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.     

The biochemistry of aromatic amines: The metabolism of 2-naphthylamine and 2-naphthylhydroxylamine derivatives

1. 2-Naphthylhydroxylamine and 2-nitrosonaphthalene were present in urine of dogs but not of guinea pigs, hamsters, rabbits or rats dosed with 2-naphthylamine. N-Acetyl-2-naphthylhydroxylamine and its O-sulphonic acid and O-glucosiduronic acid were not detected in the urine of any of these species. 2. Bile from rats dosed with 2-naphthylamine contained (2-naphthylamine N-glucosid)uronic acid and 6- and 5,6-substituted derivatives of 2-acetamidonaphthalene. 2-Amino-1-naphthyl and 2-acetamido-1-naphthyl derivatives, 2-naphthylhydroxylamine and its N-acetyl derivative or conjugates of these were not detected. Bile from a dog dosed with 2-naphthylamine contained no 2-amino-1-naphthyl derivatives. 3. 2-Naphthylhydroxylamine was metabolized by the dog, rat and guinea pig to the same products as those formed by these species from 2-naphthylamine. Rabbits formed mainly 2-amino-1-naphthyl derivatives; these are minor metabolites of 2-naphthylamine in this species. 4. (N-Acetyl-2-naphthylhydroxylamine O-glucosid)uronic acid was excreted in the urine and the bile of rats and in the urine of guinea pigs and rabbits dosed with N-acetyl-2-naphthylhydroxylamine. 5. After the administration of 2-acetamidonaphthalene, (N-acetyl-2-naphthylhydroxylamine O-glucosid)uronic acid was detected in the urine of dogs, but not in the urine of other species. The dog excreted an acid-labile cysteine derivative of 2-acetamidonaphthalene, but only traces of the corresponding mercapturic acid. 6. After dosing with N-acetyl-2-naphthylhydroxylamine-O-sulphonic acid, rats excreted derivatives of 2-amino-1-naphthol. 7. 2-Nitrosonaphthalene, N-acetyl-2-naphthylhydroxylamine, N-acetyl-2-naphthylhydroxylamine-O-sulphonic acid, 2-naphthylhydroxylamine-N-sulphonic acid, N-benzyloxycarbonyl-2-naphthylhydroxylamine and N-benzyloxycarbonyl-2-naphthylhydroxylamine-O-sulphonic acid were synthesized.     

Biotransformation of the Antimelanoma Agent Betulinic Acid by Bacillus megaterium ATCC 13368

Microbial transformation of the antimelanoma agent betulinic acid was studied. The main objective of this study was to utilize microorganisms as in vitro models to predict and prepare potential mammalian metabolites of this compound. Preparative-scale biotransformation with resting-cell suspensions of Bacillus megaterium ATCC 13368 resulted in the production of four metabolites, which were identified as 3-oxo-lup-20(29)-en-28-oic acid, 3-oxo-11α-hydroxy-lup-20(29)-en-28-oic acid, 1β-hydroxy-3-oxo-lup-20(29)-en-28-oic acid, and 3β,7β,15α-trihydroxy-lup-20(29)-en-28-oic acid based on nuclear magnetic resonance and high-resolution mass spectral analyses. In addition, the antimelanoma activities of these metabolites were evaluated with two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).     

Species differences in the metabolism of sulphadimethoxine

1. The fate of sulphadimethoxine (2,4-dimethoxy-6-sulphanilamidopyrimidine) was studied in man, rhesus monkey, dog, rat, guinea pig and rabbit. 2. About 20–46% of the dose (0·1g./kg.) of the drug is excreted in the urine in 24hr. in these species, except the rat, in which only 13% is excreted. 3. In man and the monkey sulphadimethoxine N¹-glucuronide is the major metabolite in the urine. In the rabbit and guinea pig N⁴-acetylsulphadimethoxine is the main metabolite. In the dog the drug is excreted mainly unchanged. In the rat equal amounts of the unchanged drug and its N⁴-acetyl derivative are the main products. 4. Small amounts of sulphadimethoxine N⁴-glucuronide are found in the urine of all the species. Sulphadimethoxine N¹-glucuronide occurs in small amounts in the urine of rat, dog and guinea pig; none is found in rabbit urine. 5. Sulphadimethoxine N⁴-sulphate was synthesized and found to occur in small amounts in rat urine. 6. Monkey liver homogenates fortified with UDP-glucuronic acid are able to synthesize sulphadimethoxine N¹-glucuronide with the drug as substrate. Rat liver has also this ability to a slight extent, but rabbit liver is unable to do so. 7. Sulphadimethoxine N⁴-glucuronide is formed spontaneously when the drug is added to human urine. 8. The biliary excretion of the drug and its metabolites was examined in rats. The drug is excreted in rat bile mainly as the N¹-glucuronide. The N¹- and N⁴-glucuronides administered as such are extensively excreted in the bile by rats.     

