Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic—mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65°C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene—hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography—mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-μm Nova-Pak C₁₈ column, studied by thermospray—LC—MS, and in the direct insertion probe—EI positive-ion mode.     

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.     

Determination of isoniazid and its hydrazino metabolites, acetylisoniazid, acetylhydrazine, and diacetylhydrazine in human plasma by gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric assay for isoniazid and its hydrazino metabolites in human plasma was developed. The trimethylsilyl derivatives of diacetylhydrazine and acetylisoniazid and of the benzaldehyde hydrazones of acetylhydrazine and isoniazid were separated on a 1% OV-17 column and quantitated by single ion monitoring using a LKB 9000 mass spectrometer. Deuterated analogues served as internal standards. The method is well suited for the determination of the hepatotoxic hydrazino metabolites of isoniazid in human plasma following an oral therapeutic dose of isoniazid.     

Routine screening and quantitation of urinary corticosteroids using bench-top gas chromatography—mass-selective detection

A method was developed for the routine screening, confirmation and quantitation of corticosteroids in human urine using bench top capillary gas chromatography (GC)—mass-selective detection. The free and conjugated corticosteroid fractions were isolated by liquid—liquid partition. After evaporation to dryness under vacuum the corticosteroid residues were derivatized to form the methyloxime trimethylsilyl ether derivatives. Both GC retention data and characteristic spectral data based on authentic reference standards were used for the identification and quantitation of cortisol, cortisone, tetrahydrocortisol and tetrahydrocortisone in the ppb (ng/ml) concentration range. The method is simpler and more efficient than the other GC—mass spectrometric (MS) techniques. It is also more sensitive than the liquid chromatographic—MS method.     

Screening and confirmatory analysis of β-agonists, β-antagonists and their metabolites in horse urine by capillary gas chromatography—mass spectrometry

A method for the screening and confirmatory analysis of β-agonists and -antagonists in equine urine is described. Following initial enzymic hydrolysis, the basic drugs and metabolites are extracted using Clean Screen^® DAU or Bond Elut Certify™ cartridges, and analysed as their trimethylsilyl ether or 2-(dimethyl) silamorpholine derivatives by capillary gas chromatography—mass spectrometry. The method proved to be very sensitive and selective for basic drugs. After administration of therapeutic doses of propranolol, metoprolol, timolol, isoxsuprine and clenbuterol to thoroughbred horses, the parent compound/metabolites could be detected in urine for upto 14–120 h depending on the drug.     

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.     

Characterization of prednisone, prednisolone and their metabolites by gas chromatography—mass spectrometry

Human urinary metabolites of the synthetic corticosteroids prednisone and prednisolone were detected in the course of gas chromatographic steroid profiling as methoxime-trimethylsilyl derivatives. Metabolites were provisionaly identified by combined gas chromatography—mass spectrometry. The major metabolites were 11-keto/11-hydroxy conversion products, 20-hydroxy and 4,5-dihydro analogues of the parent drugs. Cortisone, 6-hydroxy and fully saturated A-ring compounds were minor metabolites. Retention indices and mass spectral data are presented.     

Gas chromatographic—mass spectrometric method for routine monitoring of 5-fluorouracil in plasma of patients receiving low-level protracted infusions

A gas chromatographic—mass spectrometric (GC—MS) method is described which quantitates 5-fluorouracil (5-FU) plasma levels ranging from 0.5 to 50 ng/ml. The analysis uses two internal standards, 1,3-[¹⁵N₂]-5-fluorouracil and 5-chlorouracil. Extraction and derivatization of the pyrimidine bases were accomplished in a single step using acetonitrile. Compounds were analyzed as their 1,3-dipentafluorobenzyl derivatives by electron-impact MS, and the GC—MS analysis was automated with respect to sample injection and data reduction. Stability of the analysis was demonstrated by continuous unattended analysis of 5-FU in human plasma for periods of up to three days with no deterioration of the quantitative results. The method is applicable to quantitating 5-FU plasma levels in patients receiving protracted infusions of the drug for colorectal cancer or other malignancies.     

