Determination of the terfenadine metabolite azacyclonol in human serum using gas chromatography-mass spectrometry

In this work, a method for the determination of azacyclonol, a primary metabolite of terfenadine, in human serum is described. Sample preparation is carried out by liquid-liquid extraction under basic conditions. For an efficient clean up, the analytes are back-extracted into diluted hydrochloric acid and, after alkalinization, are once again extracted into the organic phase. No derivatisation step is performed. The samples are measured by gas chromatography-mass spectrometry with good selectivity. The limit of detection is 2 ng/ml. The coefficients of variation are 12.6% at 10 ng/ml and 6.44% at 200 ng/ml in the day-to-day control measurements.     

Determination of cyclophosphamide in urine by gas chromatography—mass spectrometry

A sensitive gas chromatographic method for the determination of cyclophosphamide in urine is presented. After liquid—liquid extraction with diethyl ether and derivatization with trifluoroacetic anhydride, cyclophosphamide was identified and quantified with mass spectrometry. The method is suitable for the determination of cyclophosphamide at concentrations of more than 0.25 ng/ml, which enables the uptake of cyclophosphamide during occupational activities, such as the preparation and administration of antineoplastic agents in hospitals, to be measured. Simple preparation makes the method appropriate for routine analysis.     

Determination of free salsolinol concentrations in human urine using gas chromatography—mass spectrometry

The urine concentrations of free salsolinol were determined in six healthy volunteers, using a gas chromatographic—mass spectrometric method with electron-capture negative-ion chemical ionization after derivatization with pentafluoropropionyl anhydride. The sensitivity of this method allows the quantification of salsolinol concentrations of 0.55 pmol/ml. The synthesis of [²H₄]salsolinol from dopamine and [²H₄]acetaldehyde via a Pictet—Spengler condensation is described; [²H₄]salsolinol was used as the internal standard for salsolinol quantification. The urine concentrations of free salsolinol ranged from ca. 1 to 6 pmol/ml.     

Amine metabolite profile of normal and uremic urine using gas chromatography—mass spectrometry

A method for the simultaneous analysis of phenolic amines and aliphatic amines in human urine is described. The amine metabolites in urine were extracted using Dowex 50W-X8 cationic resin, derivatized and analyzed by a gas chromatographic—mass spectrometric—computer system. The amine metabolites profile of 5 ml of urine was obtained with good gas chromatographic separation. The gas chromatographic method described here separates urinary phenolic amines, di- and polyamines and methylguanidine in a single chromatographic separation. The urinary levels of methylguanidine, putrescine, cadaverine, spermidine, p-tyramine, dopamine, and 3-methoxytyramine were quantitated by using a mass spectrometric technique. In uremic patients, only the urinary excretion of methylguanidine was increased in comparison with normal subjects, although the urinary excretion of other amines was decreased in uremic patients.     

Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography—mass spectrometry

The metabolic transformation of methandienone (I) in the horse was investigated. After administration of a commercial drug preparation to a female horse (0.5 mg/kg), urine samples were collected up to 96 h and processed without enzymic hydrolysis. Extraction was performed by a series of solid—liquid and liquid—liquid extractions, thus avoiding laborious purification techniques. For analysis by gas chromatography—mass spectrometry, the extracts were trimethylsilylated. Besides the parent compound I and its C-17 epimer II, three monohydroxylated metabolites were identified: 6β-hydroxymethandienone (III), its C-17 epimer (IV) and 16β-hydroxy-methandienone (V). In addition, three isomers of 6β,16-dihydroxymethandienone (VIa–c) were discovered. Apparently, reduction of the δ⁴ double bond of 16β-hyroxymethandienone (V) in the horse yields 16β,17β-dihydroxy-17α-methyl-5β-androst-1-en-3-one (VII). Reduction of the isomers VIa–c results in the corresponding 6β,16,17-trihydroxy-17-methyl-5β-androst-1-en-3-ones (VIIIa–c). The data presented here suggest that screening for the isomers of VI and VIII, applying the selected-ion monitoring technique, will be the most successful way of proving methandienone administration to a horse.     

