Stable isotope dilution analysis of human urinary metabolites of 17α-methyltestosterone

A method based on gas chromatography–mass spectrometry–selected-ion monitoring was developed to measure the main metabolites of 17α-methyltestosterone, 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol, in human urine. 17α-Methyl-[²H₃]-5α-androstan-3α,17β-diol and 17α-methyl-[²H₃]-5β-androstan-3α,17β-diol were used as internal standards. The methods involved purification using a Sep-Pak C₁₈ cartridge, hydrolysis by β-glucuronidase from Ampullaria and derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide/dithioerythriol/ammonium iodide. Quantitation was achieved by selected-ion monitoring of the characteristic fragment ions ([(M+H)−2×TMSOH]⁺) of the di-TMS derivatives on the chemical ionization mode. The method provides a specific, sensitive and reliable technique to determine the urine levels of 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol, and can be applied to pharmacokinetic studies of 17α-methyltestosterone.     

Estrogen metabolites in equine ovarian follicles: gas chromatographic-mass spectrometric determinations in relation to follicular ultrastructure and progestin content

Equine follicular fluid was aspirated at various developmental stages (viable, preovulatory and atretic) determined by ultrastructural study. Estrogens and progestins were analyzed by gas chromatography-mass spectrometry associated with stable isotope dilution. Progesterone and 17-hydroxyprogesterone were the principal progestins of the preovulatory and viable follicles. Among the catechol estrogens, 2-hydroxy-estradiol was particularly abundant in the preovulatory follicle and its definitive identification was made by the scan of a full mass spectrum.     

Automated chromatographic system for the simultaneous measurement of plasma pregnenolone and 17-hydroxypregnenolone by radioimmunoassay

A new, simple, rapid and highly practicable automated chromatographic system for the separation, and a sensitive radioimmunoassay system for the subsequent measurement of pregnenolone and 17-hydroxypregnenolone has been developed. Pregnenolone and 17-hydroxypregnenolone were extracted with methylene chloride and separated from cross-reacting steroids by mechanised Sephadex-LH20 multi-column chromatography. Anti-pregnenolone and anti-17-hydroxypregnenolone were obtained by immunising rabbits with pregnenolone-20-oxime-BSA and 17-hydroxypregnenolone-20-oxime-BSA. The lower detection limit of the assay is 0.15 and 0.28 nmol/l for pregnenolone and 17-hydroxypregnenolone, respectively. Normal values for this assay in young male adults, in adult females, and in prepubertal boys and girls were established as a basis for the functional diagnosis of androgen excess syndromes/steroidogenesis defects.     

Metabolism of metandienone in man: identification and synthesis of conjugated excreted urinary metabolites, determination of excretion rates and gas chromatographic-mass spectrometric identification of bis-hydroxylated metabolites

After oral administration of metandienone (17 alpha-methyl-androsta-1,4-dien-17 beta-ol-3-one) to male volunteers conjugated metabolites are isolated from urine via XAD-2-adsorption, enzymatic hydrolysis and preparative high-performance liquid chromatography (HPLC). Four conjugated metabolites are identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionization after derivatization with N-methyl-N-trimethyl-silyl-trifluoroacetamide/trimethylsilyl-imidazole (MSTFA/TMS-Imi) and comparison with synthesized reference compounds: 17 alpha-methyl-5 beta-androst-1-en-17 beta-ol-3-one (II), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (III), 17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (IV) and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (V). After administration of 40 mg of metandienone four bis-hydroxy-metabolites--6 beta,12-dihydroxy-metandienone (IX), 6 beta,16 beta-dihydroxy-metandienone (X), 6 beta,16 alpha-dihydroxy-metandienone (XI) and 6 beta,16 beta-dihydroxy-17-epimetandienone (XII)--were detected in the unconjugated fraction. The metabolites III, IV and V are excreted in a comparable amount to the unconjugated excreted metabolites 17-epimetandienone (VI), 6 beta-hydroxy-metandienone (VII) and 6 beta-hydroxy-17-epimetandienone (VIII). Whereas the unconjugated excreted metabolites show maximum excretion rates between 4 and 12 h after administration the conjugated metabolites III, IV and V are excreted with maximum rates between 12 and 34 h.     

