Drug detection in hair by chromatographic procedures

This article reviews the analysis of 31 drugs and drug metabolites in human hair by thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gas chromatography—mass spectrometry and mass spectrometry. The most important detection method after chromatographic separation of the components is the mass spectrometry because of its sensitivity and specifity. Washing steps to exclude external contamination, extraction, derivatization, stationary phases, detection modes and detection limits of the mass spectrometric and gas chromatographic-mass spectrometric procedures are presented in five tables. Additionally, a method for a gas chromatographic-mass spectrometric screening procedure is presented.     

Rapid,sensitive and specific electron capture—gas chromatographic method for the quantitation of 6-chloro-2-(1-piperazinyl)pyrazine in biological fluids

A highly specific and sensitive gas chromatographic method for the determination of 6-chloro-2-(1-piperazinyl)pyrazine (MK-212), a central serotonin-like agent, in biological fluids is described. MK-212 and a related internal standard are extracted into benzene from an alkaline solution, back-extracted into acid and then re-extracted into benzene at an alkaline pH. The amines are converted to the trifluoroacetyl derivatives (characterized by gas—liquid chromatography—mass spectrometry), chromatographed and detected with a ⁶³Ni electron capture detector. The sensitivity of the method is such that 10 ng of drug can be measured per aliquot of biological fluid. The precision and accuracy of the method are well within acceptable limits. Specificity of analysis was established by gas—liquid chromatography—mass spectrometry techniques.     

Work in progress: Analysis of the prostaglandins E by glass capillary gas chromatography with electron capture detection

Using a wide bore capillary column coated with D.E.G.S. in combination with a micro volume ⁶³Ni electron capture detector a simple and rapid method with high sensitivity was developed for the analysis of E prostaglandins.For electron capture detection the naturally occurring PGE's have to be transformed to the PGB configuration, esterified and silylated. This method with sensitivity in the low picogram range, tested with prostaglandin standards, will be used for the analysis of prostaglandins and prostaglandin metabolites in biological fluids.     

Investigation of polyamine metabolism by high-performance liquid chromatographic and gas chromatographic profiling methods

Measurements of polyamines, polyamine conjugates and their metabolites in tissues, cells and extracellular fluids are used in biochemistry, (micro)biology, oncology and parasitology. Decarboxylation of ornithine yields putrescine. Aminopropylation of putrescine yields spermidine, and aminopropylation of spermidine yields spermine. Spermidine and spermine are retroconverted to putrescine and spermidine, respectively, by initial N-acetylation and subsequent polyamine oxidation. The intermediate N-acetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine are the major urinary N-acetylpolyamines. Polyamines and N-acetylpolyamines are terminally degraded to non-α-amino acid metabolites by oxidative deamination and aldehyde dehydrogenation. Chromatography with on-line detection is the most commonly applied profiling method for polyamines, N-acetylpolyamines and their non-α-amino acid metabolites. Cation-exchange and reversed-phase high-performance liquid chromatography require pre- or post-column derivatisation, followed by UV-Vis spectrophotometric or fluorimetric detection. Isolation and derivatisation precedes gas chromatography with flame-ionisation, nitrogen-phosphorus, electron-capture or mass spectrometric detection. High-performance liquid chromatography and gas chromatography of polyamines are not competitive techniques, but rather supplementary.     

Determination of 1-methylhistamine and 1-methylimidazoleacetic acid in human urine as a tool for the diagnosis of mastocytosis

The determination of the metabolites of histamine, 1-methylhistamine (MHIS) and 1-methylimidazoleacetic acid (MIIA), in human urine is a useful tool for the diagnosis of mastocytosis. MHIS was extracted under basic conditions with chloroform and derivatized with trifluoroacetic acid anhydride, MIIA was derivatized with pentafluorobenzylbromide prior to extraction under basic conditions and the derivative was extracted with chloroform. The samples were assayed by gas chromatography–mass spectrometry. Normal concentrations of MHIS (2.01 μmol l⁻¹) and MIIA (21.3 μmol l⁻¹) in healthy volunteers’ urine samples are clearly detectable with coefficients of variation of 7.0 and 8.9%. Pathological concentrations of 10.1 and 113 μmol l⁻¹ for MHIS and MIIA, respectively, are quantifiable with coefficients of variation of 6.6 and 4.8%.     

The measurement of nanogram amounts of piperidine in tissues by gas chromatography.

A gas chromatographic method is presented which allows the determination of piperidine in tissue in nanogram quantities. Reaction of the perchloric acid tissue extracts with 3,5-dinitro-4-chloro-benzotrifluoride followed by extraction with hexane, results in a derivative of piperidine suitable for electron capture detection. The specificity of the procedure is confirmed by combined gas chromatography - mass spectrometry. Using this procedure the mean concentration of piperidine in whole mouse brain was found to be 219 pmoles per gram of tissue. No significant difference between the concentration of piperidine in the brains of active and behavioral sleeping mice could be found.     

Saturated metabolites of progesterone in human myometrium during pregnancy.