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.     

Quantification of D-amino acids in human urine using GC-MS and HPLC

Summary The relative amounts of free D-amino acids (D-AA) in the urine of seven healthy volunteers (age 27 to 49 years) were determined using chiral phase (Chirasil-L-Val) capillary gas chromatography in conjunction with selected ion monitoring mass spectrometry. The absolute amounts of free D-AA were determined by pre-column derivatization of the amino acids witho-phthaldialdehyde andN-isobutyryl-L-cysteine followed by high-performance liquid chromatographic separation and fluorescence detection of the isoindol derivatives formed. The following most abundant D-AA were found (highest and lowest absolute and relative amounts): D-Ser (379.8 — 30.1µMol/L; 56.5 — 19.0%), D-Ala (53.8 — 7.6µMol/L; 19.6 — 5.7%), D-Thr (5.8 — 0.25µMol/L; 3.4 — 1.0%), D-Val (3.7 — 0µMol/L; 4.2 — 0%), and D-Phe (3.5 — 0.35µMol/L; 4.8 — 1.4%).     

Recent methods for the determination of volatile and non-volatile organic compounds in natural and purified drinking water

Four analytical protocols for the extraction and preconcentration of organic residues in natural or purified drinking water were investigated and compared: closed loop stripping analysis; simultaneous extraction—distillation; purge and trap analysis; continuous liquid—liquid extraction. Organic extracts were submitted to a variety of separation and identification techniques. Volatiles were determined by conventional capillary column gas chromatography with tandem mass spectrometry, using triple-stage quadrupole instruments. Non-volatile and thermally labile molecules were investigated by several different techniques (high-temperature gas chromatography, capillary column supercritical fluid chromatography, pyrolysis gas chromatography—mass spectrometry, thermospray liquid chromatography with tandem mass spectrometry and conventional fast-atom bombardment with tandem mass spectrometry). Several samples recently examined in the laboratory provide examples of this multitechnique approach for a more complete knowledge of the organic carbon distribution in water-dissolved organic matter, taking into account organic substances with widely different volatilities, polarities and thermal stabilities.     

Thermospray and particle beam liquid chromatographic—mass spectrometric analysis of coumarin anticoagulants

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography—thermospray mass spectrometry (LC—TSP-MS) and liquid chromatography—electron impact mass spectrometry (LC—EI-MS) to assess the use of LC—MS methods for the determination of these compounds in biological materials. LC—TSP mass spectra showed a single [M + 1]⁺ ion with no fragmentation; LC—EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC—MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC—TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.     

Qualitative and quantitative analysis of some synthetic, chemically acting laxatives in urine by gas chromatography—mass spectrometry

A method for the qualitative and quantitative simultaneous analysis of dioxyanthraquinone, desacetyl-Bisacodyl, phenolphthalein and Oxyphenisatin in human urine using gas chromatography—mass spectrometry (GC—MS) has been developed. The compounds were extracted from urine at pH 7.5 with diethyl ether using Extrelut extraction columns, followed by evaporation and trimethylsilylation.The method used electron beam ionization GC—MS employing a computer-controlled multiple-ion detector (mass fragmentography). The recovery from urine for the various compounds was between 80% and 100%. The detection limit for these compounds was in the range 0.01–0.05 μg/ml of urine.The method proved to be suitable for measuring urine concentrations for at least four days after administration of a single oral low therapeutic dose of the laxatives to sixteen healthy volunteers.     

Determination of seven prostanoids in 1 ml of urine by gas chromatography—negative ion chemical ionization triple stage quadrupole mass spectrometry

In an isotope dilution assay, prostaglandin (PG) E₂, 6-keto-PGF_1α, thromboxane (Tx) B₂ and their metabolites PGE-M (11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF_1α, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂ were determined in urine by gas chromatography—triple stage quadrupole mass spectrometry (GC—MS—MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate—hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC—MS—MS. In each run, two or three prostanoids were determined.     