New gas chromatographic-mass spectrometric method for the determination of urinary pyrethroid metabolites in environmental medicine

We have developed and validated a new, reliable and very sensitive method for the determination of the urinary metabolites of the most common pyrethroids in one analytical run. After acidic hydrolysis for the cleavage of conjugates, the analytes cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl(2)CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl(2)CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br(2)CA), 4-fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA) were extracted from the matrix with a liquid-liquid extraction procedure using n-hexane under acidic conditions. For further clean-up, NaOH was added to the organic phase and the carboxylic acids were re-extracted into the aqueous phase. After acidification and extraction into n-hexane again, the metabolites were then derivatised to volatile esters using N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA). Separation and detection were carried out using capillary gas chromatography with mass-selective detection (GC-MS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard for the quantification of the pyrethroid metabolites. The limit of detection for all analytes was 0.05 microg/l urine. The RSD of the within-series imprecision was between 2.0 and 5.4% at a spiked concentration of 0.4 microg/l and the relative recovery was between 79.3 and 93.4%, depending on the analyte. This method was used for the analysis of urine samples of 46 persons from the general population without known exposure to pyrethroids. The metabolites cis-Cl(2)CA, trans-Cl(2)CA and 3-PBA could be found in 52, 72 and 70% of all samples with median values of 0.06, 0.11 and 0.16 microg/l, respectively. Br(2)CA and F-PBA could also be detected in 13 and 4% of the urine samples.     

The use of stable isotopes and gas chromatography/mass spectrometry in the identification of steroid metabolites in the equine

Stable isotope gas chromatography/mass spectrometry has been used successfully in the elucidation of structures of urinary steroid metabolites in the horse and in the identification of metabolites isolated from in vivo perfusion and in vitro incubation studies using equine tissue preparations. Deuterium-labeled steroids, testosterone, dehydroepiandrosterone, and 5-androstene-3 beta,17 beta-diol have been synthesized by base-catalyzed isotope exchange methods and the products characterized by gas chromatography/mass spectrometry. [16,16(-2)H2]Dehydroepiandrosterone (plus radiolabeled dehydroepiandrosterone) was perfused into a testicular artery of a pony stallion and was shown to be metabolized into 2H2-labeled testosterone, 4-androstenedione, isomers of 5-androstene-3,17-diol, 19-hydroxytestosterone, and 19-hydroxy-4-androstenedione. In further studies, equine testicular minces have been incubated with 2H2-labeled and radiolabeled dehydroepiandrosterone and 5-androstene-3 beta, 17 beta-diol. The metabolites, whose identity was confirmed by stable isotope gas chromatography/mass spectrometry, proved the interconversion of the two substrates, as well as formation of testosterone and 4-androstenedione. The aromatization of dehydroepiandrosterone was also confirmed, together with the formation of an isomer of 5(10)-estrene-3,17-diol from both substrates showing 19-demethylation without concomitant aromatization. In studies of the feto-placental unit, the allantochorion was shown to aromatize [2H5]testosterone to [2H4]estradiol, the loss of one 2H from the substrate being consistent with aromatization of the A ring. The formation of 6-hydroxyestradiol was also confirmed in this study. The same technique has been valuable in determining the structure of two metabolites of nandrolone isolated from horse urine.(ABSTRACT TRUNCATED AT 250 WORDS)     

A gas chromatographic-mass spectral assay for the quantitative determination of oleamide in biological fluids.

Oleamide is a putative endogenous sleep-inducing lipid which potently enhances currents mediated by GABAA and serotonin receptors. While a quantitative assay would aid in determining the role of oleamide in physiological processes, most of the available assays are lacking in sensitivity. We now describe a quantitative assay for measuring low nanogram amounts of oleamide in biological fluids using GC/MS in the selective ion-monitoring mode. The internal standard (13C18 oleamide) was added to known concentrations of oleamide, which were converted to the N-trimethylsilyl or N-tert-butyldimethylsilyl derivatives before analysis by GC/MS, yielding linear calibration curves over the range of 1-25 ng of oleamide when monitoring the m/z 338/356 fragments. Using this technique, oleamide levels were determined following solvent extraction of normal rat cerebrospinal fluid and plasma to be 44 and 9.9 ng/ml, respectively. This technique constitutes a sensitive and reliable method for determining low nanogram quantities of oleamide in biological fluids.     