The metabolism of arylazoisoxazolones by rats and dogs

1. When rats were given a single oral dose of the lipid-soluble fungicide 4-(2-chlorophenylhydrazono)-3-methyl[4-(14)C]isoxazol-5-one ([(14)C]drazoxolon), about 75% of the label was excreted in the urine and 13% in the faeces in 96hr. An additional 7% of the radioactivity was recovered as (14)CO(2) in 48hr. 2. About 8% of the label was excreted by rats in the bile in 0-24hr. and an additional 6% was excreted by the same route in 24-48hr. 3. When dogs were given a single oral dose of [(14)C]drazoxolon about 35% of the label was excreted in the urine and a similar amount was excreted in the faeces in 96hr. 4. The major metabolites in the urine of the rat and the dog were identified as 2-(2-chloro-4-hydroxyphenylhydrazono)acetoacetic acid (dog, 14%), the corresponding ether glucosiduronic acid (dog, 12%; rat, 13%) and ester sulphate (rat, 65%). 5. When rats were given a single oral dose of 3-methyl-4-([U-(14)C]phenylhydrazono)isoxazol-5-one about 75% of the label was excreted in the urine and 15% in the faeces in 96hr. The major metabolite in the urine was identified as the ester sulphate conjugate of 2-(4-hydroxyphenylhydrazono)-acetoacetic acid. 6. Reduction of the azo link was of minor quantitative significance. 7. These results are discussed in their relation to species differences in the toxicity of these compounds.     

The metabolic fate of triamcinolone acetonide in laboratory animals

The metabolic fate of 9-fluoro-11β,16α,17,21-tetrahydroxy-l, 4-pregnadiene-3,20-dione cyclic 16,17-acetal with 2-¹⁴C-acetone, triaacinolone acetonide (TA) was studied in rabbits, dogs, monkeys and rats and found to be qualitatively similar in all species. In the dog, rat and monkey the major excretory route was the feces irrespective of the mode of administration. In the rabbit the excreted radioactivity was equally distributed between urine and feces. The metabolites were isolated by preparative thin layer chroma tography, located by autoradiography, eluted and analyzed by MS, IR, UV and NMR. The major metabolites of triamcinolone acetonide (TA) were identified as the C-21 carboxylic acids of TA and of the 6β hydroxy-TA,(6β-OH-TA) and the previously identified (1,2) 6β-OH-TA. In addition MS and UV data indicate the presence of 9-fluoro-11β,16α, 17-trihydroxy-3,20-dioxo-1,4,6-pregnatrien-21-oic acid cyclic 16,17 acetal with 2-¹⁴C-acetone.     

Progesterone hydroxylation and side chain oxidation to acidic metabolites by the pig

The anesthetised pig excreted acidic metabolites of progesterone in the bile and urine. Liver microsomes hydroxylated progesterone at the C-21, 6 beta- and 16 alpha-position and were inhibited by Ketoconazole. The pig exhibited intraspecies variability in progesterone 21- and 6 beta-hydroxylases. Liver, adrenal and to a lesser extent kidney microsomes oxidised the alpha-ketol side-chain to a C-21-oic acid. The liver reaction gave high affinity, low Km kinetics and a Vmax of 28.6 pmol acids mm-1 mg microsomal protein-1. Both carbon monoxide and Ketoconazole were inhibitory. These results implicate the cytochrome P-450 system with the ring and side-chain oxidations of progesterone. The pig resembled the rabbit in its metabolism of progesterone and is the second species in which a microsomal alpha-ketol oxidase has been implicated in the biosynthesis of steroidal acids.     

Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated ¹⁴C-labelled eltanolone (5β-Pregnan-3α-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of ¹⁴C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC–ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography–mass spectrometry with an electron impact ion source (GC–MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).     

