RESUMEN
Several methods for the determination of collagenolytic activity were compared from the point of view of sensitivity, selectivity, simplicity and practical value for large numbers of biological samples. A labelled collagen substrate was prepared using [3H]acetic anhydride. The specificity of the assay as well as conditions allowing an optimum detection limit were investigated. The influence of low temperatures, lyophilisation and salt concentration on Clostridium histolyticum collagenase have been investigated.
Asunto(s)
Clostridium/enzimología , Colagenasa Microbiana/análisis , Frío , Colágeno/metabolismo , Estudios de Evaluación como Asunto , Liofilización , Marcaje Isotópico , Métodos , Colagenasa Microbiana/metabolismo , Cloruro de SodioRESUMEN
Gas chromatography-mass spectrometry (GC-MS) is a versatile and powerful diagnostic tool in clinical chemistry. Instrumental aspects of the technique are considered. The use of GC-MS for identification purposes is exemplified by the metabolic profiling of organic acids for the diagnosis of inborn errors of metabolism. Quantitative GC-MS is discussed with particular emphasis on the choice of the internal standard. For highest precision and accuracy, isotope dilution GC-MS is the most suitable technique; the application of ID-GC-MS to reference methods and reference materials is discussed in relation to the analysis of steroid hormones. The principles involved in devising calibration procedures and measurement protocols in quantitative GC-MS are outlined.
RESUMEN
A new, sensitive and selective detection method for vitamin K1 in human serum is reported. After the chromatographic separation, vitamin K is converted to its hydroquinone form in a wet-chemical post-column reduction reaction with tetramethylammonium octahydridotriborate. The reaction proceeds in an open tubular knitted reaction coil at elevated temperature. This system, when combined with a two-step multidimensional liquid chromatographic separation, has proven useful for the quantitative determination of serum concentrations of vitamin K1 in normal, healthy individuals.
Asunto(s)
Vitamina K/sangre , Cromatografía Líquida de Alta Presión , Humanos , Indicadores y Reactivos , Solventes , Espectrometría de Fluorescencia , TemperaturaRESUMEN
High-resolution gas chromatography (HRGC) and gas chromatography/mass spectrometry (GC/MS) are the techniques of choice to determine the retention indices of more than 200 organic acids as their trimethylsilyl (TMS) or oxime-trimethylsilyl derivatives. Several types of apolar and semipolar fused-silica capillary columns (OV-1, SE-52, and OV-1701), used to analyze and separate organic acids isolated from urine samples, are evaluated.
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Ácidos/orina , Adulto , Preescolar , Cromatografía de Gases , Ionización de Llama , Humanos , LactanteRESUMEN
A sensitive combined adsorption and reversed-phase high-performance liquid chromatographic procedure is described that permits the determination of endogenous vitamin K1 levels in serum. The separation of vitamin K1 (cis- + trans-isomers), vitamin K1 epoxide and the menaquinones MK-2, MK-4 and MK-9 by adsorption, reversed-phase and cyano-bonded phase chromatography is discussed. The methodology was further developed for the quantitative measurement of vitamin K1 in human serum. Concentrations as low as 500 pg/ml in serum could be detected. The identity of the vitamin K1 peak was confirmed by UV absorption spectrophotometry and re-chromatography. The method has been applied to the determination of serum levels in normal healthy individuals and patients treated with vitamin K1.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Vitamina K/sangre , Humanos , Vitamina K/uso terapéuticoRESUMEN
A multi-stage screening and identification scheme for the diagnosis of inborn errors in amino acid, fatty acid, carbohydrate and intermediary metabolism is described. The method is based on a computerized analysis of data obtained with dual-column gas chromatography/FID (GC/FID) and gas chromatography/mass spectrometry (GC/MS) profiling of urinary organic acids. It involves: (1) isolation of the compounds by solvent or solid-phase extraction; (2) conversion into trimethylsilyl (TMS) or TMS-oxime derivatives; (3) GC/FID analysis on SE-52 and OV-1701 capillary columns; (4) tentative identification by comparing the methylene unit (MU) values on both columns with a user-built library of reference compounds; (5) quantitative evaluation of the excretion profile; and (6) analysis by GC/MS of samples with an abnormal profile using an automated peak identification programme.
Asunto(s)
Ácidos/orina , Autoanálisis , Cromatografía de Gases , Cromatografía de Gases y Espectrometría de Masas , HumanosRESUMEN
This is a new method for isolating and concentrating urinary organic acids before gas chromatography--mass spectrometry. Sulfate and phosphate anions are removed by precipitation with Ba(OH)2, and the urine pH is adjusted to 8-8.5. The sample is then applied onto small, disposable, strong-anion-exchange columns. Neutral and basic compounds are washed out with water, which is then removed by centrifugation and by rinsing the columns with methanol and diethyl ether. The organic acids are eluted with a 4/1 (by vol) mixture of an organic solvent--either n-butanol or ethyl acetate--and formic acid containing HSO4- (0.1 mol/L) as a highly selective counter-ion, and finally with methanol alone. Sulfate ions are retained, and the eluate is evaporated before trimethylsilyl derivatization. Analytical recoveries (about 100%) of organic acids compare favorably with those obtained with solvent extraction. This convenient procedure is selective and reproducible and is a suitable alternative to the more cumbersome diethylaminoethyl-Sephadex extraction method.