Effects of environmental salinity and 17alpha-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus

Effects of environmental salinity and 17α-methyltestosterone (MT) on growth and oxygen consumption were examined in the tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to one of four groups: FW, MT treatment in FW, seawater (SW) and MT treatment in SW. All treatment groups were fed to satiation three times daily. The fish reared in SW (both control and MT-treated groups) grew significantly larger than either group in FW from day 43 throughout the experiment (195 days). The fish fed with MT added to their feed grew significantly larger than their respective controls from day 85 in FW and in SW until the end of the experiment. The routine metabolic rate (RMR) was determined monthly from month 2 (day 62) to month 5 (day 155). A significant negative correlation was seen between RMR and body mass in all treatment groups. Among fish of the same age, the SW-reared tilapia had significantly lower RMRs than the FW-reared fish. The MT-treated fish in SW showed significantly lower RMRs than the SW control group at months 3–5, whereas MT treatment in FW significantly increased the RMR at month 3. Comparison of regression lines between RMR and body mass indicates that MT treatment in FW caused a significant increase in oxygen consumption at a given mass of the fish, whereas MT treatment was without effect on RMR in SW-reared fish. These results clearly indicate that SW-rearing and MT treatment accelerate growth of tilapia, and that RMR decreases as fish size increased. It is also likely that the increased RMR and growth in MT-treated tilapia in FW may be due to the metabolic actions of MT, although the reason for the absence of MT treatment in SW is unclear.     

Biotransformation of mestanolone and 17-methyl-1-testosterone by Rhizopus stolonifer

Abstract

Microbial transformation of mestanolone (1) using the plant pathogenic fungus, Rhizopus stolonifer, resulted in the production of two known metabolites, identified as 11α-hydroxymestanolone (3) and 6α-hydroxymestanolone (4). Transformation of 17-methyl-1-testosterone (2) by R. stolonifer yielded two known metabolites, methandrostenolone (5) and 11α,17β- dihydroxy-androsta-1,4-diene-3-one (6). These transformations included α-hydroxylations at C-11 and C-6, dehydrogenation at C-4, androsta and a demethylation at C-17 positions. Structures of transformed products were determined using spectroscopic techniques.     

Biotransformation of urapidil: isolation and identification of metabolites in mouse, rat, dog and man

The identification of biotransformation products of the new antihypertensive drug urapidil in mouse, rat, dog and man has been performed by means of high-performance liquid chromatographic and mass spectrometric techniques. In urine, three metabolites were found in addition to the unchanged drug. The para-hydroxylated product (1) (6-(3-[4-(o-methoxy-p-hydroxyphenyl)piperazinyl]-propylamino)-1, 3-dimethyl-uracil), the O-demethylated compound (2) (6-(3-[4-(o-hydroxyphenyl)piperazinyl]-propylamino)-1, 3-dimethyluracil) and the uracil-N-dealkylated compound (3) (6-(3-[4-(o-methoxyphenyl)piperazinyl]-propylamino)-1-methyluracil). In urine of dog, the metabolite with the N-oxide structure (5) was also identified, but only in trace amounts (6-(3-[4-(o-methoxyphenyl)piperazinyl-N-oxide]-propylamino)-1, 3-dimethyluracil).     

Separation of testosterone metabolites in microsomal incubates using a new capillary electrophoresis assay

A capillary electrophoresis method has been developed to separate the products of liver microsomal testosterone metabolism. The microsomal mixture undergoes liquid-liquid extraction and pre-concentration, and then electrophoretic analysis takes less than 25 min including capillary conditioning steps. The development of the complex background electrolyte (Tris-HCl and borate buffers, sodium dodecyl sulfate, β-cyclodextrin and ethanol) necessary for this separation is described. A z-type capillary flow cell is used to obtain adequate detection sensitivity. The proportion in which the metabolites are produced as determined by this method allows assignment of the relative activity of cytochrome P-450 enzymes in the microsomes. The technique is useful for comparison of activity in normal and abnormal hepatic microsomes.     

