Sensitive gas chromatographic method for determination of mercapturic acids in human urine

A method was developed for sensitive determination of the specific benzene metabolite S-phenylmercapturic acid and the corresponding toluene metabolite S-benzylmercapturic acid in human urine for non-occupational and occupational exposure. The sample preparation procedure consists of liquid extraction of urine samples followed by precolumn derivatization and a clean-up by normal-phase HPLC. Determination of analytes occurs by gas chromatography with electron-capture detection. With this highly sensitive method (detection limits 60 and 65 ng/l, respectively) urinary S-phenylmercapturic and S-benzylmercapturic acid concentrations for non-occupationally exposed persons (e.g. non-smokers) can be measured precisely in one chromatographic run. Validation of the method occured by comparison with a HPLC method we have published recently.     

A new partition thin-layer chromatographic method for steroid separations

TLC plates with a 25 mu thick polyamide stationary phase were modified for the separation of neutral steroids by impregnation with propylene glycol. A mixture of tritiated 5 alpha-androstan-3 alpha,17 beta-diol, testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one and 4-androstene-3,17-dione was applied to the plate and developed in a toluene mobile phase to a height of 13.6 cm. This resulted in complete resolution of the 4 compounds as detected by a gas flow scanner or imaging analyzer. Cutting and elution of peak areas with methanol resulted in quantitative recovery of all four steroids. The thinness of the layer also permitted a 3-5% counting efficiency on scanning, resulting in good quantitation of recovery without liquid scintillation counting. The high sorptive capacity of the polyamide layer also enabled extracts of normal human serum to be defatted on the TLC plate by development with pure hexane prior to the toluene step. The new method thus offers several advantages over existing methods for steroid separations and should be adaptable to separations of other relatively non-polar compounds.     

A split stream ion exchange chromatographic method for isolating amino acids and peptides

The technique whereby a standard type single-column amino acid analyser may be modified to operate in a split stream mode is described. The column effluent is divided so that $\text{1}\text{10}$ proceeds to reaction with ninhydrin in the regular way and $\text{9}\text{10}$ proceeds to a fraction collector. Approximately 1–2 nmoles only of each amino acid is necessary for generation of the analog display. The split stream analyser has proved to be valuable in the isolation of several peptides and two glycosides.     

Enantiomeric resolution of amino acids by thin-layer chromatography

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase TLC plate, without using impregnated plates or a chiral mobile phase, is described. Amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5- -alanine amide were spotted on a reversed phase pre-coated TLC plate. Enantiomers of glutamate and aspartate were separated most effectively with solvent consisting of 25% acetonitrile in triethylamine-phosphate buffer (50 mM, pH 5.5) (v/v). Separation of - and -serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline and alanine were separated with 35% of acetonitrile solvent, and those of methionine, valine, phenylalanine and leucine with 40% of acetonitrile solvent. The possibility of using TLC for quantitative determination of amino acid enantiomers was shown by the quantitative recovery of - and -alanine from the TLC plate in the range of 0.56–4.48 nmol.     

Gas chromatographic method with mass-selective detection for the determination of 2-isopropoxyphenol in human urine

Human metabolism of the insecticide propoxur yields 2-isopropoxyphenol (IPP) which is excreted conjugated in urine. In this publication a sensitive and selective analytical method is described which permits the determination of IPP as a suitable parameter for biomonitoring. The clean-up of the hydrolysed urine samples consisted of steam distillation and solid-phase extraction using a reversed-phase column. IPP and the internal standard 2-ethoxyphenol were converted to their pentafluorobenzyl ethers. Excess of the derivatisation reagent was removed using deactivated silica gel. Separation and quantitative analysis was carried out by capillary gas chromatography and mass selective detection. Coefficients of variation were below 5% for concentrations from 6 to 300 μg/l. The detection limit was 0.5 μg/l. The method was checked by analysing six urine samples from pest controllers after indoor application of propoxur. The IPP concentrations ranged from 45 to 306 μg/g creatinine. IPP was not detected in urine specimens from 10 non-exposed persons. The sensitivity of the developed method permits the detection of latent exposure to propoxur.     

Practical gas—liquid chromatographic method for the determination of amino acids in human serum

Application of the gas—liquid chromatographic method previously reported by us was made to the analysis of the 22 amino acids including asparagine and glutamine in serum. The method permitted that aqueous serum samples obtained after deproteinization with perchloric acid were directly subjected to derivatization without any further clean-up procedure such as ion-exchange chromatography. The N-ethyloxycarbonyl methyl esters, which were prepared in the same manner as the N-isobutyloxycarbonyl methyl esters, were introduced for the determination of leucine, isoleucine, arginine and tyrosine. Both derivatives were prepared by two-step procedures involving alkyloxycarbonylation in aqueous media and esterification with diazomethane, and simultaneously analyzed by using the dual set of columns with the same thermal conditions. The advantages of this method are that the sample pretreatment and derivatization are very simple and rapid, and that both asparagine and glutamine along with other amino acids in serum can be determined.     

