A chemiluminescent immunoassay for the direct measurement of urinary 5α-androstane-3α, 17β-diol-glucuronide in urine

We described a chemiluminescent immunoassay (CIA) for 5α-androstane-3α, 17β-diol-glucuronide (3α-diol-G) in human diluted urine. This method allowed the direct measurement in 1μl of urine avoiding the hydrolysis and extraction steps for sample pretreatment commonly used in routine methods. The hapten 3α-diol-G was synthesized by a Koenigs–Knorr reaction. The immunogenic complex, 3α-diol-G conjugated to bovine serum albumin (BSA), was employed to induce the formation of specific antibodies in New Zealand rabbits. In addition, the required chemiluminescent (CL) tracer was prepared. The characteristics of the antibody was determined as regard to specificity and sensitivity and the precision of the assay methods established. In 22 hirsute women affected by policystic ovarian syndrome we found 3α-diol-G values significantly (p < 0.01) higher (146.28 ± 73.77μg/g of creatinine; mean ± SD) than those observed in normal women (72.1 ± 32.58 μg/g of creatinine; mean ± SD).     

Excretion of cholate glucuronide

[3-3H]Cholic acid glucuronide [7 alpha,12 alpha-dihydroxy-3 alpha-O-(beta-D-glucopyranosyluronate)-5 beta- cholan-24-oate] was synthesized and administered to rats prepared with either an external biliary fistula or a ligated bile duct. When bile fistula animals were given either microgram or milligram amounts of the glucuronide, biliary secretion of label was rapid and efficient: greater than 90% of the administered label was secreted within 60 min and total recovery of label in bile was 98.6 +/- 1.2%. Studies in which [14C]taurocholate was included in the dose indicated that this bile acid was secreted into bile significantly more rapidly than was the glucuronide. In animals with ligated bile ducts, urinary excretion was the major route of elimination: after 20 hr, 83.4 +/- 9.3% of the administered dose had been excreted in urine. Urinary excretion of cholate glucuronide was significantly more rapid than that of taurocholate. Gas-liquid chromatographic analysis of the methyl ester acetate derivatives of labeled compounds isolated from bile and urine by chromatography established that the bulk (greater than 70%) of the administered material was secreted in bile or excreted in urine as the intact cholate glucuronide. From these results, we conclude that the glucuronidation of cholic acid produces a derivative which is rapidly and effectively cleared from the circulation and excreted.     

Quantification of leukotriene B4 glucuronide in human urine

We have developed a method for measuring leukotriene B4 glucuronide, a marker of systemic leukotriene B4 biosynthesis, in human urine. This method involves the separation of two positional isomers of leukotriene B4 glucuronide by high-performance liquid chromatography, followed by hydrolysis with beta-glucuronidase and then leukotriene B4 quantification by enzyme immunoassay after purification by high-performance liquid chromatography. One of two positional isomers of leukotriene B4 glucuronide was predominantly present in urine. The concentration of the isomer increased in urine from aspirin-intolerant asthma patients after aspirin challenge. Urinary leukotriene E4 and leukotriene B4 glucuronide concentrations in 13 normal healthy adults were 94.6 pg/mg-creatinine (median) and 22.3 pg/mg-creatinine, respectively. Urinary LTE4 concentration increased during the first 3h after allergen inhalation in atopic patients. However, allergen-induced bronchoconstriction was not associated with an increased concentration of LTB4 glucuronide in urine. The method enabled us to precisely determine urinary leukotriene B4 glucuronide concentration.     

Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry

A simple method using liquid chromatography-linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1-3ng/mL), recovery (89-101%), intraday precision (2.0-6.8%), interday precision (3.4-9.6%) and accuracy (101-103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 degrees C during a time-storage of a week lead to the conclusion that addition of sodium azide (10mg/mL) is required for preservation of the analytes.     

Isolation and identification of the glucuronide of 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino)benzoic acid from rabbit urine

The metabolic profile of 3H-1,2-dihydro-2-(4-methylphenylamino)methyl-1-pyrrolizinone (SFZ-47), a putative non-steroidal anti-inflammatory pro-drug, has been studied in rabbit urine. Semi-preparative reversed-phase HPLC of 24 h urine from two rabbits given single oral doses of SFZ-47 (200 mg) allowed the separation of SFZ-47 together with the oxidative metabolite 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino)benzoic acid (SFZ-47-COOH) and its glucuronide conjugate. The glucuronide was characterized by ESI-MS(n) and (1)H NMR and shown to be the 1-O-acyl beta-D-glucuronide conjugate of SFZ-47-COOH. The method gave excellent resolution of the glucuronide from endogenous constituents in urine and may be suitable for the preparation of glucuronide metabolites of other drugs.     

Time-course relationships between serum LH, serum progesterone and urinary preganediol concentrations in normal women

A specific and sensitive gas chromatographic method was used to investigate the concentration of pregnanediol glucuronide in urine in relation to the time of ovulation. Serum LH and progesterone concentrations in the same subjects were used as evidence for the occurrence of ovulation. The urinary concentration of pregnanediol glucuronide in 24-hour collections and in overnight specimens increased 2-fold or more from the day of the midcycle LH peak to the time of predicted ovulation (24-48 hour after the LH peak) in parallel with the rise in serum progesterone concentration.     