Biotransformation of isoeugenol to vanillin by a newly isolated Bacillus pumilus strain: identification of major metabolites

A bacterial strain S-1 capable of transforming isoeugenol to vanillin was isolated. The strain was identified as Bacillus pumilus based on biochemical tests, cellular fatty acid composition, riboprint pattern and 16S rRNA gene sequence analyses. In the biotransformation of isoeugenol, vanillin was the main product. With the growing culture of B. pumilus S-1, 10 g l⁻¹ isoeugenol was converted to 3.75 g l⁻¹ vanillin in 150 h, with a molar yield of 40.5% that is the highest up to now. Dehydrodiisoeugenol, a dimer of isoeugenol, was separated by preparative thin layer chromatography and identified by gas chromatography–mass spectrometry. Based on the accurate masses obtained from gas chromatography–high resolution mass spectrometry, two key intermediates, isoeugenol-epoxide (IE) and isoeugenol-diol (ID), were identified by mass spectra interpretations. The biotransformation with resting cells showed that vanillin was oxidized to vanillic acid and then to protocatechuic acid before the aromatic ring was broken. These findings suggest that isoeugenol is degraded through an epoxide-diol pathway.     

Biotransformation of 3b-Hydroxyandrost-5-en-17-one by Cell Suspension Cultures of Catharanthus roseus

Catharanthus roseus (L.) G. Don cell suspension cultures were used to transform 3b-hydroxyandrost-5-en-17-one, the products were isolated by chromatographic methods. Their structures were established by means of NMR and MS spectral analyses. Nine metabolites were respectively elucidated as: androst-4-ene-3,17-dione (Ⅰ), 6a-hydroxyandrost-4-ene-3,17-dione (Ⅱ), 6a,17b-dihydroxyandrost-4-en-3-one (Ⅲ), 6b-hydroxyandrost-4-ene-3,17-dione (Ⅳ), 17b-hydroxyandrost-4-en-3-one (Ⅴ), 15a,17b-dihydroxyandrost-4-en-3-one (Ⅵ), 15b,17b-dihydroxyandrost-4-en-3-one (Ⅶ), 14a-hydroxyandrost-4-ene-3,17-dione (Ⅷ), 17b-hydroxyandrost-4-ene-3,16-dione (Ⅸ). It is the first time to obtain the above compounds by biotransformation with Catharanthus roseus cell cultures.     

氧化葡萄糖酸菌转化制备米格列醇关键中间体

米格列醇作为一种新型α－葡糖苷酶抑制剂,能够有效治疗Ⅱ型糖尿病,并已迅速成为首选药物。葡萄糖酸菌细胞膜上含有多种脱氢酶,能够催化一系列重要产物的微生物转化,米格列醇就是其中之一。本文将详细说明不同的微生物转化过程,并进行比较。     

17-Hydroxylase/C17,20-lyase (CYP17) is not the enzyme responsible for side-chain cleavage of cortisol and its metabolites

The question addressed in this study was the nature of the enzyme required to remove the side-chain of 17-hydroxycorticosteroids, leading in the case of cortisol to the excretion of 11β-hydroxyandrosterone, 11-oxo-androsterone and the corresponding etiocholanolones. We questioned whether it could be CYP17, the 17-hydroxylase/17,20-lyase utilized in androgen synthesis. The conversion of exogenous cortisol to C₁₉ steroids in patients with complete 17-hydroxylase deficiency (17HD) was studied rationalizing that if CYP17 was involved no C₁₉ steroids would be formed. The urinary excretion of the four 11-oxy-C₁₉ steroids as well as many of the major C₂₁ cortisol metabolites were measured by GC/MS. Our results showed that the conversion of cortisol to C₁₉ steroids was normal in 17HD indicating that a currently unidentified enzyme must be responsible for this transformation.