Gas chromatographic method for the determination of testosterone in urine

A method is described for measuring testosterone in the urine using glucuronidase hydrolysis, continuous extraction with methylene dichloride, and chromatography on silica gel column as well as thin-layer and gas chromatography. This method proved to be practical for processing large numbers of samples and gave consistently reproducible results in the more than 600 specimens tested in the course of clinical evaluation of patients.     

A convenient manual trinitrobenzenesulfonic acid method for monitoring amino acids and peptides in chromatographic column effluents.

The trinitrobenzenesulfonic acid (TNBS) method of R. Fields (1971, Biochem. J., 124, 581–590) has been modified for the manual detection of amino acids and peptides in chromatographic column effluent by changing the reaction conditions to 1 mm TNBS in 0.4 m potassium borate buffer, pH 9.2, at room temperature for 30 to 50 min. The reaction with amines and the spontaneous hydrolysis of TNBS are stopped by neutralization to pH 6.25 with sodium monobasic phosphate (0.33 m). Sodium sulfite (3 mm) is added to increase the absorptivity of the product. The TNBS reagent blank is less than 0.100 A₄₂₀ after 50 min of reaction. Since the ΔA₄₂₀ of the reagent blank is ～0.002/min before quenching the reaction, and zero afterward, the time required for reaction and for absorbance measurements need not be controlled precisely. Alkaline hydrolysis of peptides is carried out prior to detection to increase the sensitivity of the method. This procedure is convenient for the manual determination of 5 to 100 nmol of amino acids in the 50–100 samples required to define a chromatographic elution profile.     

New strategy for the characterization of amino acids by thin-layer electrophoresis and thin-layer chromatography using acidic mobile phases

Rapid, easy, and reliable identification procedures for the common amino acids (1- to 10-nmol amounts) based upon an association of low-voltage electrophoresis (1.04 formic acid) and chromatography (propanol or isopropanol/formic acid/water, 75/5/20, v/v) on a cellulose thin layer and the use of a well-adapted mixture of structural analogs of amino acids are described. Applications of these procedures are also given.     

Gas chromatographic determination of free amino acids and amino acids attached to transfer RNA in biological samples

A method was developed to analyze quantitatively free amino acids and amino acids attached to transfer RNA (tRNA) in tissue samples by gas chromatography. Free amino acids were purified by ion-exchange chromatography after deproteinization. Total cellular aminoacyl-tRNA was extracted from rabbit reticulocytes and liver by a modified phenol extraction method under conditions which were designed to prevent deacylation of the attached amino acids. After deacylation and separation from tRNA by pressure ultrafiltration, eighteen amino acids were determined by gas chromatography as their N-heptafluorobutyryl isobutyl derivatives.     

Evaluation of a centrifuge method and thin-layer preparation in urine cytology

OBJECTIVE: To evaluate and compare the quality and cost of urine cytology using the Cytospin method (Shandon, ThermoElectron Corporation, Waltham, Massachusetts, U.S.A.) and the AutoCyte PREP (TriPath Imaging, Burlington, North Carolina, U.S.A.) in a general laboratory. STUDY DESIGN: A study of differences between the Cytospin method and AutoCyte PREP in the areas of specimen preparation, staining, number and quality of diagnostic cells, background, screener preference, and cost was undertaken over a 3-month period in 2000. Sixty fresh voided urine samples from 25 patients with known transitional cell carcinoma were prepared by the Cytospin method and the AutoCyte PREP according to the manufacturers' instructions. RESULTS: The Cytospin method had longer preparation time but shorter screening time than the AutoCyte PREP. The number of diagnostic cells was higher in the Cytospin method. Fixation quality and staining clarity were better in the Cytospin method. Qualitative assessment of cell arrangements, cell and nuclear size and shape, nuclear/cytoplasmic ratio and nuclear membrane irregularity showed no significant differences between the 2 methods. Cellular details and nuclear chromatin patterns were clearer and better preserved in the Cytospin method, but the AutoCyte PREP showed less blood and inflammatory cells and debris. CONCLUSION: In the majority of cases the screeners preferred the Cytospin method due to its better overall cytologic quality. However, the amount of blood, inflammation and debris was much lower in the AutoCyte PREP. This reduced the need to make a second, diluted specimen and made turnaround time faster. The AutoCyte PREP was 7 times more expensive than the Cytospin method.     

Comparison of a novel thin-layer chromatographic—fluorescence detection method with a spectrofluorometric method for the determination of 7-hydroxycoumarin in human urine

A novel method for the determination of 7-hydroxycoumarin in human urine which combines thin-layer chromatography (TLC) with fluorescence detection (FD) has been devised. The limit of detection (1 ng/ml) enables determination of 7-hydroxycoumarin after both administration of coumarin and environmental exposure to this fragrance material. When compared to a spectroflourometric method of analysis, the TLC—FD method proved to be more selective for the analysis of 7-hydroxycoumarin in human urine.