Improved screening method for beta-blockers in urine using solid-phase extraction and capillary gas chromatography—mass spectrometry

An improved screening method for beta-blockers in urine is proposed, involving enzymatic hydrolysis, solid-phase extraction and capillary gas chromatography—mass spectrometry. Several extraction methods for beta-blockers, such as conventional liquid—liquid and solid-phase extraction procedures, have been evaluated for at least eight beta-blockers. Additionally, the gas chromatographic properties and mass fragmentation of the trimethylsilyl—trifluoroacetyl, trifluoroacetyl and cyclic n-butylboronate derivatives of beta-blockers have been compared and evaluated with respect to their efficiency for screening urine. The resulting screening method proved to be a specific and sensitive procedure, enabling these analytes to be detected and identified up to 48 h after the administration of a dosage, usually encountered in doping cases.     

Simultaneous analysis of phenylglycols and phenylethanols in human urine by gas chromatography—mass spectrometry

A method is described for the determination of the neutral metabolites formed from catecholamines and various other structurally related phenylethylamines by using gas chromatography—chemical ionization—mass spectrometry. These metabolites (phenylglycols and phenylethanols) were extracted from urine specimens and converted to pentafluoropropionyl derivatives which were separated on either 3% OV-1, 3% SP-2250, or 3% QF-1 packed columns. Our results demonstrate the presence in human urine of p-hydroxyphenylglycol, a metabolite of octopamine. One patient excreted 13 and 91 μg/day of free and total (free + conjugated) p-hydroxyphenylglycol, respectively. Treatment with a monoamine oxidase inhibitor reduced the excretion of total p-hydroxyphenylglycol to 30% of baseline level.     

Measurement of testosterone and pregnenolone in nails using gas chromatography–mass spectrometry

An efficient method for the determination of testosterone and pregnenolone in human nails using gas chromatography–mass spectrometry (GC–MS) with d₃-testosterone as an internal standard is described. The method involves alkaline digestion and liquid–liquid extraction, with subsequent conversion to mixed pentafluoropenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive analysis in the selected-ion monitoring (SIM) mode. The limit of detection (LOD) and limit of quantification (LOQ) were lowered to 0.1 and 0.2 pg/g, respectively, when 100 mg of nail-clippings were used. The mean recoveries of testosterone and pregnenolone were 89.8 and 86.7%, respectively, while good overall precision (% C.V.; 4.5–9.5) and accuracy (% bias; 3.9–8.4) were demonstrated. Linearity as a correlation coefficient was 0.9913 (testosterone) and 0.9965 (pregnenolone). When applied to fingernail and toenail samples from seven healthy men and nine healthy women, testosterone and pregnenolone were positively detected in the concentration range of 0.24–5.80 ng/g. The levels of two steroids studied in the nails were found to be higher in the male subjects than in the female subjects, and except for the toenails of the females, the levels of testosterone were higher than those of pregnenolone.     

Isatin (indole-2,3-dione) in urine and tissues: Detection and determination by gas chromatography—mass spectrometry

A simple procedure based upon capillary column gas chromatography-mass spectrometry (GC—MS) is described for the detection and determination of isatin (indole-2,3-dione) in body fluids and tissues. After addition of 5-methylisatin as internal standard to urine or tissue homogenates, organic extracts are dried and derivatized successively with hydroxylamine hydrochloride and the reagent N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). The tert.-butyldimethylsilyl derivatives obtained show good GC—MS properties and allow quantification by selected-ion monitoring of m/z 333 (isatin) and m/z 347 (internal standard). Adult and newborn human urine output values lie in the ranges 0.4–3.2 mg/mmol of creatinine (5–30 mg per 24 h) and 0.002–0.518 mg/mmol of creatinine, respectively. There is a discontinuous regional distribution in rat tissues. The GC—MS properties of a number of derivatives formed by successive reaction of isatin with hydroxylamine hydrochloride (or methoxyaminehydrochloride or ethoxyamine hydrochloride) and MTBSTFA, bis(trimethylsilyl)trifluoroacetamide, pentafluoropropionic anhydride or pentafluorobenzyl bromide are also described.     

Determination of the anti-ulcer agent geranylgeranylacetone in serum by gas chromatography—mass spectrometry

A highly specific and sensitive method for the determination of the anti-ulcer drug geranylgeranylacetone (GGA) in human serum is described. The extract from serum with hexane was saponified with potassium hydroxide and subjected to silica gel column chromatography to remove interfering substances. GGA in the partially purified extract was then reacted with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine and measured by selected ion monitoring using gas chromatography—mass spectrometry. A low detection limit (1 ng/ml) and high precision were obtained.     

Determination of ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d₃-ephedrine as internal standard. Calibration curves, with r²>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.     