A secondary goal was to determine to what extent 11-oxy-C₁₉ steroids were metabolites of cortisol or adrenal synthesized 11β-hydroxyandrostenedione. Since cortisol-treated 17HD patients cannot produce androstenedione, all C₁₉ 11-oxy-metabolites excreted must be derived from exogenous cortisol. The extent to which 17HD patients have lower relative excretion of C₁₉ steroids should reflect the absence of 11β-hydroxyandrostenedione metabolites. Our results showed almost all of 11-oxo-etiocholanolone and 11β-hydroxyetiocholanolone were cortisol metabolites, but in contrast the excretion of 11β-hydroxyandrosterone was less than 10% that of normal individuals, indicating that in excess of 90% must be a metabolite of 11β-hydroxyandrostenedione.     

靶向ADAM17基因RNA干扰慢病毒载体的构建及慢病毒包装

目的构建靶向ADAM17基因RNA干扰（RNAi）慢病毒载体及包装慢病毒。方法根据人ADAM17mRNA序列设计4个靶序列,合成4对寡核苷酸序列,同时合成1对阴性对照寡核苷酸序列;将以上5对寡核苷酸序列退火后连入pLVTHM质粒,经酶切和测序鉴定。将重组慢病毒质粒转染至A549细胞,以Real-time PCR检测A549细胞中ADAM17 mRNA表达。将干扰效果最佳的质粒载体和包装质粒共转染至293T细胞,包装产生病毒颗粒。以流式细胞术检测重组慢病毒的滴度。结果酶切和测序证实干扰靶序列已被准确克隆到pLVTHM质粒载体。pLVTHM-ADAM17-siRNA1-4均可显著抑制A549细胞ADAM17 mRNA的表达,其中pLVTHM-ADAM17-siRNA4的抑制效果最佳。LV-ADAM17-siRNA4重组慢病毒的滴度为2.16×108TU/ml。结论成功构建了靶向人ADAM17基因RNAi慢病毒载体及包装了重组慢病毒。     

Expression and Identification of a Novel Apoptosis Gene Spata17 （MSRG-11） in Mouse Spermatogenic Cells

In this study,anti-spermatogenesis-associated 17 (Spatal7) polyclonal antibody was preparedby immunizing New Zealand white rabbits with a synthesized peptide corresponding to the amino acid se-quence 7-23 of the mouse Spata17 protein.Immunohistochemical analysis revealed that Spata17 proteinwas most abundant in the cytoplasm of round spermatids and elongating spermatids within seminiferoustubules of the adult testis.The expression of Spata17 mRNA in cultured mouse spermatogonia (GC-1) cellswas almost undetectable.In an experimental unilateral cryptorchidism model of an adult mouse,the expres-sion of Spata17 mRNA had no obvious difference with the normal testis until postoperation day 1,butgradually decreased from day 3 and was almost undetectable on day 17.Immunohistochemical analysisrevealed that the protein was almost undetectable within seminiferous tubules of an experimental unilateralcryptorchidism model of the adult testis on postoperation day 8.Flow cytometry analysis showed that theexpression of Spatal7 protein in the GC-1 cell line could accelerate GC-1 cell apoptosis.The effect increaseswith the increasing of the transfected dose of pcDNA3.1 (-)/Spata17.By Hoechst 33258 staining,a classicalway of identifying apoptotic cells,we further confirmed that the apoptosis was induced by expression ofSpata17 in transfected GC-1 cells.     

Simultaneous determination, in calf urine, of twelve anabolic agents as heptafluorobutyryl derivatives by capillary gas chromatography-mass spectrometry