A method for the separation and quantitative determination of epimeric 3alpha/beta-hydroxy-5alpha/beta-pregnan-20-ones by gas liquid chromatography and electron capture detection is presented. Reliability of the method and applicability to biological material was tested. In one total human uterus and 20 samples of myometrium the concentration of epimeric pregnanol-ones was determined. 3beta-hydroxy-5alpha-pregnan-20-one and 3alpha-hydroxy-5alpha-pregnan-20-one were present in similar quantitative range as progesterone and 20alpha-hydroxy-4-pregnen-3-one. A correlation between progesterone and concentration of metabolites could not be established. 3beta-hydroxy-5beta-pregnan-20-one was not detected in the tissue.     

Simultaneous gas chromatographic determination of lorcainide hydrochloride and three of its principal metabolites in biological samples

A method is described for the determination of the antiarrhythmic drug lorcainide hydrochloride and its three main metabolites in plasma, urine, faeces and tissues from man and animals. The procedure involves the extraction of the parent drug, its metabolites and the internal standard from the biological materials at different alkaline pH values, back-extraction into sulphuric acid and re-extraction into the organic phase (heptane—isoamyl alcohol). After silylation of the different phenolic and the N-dealkylated metabolites, analyses were carried out by automated gas—liquid chromatography with electron-capture detection. The method has a sensitivity limit of 5 ng for lorcainide, and 10–20 ng for the various metabolites, per millilitre of plasma.The method was applied to urine, faeces, plasma and tissue samples from man and animals. It was also suitable for automatic sample analysis.     

Synthesis of releasable electrophore tags for applications in mass spectrometry

Releasable electrophore mass tags (electrophore tags) are compounds for use as labels in ligand assays such as hybridization assays and immunoassays. In such assays, the electrophore-tagged reagent (e.g., DNA probe or antibody) is quantified at the conclusion of the assay by cleaving a bond in the attached tag so that the electrophore part can be brought into the gas phase (usually thermally) for detection by electron capture mass spectrometry (EC-MS) or a related technique. Interest in these tags is promoted mainly by their potential to provide highly sensitive and multiplexed assays. The high multiplexing arises from the opportunity to measure many such tags simultaneously in the mass spectrometer, where each tag has an electrophore part with a unique mass. In this study five precursors of electrophore mass tags are presented. Each precursor can lead to a large library of electrophore tags in a practical way, since each precursor can be converted to many different electrophore tags by reaction with commonly available phenols that provide a variation in mass. The phenol-reactive part of the tag is either a polyfluorobiphenyl or a benzyl chloride moiety. Representative library compounds are prepared and detected in an inert ester form by gas chromatography electron capture mass spectrometry (GC-EC-MS). Further, one tag is conjugated to DNA, and the resulting product is detected by laser-induced electron capture time-of-flight mass spectrometry on a silver surface. A calculation by the semiempirical method AM1 for an ion formed by one of the electrophores suggests that ring rotation promotes dissociative electron capture. The features of practical synthesis, simple composition, physicochemical stability, high multiplicity, high sensitivity, and potential for high throughput detection make releasable electrophore mass tags attractive for highly multiplexed assays. This includes their use in SNP assays or dideoxy DNA sequencing for detection of mutations in individuals, where the combination of high accuracy and speed is essential.     

Quality control for gas chromatography of prostaglandins

Inaccuracy in analytical results may be due to inadequate monitoring for deficiencies in gas chromatographic determination of prostaglandins.The reasons for discrepancies in the results obtained with gas chromatography with electron capture detection were investigated.     

Quantitative determination of 6-keto-PGF1α released by rat aorta: Comparison of mass fragmentography and high resolution gas chromatography with electron capture detection

Mass fragmentography (MF) and high resolution gas chromatography with electron capture detection (HRGC-ECD) were used for measuring 6-keto-PGF_1α, the stable hydrolysis product of prostacylin (PGI₂) released by fresh rings of rat aorta, incubated in the absence of the precursors arachidonic acid or prostaglandin endoperoxide (PGH₂). The incubation medium was acidified, extracted, chromatographed on silicic acid column and derivatized. Comparable results were obtained analyzing each sample by MF and HRGC-ECD. Both methods proved to be suitable in terms of sensitivity and specificity for the measurement of 6-keto-PGF_1α produced by individual rat aortae.     

Microwave-assisted derivatization of 2,5-hexanedione in urine: evaluation using GC-MS and GC-ECD

2,5-Hexanedione, the main metabolite of n-hexane, can be responsible for axonal degeneration symptoms via formation of pyrrol-adducts with several amino acids. In order to make it amenable to gas chromatographic analysis, a protocol including microwave assisted derivatization is presented and compared to state-of-the-art technique of urine analysis. The applied methodology includes derivatization with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine, extraction of the oximes and final analysis using either GC-MS or GC-muECD. Furthermore, the mass spectra of derivatized 2,5-hexanedione and 5-hydroxy-2-hexanone as well as preliminary excretion kinetics are provided. Orthogonal regression methodology demonstrated superior sensitivity for the microwave heating. Limits of detection were calculated to be approximately 20 ng mL(-1) with both MS and electron capture detection, the decompositon of excess derivatizing agent using sulfuric acid, following the reaction is beneficial. A matrix effect caused by urine was not observed, a calibration in aqueous matrix ensures accurate results therefore. Microwave heating yields excellent results regarding recovery, sensitivity and the time needed for sample preparation, furthermore, it is demonstrated that both mass selective as well as electron capture detection are of comparable suitability for this task.     

