiospecific HPLC analysis of amino compounds in biogenic matrices on achiral stationary phases. The new reagents were tested in the derivatization of β-adrenoceptor antagonists and anti-arrhythmic agents and allowed derivatization in the presence of water. (+)- and (−)-NAB-C were compared with other reagents with a 6-methoxy-2-naphthyl moiety as a chromophore. The reagents were suitable for the analysis of nanogram amounts of, for example, metoprolol enantiomers in plasma, a prerequisite for application in pharmaco- or toxicokinetic studies.     

4-Bromomethyl-7-methoxycoumarin and analogues as derivatization agents for high-performance liquid chromatography determinations: a review.

A major part of modern analytical problem solving deals with the trace level determination of organic compounds and contaminants in biomedical, food and environmental samples. In the analysis of these samples chromatographic techniques play a predominant role. Unfortunately, however, even the combined force of an efficient separation plus a sophisticated mode of detection does not always create sufficient selectivity and/or sensitivity for the final goal to be attained. In such cases, special attention has to be devoted to derivatization or conversion of the analyte(s) of interest (for improved detection selectivity and/or sensitivity) and sample pretreatment (for trace enrichment and clean-up). The above is especially true when, as is often the case today, relatively polar drugs, endogenous compounds, additives or environmental pollutants and/or their (bio)-degradation products have to be determined. For such classes of compounds high-performance column liquid chromatography (HPLC) generally is the preferred method of separation. Reversed-phase HPLC with fluorescence detection is a powerful means of analysis for compounds which possess native fluorescence. They are, however, relatively few in number. In order to make the method useful for a much wider range of analytes, one can therefore resort to derivatization (labelling) or other means of analyte conversion to obtain highly fluorescent reaction products, which can then be detected with the required selectivity and sensitivity. 4-Bromomethyl-7-methoxycoumarin is often used as fluorescent label for the determination of compounds possessing a carboxylic group. About 8% of the biologically interesting analytes--ranging from polar amino acids and peptides to apolar fatty acids--possess such a group.(ABSTRACT TRUNCATED AT 250 WORDS)     

β-受体阻滞剂的分析研究

本文叙述了用高效毛细管气相色谱/氮磷检测器及气相色谱/质量选择检测器对13种β-受体阻滞剂的分析研究。样品用不同衍生化方法作了比较,初步结果表明:衍生化效果TFAA与MBTEA无很大差异;而MSTFA比上述两者效果均佳。本文的研究为β-受体阻滞剂的体液气相色谱研究提供了可行的方法。     

A solid phase extraction technique for the isolation and identification of opiates in urine.

A quantitative method was developed for the simultaneous analysis of morphine, codeine, hydromorphone, hydrocodone, and oxycodone in urine by gas chromatography/mass spectrometry. Samples were hydrolyzed with beta-glucuronidase and then extracted by solid phase extraction on Bond Elute Certify cartridges at pH 6.8. Nalorphine was used as the internal standard. The opiates were analyzed by full-scan electron impact GC/MS after derivatization with acetic anhydride-pyridine. The standard curves for all five drugs were linear between 50 and 1000 ng/mL, with correlation coefficients exceeding 0.99. Coefficients of variation were less than 7%. The method was applied to the analysis of postmortem urines positive by EMIT opiate assay, and the effect of the hydrolysis procedure on recovery of each drug was measured. The results indicate that the hydrolysis procedure is effective in increasing the recovery of all five drugs from urine. The described method enables the laboratory to identify the five opiates most commonly encountered in forensic and clinical laboratories. Its sensitivity for all five drugs is well below GC/MS cutoffs for codeine and morphine employed in NIDA laboratories, and it provides for conclusive full-scan drug identification.     

麻醉性镇痛剂及中枢神经刺激剂的分析研究

本文叙述了用高分辨气相色谱法/火焰离子化检测器,气相色谱/氮磷检测器以及气相色谱/质量选择检测器对12种麻醉性镇痛剂和2种中枢神经刺激剂的分析研究。样品用不同衍生化方法作了比较。初步结果表明:衍生化效果TFAA比MBTFA好,但与MSTFA无明显区别。本法对所有14种药物均能获得满意的分离,为筛选这些药物提供了可靠、快速而简易的方法。     

Determination of carbisocaine, heptacaine and pentacaine in plasma by capillary gas chromatography with nitrogen-selective detection

A gas-liquid chromatographic method is described for the determination of the local anaesthetics carbisocaine, heptacaine and pentacaine in plasma. A C(18) solid-phase extraction was used in a modification to increase selectivity. Following on-column derivatization with trimethylanilinium hydroxide, the analytes were determined by means of capillary gas chromatography and nitrogen-phosphorus selective detection. In comparison with flame ionization detection, the sensitivity of NPD was 20 times higher with a limit of determination in plasma of 10 ng ml(-1).     