A method to determine twelve anabolic hormones (diethylstilbestrol, hexestrol, dienestrol, 17β-estradiol, 19-nortestosterone, testosterone, 1-dehydrotestosterone, 17α-methyltestosterone, progesterone, estrone, 17α-ethynilestradiol, and trenbolone) is presented. Urine samples were extracted with octadecylsilica columns and clean-up was performed in two steps with basic alumina and silica solid-phase extraction cartridges. The extracts obtained were derivatized with heptafluorobutyric anhydride and analyzed by GC-MS. Stability of derivatives was good and compounds having keto groups produced enol derivatives that were stable also. SIM mode was applied to increase the sensitivity and, when possible, the higher m/z ions were selected to improve identification. Repeatability of the chromatographic analysis was evaluated on the basis of area repeatability, and the coefficient of variation obtained was lower than 13%. Absolute recoveries were in the range 35–60% (dehydrotestosterone and estrone <20%) with coefficients of variation between 14 and 37% for the whole procedure. [²H₃]Testosterone and [²H₈]diethylstilbestrol were evaluated to improve quantitative data. The recovery of [²H₃]testosterone was found to be equal to or slightly higher than that of the other hormones, but the recovery of [²H₈]diethylstilbestrol was lower than any other. [²H₃]Testosterone was the most suitable for use as an internal standard, as its addition at the beginning of analytical procedure, corrected recovery results and greatly improved precision. Corrected recoveries from urine ranged from 72–110%, and coefficients of variation ranged from 6–15%, except for testosterone which yielded slightly higher values. The limit of detection was 0.5 ng/ml for all the compounds studied.     

Quantification of the neuromuscular blocking agent rocuronium and its putative metabolite 17-desacetylrocuronium in heparinized plasma by capillary gas chromatography using a nitrogen sensitive detector

We have developed a sensitive and specific capillary GC (cGC) assay for the quantification of the quarternary aminosteroidal compound rocuronium (roc), a neuromuscular blocking agent, and its putative metabolite 17-desacetylrocuronium (17OH-roc), using 3-desacetylvecuronium (3OH-vec) as an internal standard (I.S.). This novel method has been applied to a pharmacokinetic study with roc, monitoring sixty patients who were classified according to four different body mass index (BMI) groups. The isolation of these drugs from plasma was carried out using a dichloromethane liquid-liquid extraction after ion-pairing of the positively charged ammonium compounds with iodide. To achieve thermal stability, tert.-butyldimethylsilyl-ethers were formed at the 3OH- and 17OH-steroidal positions by reaction with N-methyl-N-(tert.-butyldimethylsilyl)-trifluoroacetamide at 70°C overnight. An automated cGC system fitted with a nitrogen sensitive detector with a specially prepared glass phase bead and a computer controlled data handling system was used to analyze and quantify the compounds, which were separated on a DB1 capillary column with helium as the carrier gas and a temperature program ranging from 120 to 300°C. The method is linear for 50-6400 ng/ml for roc and 80-6400 ng/ml for 17OH-roc. The detection limits were 10 ng/ml for roc and 50 ng/ml for 17OH-roc. The lower limit of quantification was 50 ng/ml for roc and 80 ng/ml for 17OH-roc. Intra-assay coefficients of variation (C.V.s) were 10% and 15% and the inter-assay C.V.s 8-18% and 16-21% for roc and 17OH-roc, respectively.     

Accurate assignment of ethanol origin in postmortem urine: liquid chromatographic-mass spectrometric determination of serotonin metabolites

Toxicological examination of fatal aviation accident victims routinely includes analysis of ethanol levels. However, distinguishing between antemortem ingestion and postmortem microbial formation complicates all positive ethanol results. Development of a single analytical approach to determine concentrations of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA), two well-known metabolites of serotonin, has provided a convenient, rapid and reliable solution to this problem. Antemortem ethanol leads to an elevation in the 5-HTOL/5-HIAA ratio for 11-19 h after acute ingestion. The liquid-liquid extracts of postmortem urine samples were subjected to liquid chromatography-mass spectrometry (LC-MS) for the simultaneous quantitation of these two analytes, yielding detection limits of 0.1 ng/ml for each. Examination of the 5-HTOL/5-HIAA ratio was undertaken for 44 urine samples known to be antemortem ethanol-positive or antemortem ethanol-negative. Recent ethanol ingestion was conveniently and accurately separated using a 5-HTOL/5-HIAA ratio of 15 pmol/nmol, a value previously suggested using human volunteers. All 21 ethanol-negative postmortem samples were below this cutoff, while all 23 ethanol-positive postmortem samples were above this cutoff. Thus, we recommend the employment of this cutoff value, established using this straightforward LC-MS procedure, to confirm or deny recent antemortem ethanol ingestion in postmortem urine samples.