Determination of pethidine and its major metabolites in human urine by gas chromatography

Procedures based on gas chromatography were established to determine pethidine and its major metabolites in human urine. The chromatographic system consisted of a glass column packed with 3% (w/w) SP2250 on Chromosorb W (80–100 mesh) linked to a nitrogen—phosphorus detector. Diethyl ether was used as the extraction solvent. Pethidinic and norpethidinic acids, and their conjugated metabolites (after β-glucuronidase treatment) were determined after conversion into pethidine and norpethidine by acid-catalysed esterification. The retention times of pethidine, norpethidine and chlorpheniramine (internal standard) were 3.3, 4.5 and 7.5 min, respectively. The amount of unchanged drugs and metabolites excreted varied considerably among the subjects. The mean 24-h urinary recoveries in eight patients of pethidine, norpethidine, pethidinic acid, norpethidinic acid, and the glucuronides of pethidinic and norpethidinic acids were 6.62 ± 5.05, 4.33 ± 1.19, 18.9 ± 6.29, 9.10 ± 4.26, 15.1 ± 3.02 and 7.57 ± 2.28%, respectively. This indicates that the major metyabolic pathways of pethidine in the eight patients were hydrolysis followed by conjugation. Over 60% of the dose was accounted for in 24 h after intramuscular administration of 1 mg/kg pethidine.     

Species differences in the aromatization of quinic acid in vivo and the role of gut bacteria

1. The fate of (−)-quinic acid has been investigated in 22 species of animals including man. 2. In man and three species of Old World monkeys, i.e. rhesus monkey, baboon and green monkey, oral quinic acid was extensively aromatized (20–60%) and excreted in the urine as hippuric acid, which was determined fluorimetrically. 3. In three species of New World monkeys, i.e. squirrel monkey, spider monkey and capuchin, in three species of lemurs, i.e. bushbaby, slow loris and tree shrew, in the dog, cat, ferret, rabbit, rat, mouse, guinea pig, hamster, lemming, fruit bat, hedgehog and pigeon, oral quinic acid was not extensively aromatized (0–5%). 4. In the rhesus monkey, injected quinic acid was not aromatized, but largely excreted unchanged. 5. In rhesus monkeys pretreated with neomycin to suppress gut flora, the aromatization of oral quinic acid was considerably suppressed. 6. In rats and rhesus monkeys [¹⁴C]quinic acid was used and this confirmed its low aromatization in rats and its high aromatization in the monkeys. 7. Shikimic acid given orally was excreted as hippuric acid (26–56%) in rhesus monkeys, but not in rats. 8. The results support the view that quinic acid and shikimic acid are aromatized by the gut flora in man and the Old World monkeys.     

Identification of the major urinary metabolite of alprenolol in man, dog and rat

After oral administration of ³H-alprenolol to man, dog and rat, urinary metabolites of the drug have been separated by ion-exchange chromatography on Bio-Rex 70, a carboxylic acrylate resin. The major metabolite has been identified by GC-MS as 4-hydroxyalprenolol. Occurring in the urine largely in a conjugated form, it represents about 40 % of the excreted amount in man and dog and about 30 % in rat. Including alprenolol, which also appears largely as a conjugate, about 80 % of the amount of radioactivity excreted in human urine can be accounted for.     

Simplified method for simultaneous determination of diazepam and its metabolites in urine by thin-layer chromatography and direct densitometry

A direct densitometric method for determination of diazepam and its metabolites in urine was developed. The proposed procedure involves acid hydrolysis of urine specimens, thereby converting diazepam and its metabolites into benzophenones [2-methylamino-5-chlorobenzophenone (MACB) and 2-amino-5-chlorobenzophenone (ACB)]. It is followed by extraction with chloroform—isopropanol (3:1, v/v). The two benzophenones were separated on thin-layer chromatography plates using hexane—diethyl ether—acetic acid (80:10:10) as a mobile phase. Quantitation of the MACB and ACB spots was achieved by direct ultraviolet densitometry. The limit of detection was 0.5 μg per ml of urine for both benzophenones. The proposed method is simple, rapid, reproducible and has been found to be effective for direct determination of diazepam and its metabolites in urine.     