Asunto(s)
Compuestos de Bario , Ácidos Carboxílicos/orina , Cromatografía por Intercambio Iónico/métodos , Acetatos , Ácido Acético , Adulto , Bario , Niño , Cromatografía de Gases , Formiatos , Cromatografía de Gases y Espectrometría de Masas , Glutaratos/orina , Humanos , Concentración de Iones de Hidrógeno , Hiperoxaluria/orina , Lactante , Errores Innatos del Metabolismo/orina , Concentración Osmolar , Fosfatos/orina , Solventes , Sulfatos/orina , Ácidos SulfúricosRESUMEN
Stomach, small intestine contents, blood, liver, kidney and urine of a 28-years old man, were analyzed for residues of Endosulfan (6, 7, 8, 9, 10, 10-hexachloro-1, 5, 5a, 6, 9, 9a-hexahydro-6, 9-methano-2, 4, 3-benzo(e)dioxathiepin 3-oxide). The analysis results showed the presence of high concentrations of the two endosulfan isomers in all samples. Since also alcohol was present in all the tissues analyzed, it was concluded that the victim died of a combined endosulfan-alcohol poisoning. No other drugs were found.
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Endosulfano/envenenamiento , Adulto , Intoxicación Alcohólica , Cromatografía de Gases , Interacciones Farmacológicas , Endosulfano/sangre , Endosulfano/metabolismo , Humanos , Masculino , SuicidioRESUMEN
Chromatographic procedures have been developed for determination of the stabilizers N-acetyl-DL-tryptophan and octanoic acid in human albumin solutions. N-Acetyl-DL-tryptophan and the internal standard, N-formyl-DL-tryptophan, were separated by liquid chromatography on a reversed-phase column with UV detection at 280 nm. Deproteinization and extraction were carried out with methanol. The extraction recovery at the level of 4.9 mM was 92.5 +/- 2.5% (S.D.) (n = 10), and the average coefficient of variation (C.V.) for replicate analyses of albumin solutions (mean = 2.57, 10.44 and 17.10 mM) was 1.10% (n = 27). Octanoic acid was determined gas chromatographically as its methyl ester, with nonanoic acid as the internal standard. The sample pretreatment included acidification, extraction with hexane and derivatization with methanol-sulphuric acid. The relative recovery from albumin solutions was 89.7 +/- 5.8% (S.D.) (n = 6), and replicate determinations of the compound yielded a C.V. of 5.5% (mean = 14.82 mM, n = 9).
Asunto(s)
Albúminas/análisis , Caprilatos/análisis , Triptófano/análogos & derivados , Cromatografía de Gases , Cromatografía Liquida , Estabilidad de Medicamentos , Humanos , Factores de Tiempo , Triptófano/análisisRESUMEN
A new method, based upon a selective extraction and gas chromatographic/mass spectrometric analysis, was developed to monitor the effect of combined haemoperfusion-haemodialysis treatment in a case of propericiazine poisoning. The method relies on the selected ion monitoring of the acetate derivatives of propericiazine and its internal standard fluphenazine, after their extraction from 1 ml of alkalinized plasma with n-hexane:isopropanol (8:2, v/v), back-extraction into an acidified water phase, realkalinization and extraction with n-hexane: isopropanol, derivatization with acetic anhydride and gas chromatography on a short (12 m) OV-101 fused silica capillary column. The described procedure is specific and provides between-assay variability of 4.8% CV at 5 micrograms 1(-1) plasma concentration. The method enables quantification down to 1 microgram 1(-1) and hence demonstrates sufficient sensitivity to permit pharmacokinetic or drug monitoring studies.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Fenotiazinas/sangre , Femenino , Hemoperfusión , Humanos , Persona de Mediana Edad , Fenotiazinas/envenenamiento , Diálisis Renal , Intento de SuicidioRESUMEN
Owing to the lack of sensitivity and/or selectivity of the existing chemical assays, vitamin K deficiency has always been diagnosed indirectly by measuring its effect on blood coagulation. We used our recently developed multidimensional liquid chromatographic assay for what is, to the best of our knowledge, the first systematic investigation of physiological vitamin K levels in human blood. It allowed the unequivocal demonstration of trans-phylloquinone (vitamin K1) and its quantification down to a level of 0.5 nanogram per milliliter of serum (ng/ml. In healthy adults, a mean serum concentration of 2.6 ng/ml was found, with a normal range of 0.9 to 7.8 ng/ml. These values apparently are distributed in a log-normal way.