Determination of estradiol 2- and 4-hydroxylase activities by gas chromatography with electron-capture detection

A highly sensitive assay has been developed for measuring the rate of formation of 2-hydroxyestradiol and 4-hydroxyestradiol from estradiol by microsomal preparations. Catechol estrogens were converted to heptafluorobutyryl esters, which were separated by capillary column gas chromatography and quantified using electron-capture detection. 2-Hydroxyestradiol 17-acetate was used as an internal standard. The identity of catechol estrogen derivatives was verified by gas chromatography—mass spectrometry using negative-ion chemical ionization. Estrogens were identified by negative molecular ions and/or by characteristic fragments. This procedure permits quantification of catechol estrogens at the subpicogram level. The assay was validated by comparing estrogen 2- and 4-hydroxylase activities in microsomes from hamster and rat liver with values reported previously.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic—mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using ¹⁵O-labelled amino acids and gas chromatography—mass spectrometry.     

Simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma by gas chromatography—mass spectrometry as indices of cholesterol and bile acid biosynthesis

A very sensitive and specific method for the simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma is described. The assay is based on isotope dilution mass spectrometry: the extracts from plasma were treated with benzylamine followed by dimethylethylsilylimidazole, then the resulting dimethylethylsilyl ether derivatives of mevalonylbenzylamide and 7α-hydroxycholesterol were determined by gas chromatography—mass spectrometry using high-resolution selected-ion monitoring. Simple regression analysis revealed significant correlations between the plasma level of mevalonate and the hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) (r = 0.83, P < 0.01) and between the plasma level of free 7α-hydroxycholesterol and the hepatic activity of cholesterol 7α-hydroxylase (EC 1.14.13.7) (r = 0.76, P < 0.05).     

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.     

Identification by gas chromatography and mass spectrometry of lipids from the rat Harderian gland

Lipids from rat Harderian glands were extracted with ethyl acetate, hydrolysed with base and examined by gas chromatography (GC) and gas chromatography—mass spectrometry (GC—MS) as trimethylsilyl (TMS), [²H₉]TMS, methyl ester—TMS, picolinyl, nicotinate and nicotinylidene derivatives. The latter three derivatives were used to reveal the structures of the alkyl chains of fatty acids, alcohols and glycerol ethers, respectively. Forty-eight compounds were identified, representing about 97% of the total extracted lipids as measured by GC peak areas. The major constituents were fatty acids with chain lengths from 12 to 22 carbon atoms (mainly C₁₈ and C₂₀) and fatty alcohols (C₁₆ to C₂₆) derived from wax esters. Most of these acids and alcohols were unsaturated in the ω-7 position and were accompanied by smaller amounts of the saturated and ω-5 monounsaturated analogues. Glycerol ethers were also identified for the first time in this secretion; the ether chains contained from 14 to 19 carbon atoms (mainly 16) and were straight-chain saturated, unsaturated (ω-5 and ω-7) and branched (iso). The only sterol found was cholesterol amounting to 1.24% of the total extract.     

Simultaneous detection of methylphenidate and its main metabolite, ritalinic acid, in doping control

Two analytical methods for the simultaneous detection in urine of methylphenidate and its main metabolite, ritalinic acid, are described. Both procedures are based on solid-phase extraction of urine samples on Bond Elut Certify columns, and capillary gas chromatographic—mass spectrometric detection of O-trimethylsilyl, N-trifluoroacetyl derivatives. The former method is used as a general screening procedure for the detection of basic polar nitrogen-containing compounds in urine such as stimulants, narcotic and adrenergic drugs. The latter procedure is proposed as a specific method to confirm methylphenidate ingestion. The two methods are sensitive enough to detect methylphenidate and ritalinic acid in urine at least for 24 h after administration of a therapeutic dose (20 mg oral dose) of methylphenidate.