颠茄类生物碱的毛细管气相色谱和色—质联用分析

本文用毛细管气相色谱和气相色谱-质量选择检测器分析颠茄生物碱类(莨菪碱、东莨菪碱、山莨菪碱和樟柳碱)的研究。对颠茄生物碱与不同硅烷化试剂、反应能力、反应时间以及反应温度作了探索。结果表明,MSTFA是最好的硅烷化试剂。文内给出了这些生物碱的原型和硅烷化衍生物的保留值和它们的特征性碎片离子。这些数据有助于该类生物碱的鉴定。方法已初步应用于这些生物碱在生物合成中的转化研究。此外,还讨论了颠茄生物碱类TMS衍生化的优点。     

High-performance liquid chromatographic analysis of cyclophosphamide.

A reversed-phase high-performance liquid chromatographic technique is described for the analysis of cyclophosphamide in the presence of its hydrolysis products. The drug was quantified using a UV detector at a low wavelength. A single band was observed for the intact drug, which was well separated from tis hydrolysis product(s). Quantificaiton was obtained with adequate precision by the use of an injector loop or a suitable internal standard (hydrocortisone). The technique requires no extraction of the drug from aqueous solution or derivatization for analysis. The method was applied to partially hydrolyzed and to known solutions of cyclophosphamide. With suitable modification, the method may be useful for analysis of dosage forms but probably lacks the sensitivity necessary for analysis of the drug in biological samples.     

Liquid chromatographic analysis of monosaccharides with phenylisocyanate derivatization.

Because of the lack of sensitivity in carbohydrate analysis, HPLC pre-column derivatization techniques which give strongly UV absorbing compounds have been reported. These techniques have the disadvantage of leading to several chromatographic peaks from each reducing sugar. To enhance both the sensitivity and the selectivity of such specific separation problems, a simplex procedure was applied to optimize the phenylisocyanate derivatization of monosaccharides: a major and stable derivative was formed under the optimal conditions established. The method was extended to deoxysugars and methylglycosides. The limit of detection was 0.2-1 ng for all sugars tested.     

Enantiomeric separation of chiral carboxylic acids, as their diastereomeric carboxamides, by thin-layer chromatography

A thin-layer chromatographic (TLC) method is described for the enantiomeric separation of chiral carboxylic acids using chiral derivatization and non-chiral TLC conditions (ordinary plates and mobile phases) to separate the diastereomeric carboxamides obtained. New chiral derivatizing agents, "levobase" (1R, 2R)-(-)-1-(4-nitrophenyl)-2-amino- 1,3-propanediol, and "dextrobase" (the enantiomer of levobase) are used for carboxamide formation in the presence of dicyclohexylcabodiimide as coupling agent. The procedure is very simple and convenient to carry out. Good resolution is obtained for a wide range of carboxylic acid enantiomeric pairs containing one to two chiral centres.     

Analysis of post mortem brain tissue using EMIT

EMIT, normally used on urine or serum for the detection of drugs of abuse, has been utilized for the analysis of drugs in aqueous brain extracts. A modified Stas-Otto procedure performed on the brain tissue produced a liquid containing no interfering substances. The detection limits proved to be at least as sensitive as the chromatographic screening techniques normally applied to larger portions of the final aqueous filtrate. Out of 166 cases, 50 positive findings were determined. Two glutethimide cases gave positives for the barbiturate assay and a fatal overdose of amitriptyline appeared positive when tested with the benzodiazepine reagents. All other positive findings correlated well with the chromatographic findings.     

RP-HPLC法测定赖氨肌醇维B12口服溶液中盐酸赖氨酸的含量

目的 建立反相高效液相色谱检测方法赖对氨肌醇维B12口服溶液中盐酸赖氨酸的含量的方法.方法 优选盐酸横氨酸组份的柱前衍生条件及色谱条件.结果 在选定的柱前衍生条件和色谱条件下,盐酸赖氨酸的分析不受辅料和其他物质的干扰.结论 本方法专属性强,灵敏度高,可用于赖氨肌醇维B12口服溶液中盐酸赖氨酸的含量测定.     

清开灵注射液生产中板蓝根提取液氨基酸分析

目的分析清开灵注射液生产过程中所用板蓝根提取液的氨基酸的成分。方法柱前衍生化反相高效液相色谱法,色谱柱为ZorbaxEclipse氨基酸分析柱,邻苯二甲醛（OPA）和9-芴甲基氯甲酸酯（FMOC）使氨基酸在柱前发生衍生化以用于色谱分析。结果板蓝根提取液中至少含有15种氨基酸,其中精氨酸、脯氨酸、苏氨酸、缬氨酸和丙氨酸的含量最高,板蓝根提取液是清开灵注射液中精氨酸的主要来源。结论为强化清开灵注射液生产的过程控制和提高产品的质量标准提供了研究思路。