Determination of kynurenine by a simple gas—liquid chromatographic method applicable to urine,plasma, brain and cerebrospinal fluid

A simple, sensitive and specific method for the determination of kynurenine is described. This is based on alkaline cleavage of kynurenine, followed by solvent extraction, trifluoroacetylation and gas—liquid chromatography with electron capture detection. Using this method kynurenine has been determined in urine and plasma, and for the first time in brain and cerebrospinal fluid. Increases in kynurenine in brain, plasma and urine are demonstrated following tryptophan administration to man and rat.     

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers^1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer^3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)^1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis⁹. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity^10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids¹². This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites¹³.Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.     

The measurement and mass spectral identification of indole-3-pyruvate from tomato shoots

Endogenous indole-3-pyruvate has been identified by full-scan combined gas chromatography-mass spectrometry and measured using gas chromatography with an electron capture detector. High specific-activity [5-3H]indole-3-pyruvate was synthesized from [5-3H]tryptophan and used as an internal standard. In order to allow purification of the labile indole-3-pyruvate it was stabilised by the formation in the crude extract of its pentafluorobenzyl oxime derivative. This derivative also allowed sensitive detection and measurement of indole-3-pyruvate in the picogram range using a gas chromatograph with an electron capture detector. Endogenous levels were found to be between 8-10 ng/g f.wt. of tomato shoots which is comparable to that of the indole-3-acetic acid pool size, 11 ng/g f.wt., in this tissue.     

Determination of sulfinpyrazone and two of its metabolites in human plasma and urine by gas chromatography and selective detection

A selective and sensitive gas chromatographic method for simultaneous determination of sulfinpyrazone and two of its metabolites (the para-hydroxylated metabolite and the sulfone metabolite) in biological fluids using alkali flame ionization detection (AFID), electron capture detection (ECD) and mass fragmentographic detection is described. The compounds are extracted from the samples, methylated and separated on 2% OV-17 or 8% OV-225 columns. Phenylbutazone is used as internal standard. Standard curves are linear. The coefficient of variation at 10 μg/ml of sulfinpyrazone in plasma was shown to be 1.8% (AFID), and the detection limits were 0.1 μg/ml (AIFD) and 10 ng/ml (ECD). Mass spectra of the methylated compounds are shown and serum concentration curves after oral administration of 100 mg sulfinpyrazone to two persons are determined together with the excreted amounts of drug and metabolites.     

Specific determination of threonine in biological samples by gas chromatography with electron capture detection

Threonine was oxidized into acetaldehyde at 0 degrees C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 microl sample solution and the determination limit of threonine was 1 nmol in 200 microl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.     

Simultaneous determination of tryptophan and its metabolites in mouse brain by high-performance liquid chromatography with fluorometric detection

A new method for the determination of tryptophan and its metabolites in a single mouse brain using high-performance liquid chromatography (HPLC) with fluorometric detection is described. Tryptophan, serotonin, 5-hydroxyindoleacetic acid, indoleacetic acid, and tryptophol were clearly separated by a C8 reverse-phase column. Tissue preparation is performed only to centrifuge homogenates of brain prior to the injection to HPLC. The sensitivity is in the range from 10 to 15 pg.     

Capillary gas chromatography using electron capture or selected ion monitoring detection for the determination of muramic acid,diaminopimelic acid and the ratio of d/l-alanine in bacteria

Gas chromatographic analyses of muramic acid, diaminopimelic acid and D-alaline, which are specific components of the bacterial cell wall, have been performed using electron capture or selected ion monitoring detection. Intact cells or peptidogylycan preparations were hydrolyzed in HCl and DCl. After purification by cation exchange chromatography, followed by conversion to the N-heptafluobutyrliso-butyl esters, the components were separated on a 25 m fused silica column coated with SE-54 or on a chiral glass capillary column.The detection limits for muramic acid and diaminopimelic acid were about 10 pg using either detection method and the procedure has the potential sensitivity for detecting about 3 × 10⁵ bacterial cells, e.g., Escherichia coli.Mass spectrometric determination of the d/l ratio of alamine in intact cells of Group A streptococci, type M 15 and in peptidogylcan preparations thereof indicated the proportions 10.2% and 10.5% of D-alanine, respectively. The values uncorrected for racemization during acid hydrolysis were 10.3% and 10.7%, respectively.     

A highly sensitive method for quantitative determination of abscisic Acid

An abscisic acid derivative was formed by reaction with pentafluorobenzyl bromide which allowed highly sensitive detection by gas-liquid chromatography with electron capture detection. In comparison to the methyl ester derivative, the pentafluorobenzyl derivative of abscisic acid was four times more sensitive to electron capture detection and was stable at room temperature in the presence of ultraviolet light. Derivatization was rapid and the molecular weight of the new compound was confirmed by gas-liquid chromatography-mass spectrometry.