New insights into the bioavailability of red raspberry anthocyanins and ellagitannins

Red raspberries, containing ellagitannins and cyanidin-based anthocyanins, were fed to volunteers and metabolites appearing in plasma and urine were analysed by UHPLC-MS. Anthocyanins were not absorbed to any extent with sub nmol/L concentrations of cyanidin-3-O-glucoside and a cyanidin-O-glucuronide appearing transiently in plasma. Anthocyanins excreted in urine corresponded to 0.007% of intake. More substantial amounts of phase II metabolites of ferulic acid and isoferulic acid, along with 4′-hydroxyhippuric acid, potentially originating from pH-mediated degradation of cyanidin in the proximal gastrointestinal tract, appeared in urine and also plasma where peak concentrations were attained 1–1.5 h after raspberry intake. Excretion of 18 anthocyanin-derived metabolites corresponded to 15.0% of intake, a figure substantially higher than obtained in other anthocyanin feeding studies. Ellagitannins pass from the small to the large intestine where the colonic microbiota mediate their conversion to urolithins A and B which appeared in plasma and were excreted almost exclusively as sulfate and glucuronide metabolites. The urolithin metabolites persisted in the circulatory system and were excreted in urine for much longer periods of time than the anthocyanin metabolites although their overall urinary recovery was lower at 7.0% of intake. It is events originating in the proximal and distal gastrointestinal tract, and subsequent phase II metabolism, that play an important role in the bioavailability of both anthocyanins and ellagitannins and it is their metabolites which appear in the circulatory system, that are key to elucidating the mode of action(s) underlying the protective effects of these compounds on human health.     

Determination of histamine, methylhistamines and histamine—o-phthaldialdehyde complexes by two high-performance liquid chromatographic procedures : Application to biological samples

Two high-performance liquid chromatographic procedures were proposed to measure histamine. The first, with UV detection and a strong acid cation exchanger (Partisil 10, SCX Whatman), made it possible to isolate histamine and some methylated derivatives. The second, with a C₁₈ sorbent (μBondapak, Waters, 10 μm particle size) eluted with ion-pairing phases, made it possible to isolate the histamine—o-phthaldialdehyde complexes. This last procedure allied with a chromatographic purification step gave lower or identical amounts of histamine than those described in human urine (16 ± 7 μg per 24 h), canine whole blood (1.5 ± 1 ng/ml) and human gastric juice (2.3 ± 1.4 ng/ml). The two procedures gave the concentration of a histamine-like compound isolated from the antral mucosa.     

The metabolism of labelled hexadecyl sulphate salts in the rat, dog and human.

The metabolic fate of [1-14-C]hexadecylsulphate and hexadecyl[35-S]sulphate, administered intravenously as the sodium and trimethylammonium salt to dogs and orally as the erythromycin salt to dogs, rats and humans, was studied. Studies with rats indicated that the compounds were well absorbed and rapidly excreted in the urine. However, after oral administration of the 14-C-and 35-S-labelled hexadecyl sulphate erythromycin salt to dogs, considerable amounts of radioactivity were excreted in the faeces as unmetabolized hexadecyl sulphate. Studies with two humans showed that orally administered erythromycin salt of [1-14C]hexadecyl sulphate was well absorbed in one person but poorly absorbed in the other. Radioactive metabolites in urine were separated by t.l.c. in two solvent systems. The main metabolite of hexadecyl sulphate in the dog, rat and human was identified as the sulphate ester of 4-hydroxybutyric acid. In addition, psi-[14-C]butyrolactone as a minor metabolic product of [1-14-C]hexadecyl sulphate was also isolated from the urine of rat, dog and man. However, there was still another metabolite in dog urine, which comprised about 20% of the total urinary radioactivity and carried both 14-C and 35-S labels. This metabolite was absent from rat urine. The metabolite in dog urine was isolated and subsequently identified by t.l.c. and g.l.c. and by isotope-dilution experiments as the sulphate ester of glycollic acid. Small amounts (about 5% of the total recovered radioactivity in excreta) of labelled glycollic acid sulphate were also found in human urine after ingestion of erythromycin [1-14-C]hexadecyl sulphate.     

Simultaneous determination of glucuronides of trimetozine in human urine by gas chromatography

A gas chromatographic method for the simultaneous determination of four glucuronides (metabolites) of trimetozine excreted in human urine is described. The methodinvolves pretreatment of the urine specimen [i.e. removal of interfering substances by solvent extraction, desalting on an ion-exchange (Amberlite XAD-2) column], and permethylation of glucuronides by reaction with methylsulfinyl carbanion and methyl iodide. The permethylated derivatives were submitted to gas chromatographic separation on an OV-17 column, and their structures were investigated by subsequent gas chromatographic—mass spectrometric analysis. The minimum detectable concentration of each glucuronide is 5 μg/ml when 1 ml of urine is used. The utility of the present method is successfully demonstrated by determining the urinary concentration of four glucuronides following oral administration of trimetozine to human subjects.     

Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography—mass spectrometry using their N-labelled analogs

For the quantification of nitrite and nitrate, the stable metabolites of -arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic—mass spectrometric (GC—MS) method in which [¹⁵N]nitrite and [¹⁵N]nitrate were used as internal standards. Endogenous nitrite and [¹⁵N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [¹⁵N]nitrate they were reduced to nitrite and [¹⁵N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-μl aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [¹⁵N]nitrite ([¹⁵N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 ± 0.25 of nitrite and 109.5 ± 61.7 of nitrate (mean ± S.D., μmol/mmol creatinine) with a mean 24-h output of 5.7 μmol for nitrite and 1226 μmol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean ± S.D., μmol/l) 3.6 ± 0.8 and 68 ± 17, respectively.     

Studies on the metabolism of 2,4′-isobutylphenylpropionic acid (ibuprofen) by gas chromatography and mass spectrometry : Dialysis fluid, a convenient medium for studies on drug metabolism

2,4′-Isobutylphenylpropionic acid (ibuprofen) has previously been demonstrated to yield four urinary metabolites, formed by ω1-, ω2- and ω3-hydroxylation and by a further oxidation of the primary alcohol of the ω1-hydroxylated metabolite to a carboxyl group. By synthesis and gas chromatography—mass spectrometry the suggested structure of the ω3-hydroxylated metabolite was verified in the present study. Moreover, a new metabolite, 2,4′-carboxyphenylpropionic acid, was demonstrated to be present in substantial amounts in dialysis fluid from a nephrectomized patient. In such patients ingested drugs cannot be excreted in the urine, but are metabolized to end products. Thus, dialysis fluid may be a convenient medium for studies on drug metabolism.     

Corticosteroid side-chain oxidations--I. Structural effects on the excreted isotope ratios of 4-14C- and 21-3H-labelled corticosteroid metabolites in rabbit urine

The metabolic fates of 4-14C- and 21-3H-labelled corticosteroids have been investigated in the rabbit by analysis of the normalized isotope ratios of neutral and acidic metabolites excreted in the urine. Isotope ratios of excreted radioactivity declined in the order cortisol (F) greater than corticosterone (B) greater than 11-desoxycortisol (S) greater than deoxycorticosterone (DOC). Steroid acids, isolated in alumina fraction C, represented 19.0, 15.0, 9.7 and 2.7% of the doses of DOC, B, S and F, respectively, and the isotope ratios declined in the order F greater than B greater than S greater than DOC. HPLC of steroid acid methyl ester derivatives indicated generally low isotope ratios for DOC and S steroid acids, consistent with complete side-chain oxidation to 20-oxo-21-oic acids and/or 17-carboxylic acids. Several B metabolite methyl esters peaks also exhibited low isotope ratios, but both B and F metabolites gave methyl esters that retained significant tritium consistent with the presence of 20-hydroxysteroid acids. The 21-hydroxy-steroid metabolite fractions had isotope ratios of F = S greater than B greater than DOC. HPLC showed that 20-oxo (tetrahydro) metabolites of B and F had reduced isotope ratios unlike the C-20 reduced (hexahydro) metabolites of DOC and S. It may be concluded that the metabolic fate of the corticoid side-chain in the rabbit is dependent on the steroid structure and may result in the excretion of both 20-oxo and 20-hydroxysteroid acids.     

Simultaneous determination of thioTEPA, TEPA and a novel, recently identified thioTEPA metabolite, monochloroTEPA, in urine using capillary gas chromatography

An assay for the simultaneous quantitative determination of thioTEPA, TEPA and the recently identified metabolite N,N′-diethylene-N″-2-chloroethylphosphoramide (monochloroTEPA) in human urine has been developed. MonochloroTEPA was synthesized by incubation of TEPA with sodium chloride at pH 8. Thus, with this assay monochloroTEPA is quantified as TEPA equivalents. Analysis of the three analytes in urine was performed using gas chromatography with selective nitrogen–phosphorous detection after extraction with a mixture of 1-propanol and chloroform from urine samples. Diphenylamine was used as internal standard. Recoveries ranged between 70 and 100% and both accuracy and precision were less than 15%. Linearity was accomplished in the range of 25–2500 ng/ml for monochloroTEPA and 25–5000 ng/ml for thioTEPA and TEPA. MonochloroTEPA proved to be stable in urine for at least 4 weeks at −80°C. ThioTEPA, TEPA and monochloroTEPA cummulative urinary excretion from two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples and that the excreted amount of monochloroTEPA exceeded that of thioTEPA on day 2 to 5 of urine collection.