Detection of the illegal use of ethinylestradiol in cattle urine by gas chromatography—mass spectrometry

A method for the detection of ethinylestradiol in cattle urine is described, based on enzymic hydrolysis of the sample, clean-up by means of disposable octadecyl and amino solid-phase extraction columns, fractionation by reversed-phase high-performance liquid chromatography, and detection by gas chromatography—mass spectrometry (selected-ion monitoring). Identification is based on both gas chromatographic and mass spectrometric data. The method has been tested on urine samples for a collaborative study and all the results found were correct.     

Determination of clenbuterol in urine as its cyclic boronate derivative by gas chromatography—mass spectrometry

A rapid and reliable gas chromatographic—mass spectrometric method for the determination of clenbuterol in urine is described. Penbutolol was used as internal standard. Four derivatization procedures have been tested, of which 1-butaneboronic acid gave the best results. The method includes extraction of the alkalinized urine (3 ml) with tert.-butyl methyl ether—n-butanol (9:1), derivatization with 1-butaneboronic acid (15 min at room temperature), and analysis in the selected-ion monitoring mode of the derivatives of clenbuterol at m/z 243, 327 and 342 and of penbutolol at m/z 342 and 357. The detection limit is 0.5 ng/ml and the recovery better than 90%.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Analysis of profiles of conjugated steroids in urine by ion-exchange separation and gas chromatography—mass spectrometry

A simplified, flexible method for the analysis of metabolic profiles of steroids in urine is described. Solid extraction with Amberlite XAD-2 or Sep-Pak C_{1 g} cartridges is followed by group fractionation of unconjugated neutral and phenolic steroids, monoglucuronides, monosulphates and disulphates on the lipophilic strong anion exchanger triethylaminohydroxyproply Sephadex LH-20 (TEAP-LH-20). Following brief enzymatic hydrolysis or solvolysis the steroids are purified on TEAP-LH-20. O-Methyloxime and trimethylsilyl ether derivatives are prepared and purified by filtration through Lipidex 5000, and are then analyzed by glass capillary column gas—liquid chromatography and gas chromatography—mass spectrometry.Between 2 and 5 ml of urine are used for a comprehensive analysis. Unconjugated neutral and phenolic steroids are isolated in half a day, corresponding steroids in the conjugate fractions in two days. No fraction containing steroids is discarded, but the analysis can be limited to a selected fraction.     

Identification of some abnormal metabolites in psoriatic nail using gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric analysis was used to separate and identify abnormal compounds in the nail of psoriatic patients. The nail was extracted with heated ethanol, and the extract was analyzed with and without trimethylsilylation. Tetradecanoic acid octadecyl ester, hexadecanoic acid octadecyl ester and octadecanoic acid octadecyl ester were first identified in the psoriatic nail, but were not detected in normal nail.     

Analysis of procarbazine and metabolites by gas chromatography—mass spectrometry

Twelve compounds representing procarbazine, seven metabolites, and an internal standard were analyzed by gas chromatography—mass spectrometry on a 3% OV-1 column. Procarbazine and four metabolites were derivatized with acetic anhydride.A sensitive, specific and quantitative assay was established by selected ion monitoring using a synthetic analogue of the drug as an internal standard. The limits of detection were approximately 1 ng/ml of plasma while the limits of quantitation were 10 ng/ml of plasma.Studies on the degradation of procarbazine - HCl in 0.05 M phosphate buffer (pH 7.4) were compared to in vivo studies. At 1 h after incubation of procarbazine - HCl in buffer, the azo and aldehyde metabolites were detected in the highest concentrations representing 27.2% and 20.3% of total drug and metabolites. In the in vivo studies, analyses of rat plasmas indicated that 1 h after an oral dose of procarbazine - HCl, the aldehyde metabolite represented 72% of the total drug and metabolites, and that relatively little of the azo metabolite was present.     

Identification of two Thormählen-positive compounds from melanotic urine by gas chromatography—mass spectrometry

The isolation of two Thormählen-positive compounds from the urine of a patient with malignant melanoma and the elucidation of their structure by gas chromatography—mass spectrometry is described. The compounds were isolated using a poly-N-vinylpyrrolidone column and separated by preparative thin-layer chromatography. After elution they were analyzed by gas chromatography and gas chromatography—mass spectrometry as their trimethylsilyl derivatives and after hydrolysis also as their tert.-butyldimethylsilyl derivatives. The results showed the main Thormählen-positive compound A to be the glucuronide of 5-hydroxy-6-methoxyindole, whereas the minor compound AX appeared to be the glucuronide of its isomer 6-hydroxy-5-methoxyindole.