The Synthesis of the Glucuronide Metabolite of UK-157,147 Using Immobilised Uridine 5′-Diphosphoglucuronyl Transferase and Traditional Organic Chemistry Techniques (Imidate Method)

UK-157,147 (systematic name (3S,4R)-[6-(3-hydroxyphenyl)sulfonyl]-2,2,3-trimethyl-4-(2-methyl-3-oxo-2,3-dihydropyridazin-6-yloxy)-3-chromanol), a potent potassium channel opener designed for rapid systemic clearance, is metabolised in vivo to a glucuronide metabolite. A standard of this metabolite of UK-157,147 was required for biological testing to confirm the absence of pharmacological activity and assay development. The glucuronide conjugate of UK-157,147 was initially synthesised using a bioreactor containing immobilised microsomes as a source of uridine 5-diphosphoglucuronyl transferase (E.C.2.4.1.17). At a later date, larger amounts of the glucuronide metabolite were synthesised by traditional organic chemistry techniques using the imidate method. The identity of the chemically and biocatalytically produced glucuronide conjugate sample was proven by LC-MS and LC-NMR-MS.     

Simultaneous determination of nicotine and eight nicotine metabolites in urine of smokers using liquid chromatography-tandem mass spectrometry

A method based on liquid chromatography tandem mass spectrometry (LC-MSMS) applying atmospheric pressure chemical ionisation (APCI) in the positive ion mode was developed for the direct determination of nicotine, cotinine, trans-3'-hydroxycotinine, their corresponding glucuronide conjugates as well as cotinine-N-oxide, norcotinine, and nicotine-N'-oxide in the urine of smokers. The assay involves filtration of crude urine, fast liquid chromatography on a reversed-phase column and mass-specific detection using MSMS transitions. Deuterium-labeled nicotine, cotinine, and trans-3'-hydroxycotinine were used as internal standards. Glucuronides used as reference material were either chemically (cotinine-N-glucuronide) or enzymatically synthesized (nicotine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide). Precision for the major nicotine analytes at levels observable in urine of smokers was better than 10%. Accuracy expressed in recovery rates in urine matrix for nicotine, cotinine, trans-3'-hydroxycotinine, and cotinine-N-glucuronide ranged from 87 to 113%. Quantitative results for the three glucuronides in urine samples of 15 smokers were compared to an indirect method in which the aglycons were determined with gas chromatography and nitrogen-selective detection (GC-NPD) before and after enzymatic splitting of the conjugates. Good agreement was found for cotinine-N-glucuronide (coefficient of variation, CV: 9%) and trans-3'-hydroxycotinine-O-glucuronide (CV: 20%), whereas the accordance between both methods was moderate for nicotine-N-glucuronide (CV: 33%). The described LC-MSMS method allows the simultaneous determination of nicotine and eight of its major metabolites in urine of smokers with good precision and accuracy. Since the method requires a minimum of sample clean-up and a very short time for chromatography (3 min), it is suitable for determining the nicotine dose in large-scale human biomonitoring studies.     

Direct measurement of the glucuronide conjugate of 1-hydroxypyrene in human urine by using liquid chromatography with tandem mass spectrometry

To evaluate human exposure to polycyclic aromatic hydrocarbons (PAHs), we developed a rapid, simple and sensitive method for determining 1-hydroxypyrene-glucuronide (1-OHP-G) in human urine. To improve precision, a deuterated glucuronide was used as an internal standard. The method requires only 1 mL of urine. The urine was treated with a mixed-mode anion-exchange and reversed-phase solid-phase extraction cartridge (Oasis MAX). The analytes were analyzed with a C(18) reversed-phase column with a gradient elution, followed by tandem mass spectrometry with electrospray ionization in negative ion mode. The detection limit of 1-OHP-G (corresponding to a signal-to-noise ratio of 3) was 0.13 fmol/injection. Urinary concentrations of 1-OHP-G determined by this method were strongly correlated (r(2)=0.961) with concentrations of 1-hydroxypyrene by conventional HPLC with fluorescence detection.     

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated frrm normal human urine was acheived by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urin.     

The development of a radioimmunoassay for formoterol

The development of a radioimmunoassay (RIA) for the beta 2-stimulant formoterol is described. The sensitivity of the method is 0.1 ng/ml in plasma and urine, when a 1-ml sample is used. The cross-reactivity of the antiserum with formoterol glucuronide was 30%. Since formoterol is metabolized extensively to formoterol glucuronide in rats, dogs and humans, extraction with ethyl ether prior to the radioimmunoassay was carried out. Satisfactory agreement was obtained for levels of formoterol in plasma and urine when they were determined by RIA and gas chromatography-mass spectrometry. The concentration of formoterol was determined in dog plasma and human urine after oral administration of formoterol fumarate to dogs (61 mcg/kg) and humans (40 mcg).     

The metabolism of tetralin

1. [1-(14)C]Tetralin was synthesized and fed to rabbits. 2. Of the radioactivity, 87-90% was excreted in the urine within two days and 0.5-3.7% on the third day. The faeces contained 0.6-1.8%. No radioactivity was found in the breath and negligible amounts were retained in the tissues. About 90-99% of an administered dose was accounted for. 3. The main metabolite in the urine was the glucuronide of alpha-tetralol (52.4%). Other conjugated metabolites were beta-tetralol (25.3%), 4-hydroxy-alpha-tetralone (6.1%), cis-tetralin-1,2-diol (0.4%) and trans-tetralin-1,2-diol (0.6%). 4. beta-Tetralone, alpha-naphthol, 1,2-dihydronaphthalene and naphthalene, previously reported as metabolites, are artifacts, and tetralin, alpha-tetralone, beta-naphthol, 5-hydroxytetralin, and 6-hydroxytetralin are not metabolites. 5. The major metabolite of tetralin, alpha-tetralol and alpha-tetralone is the glucuronide of alpha-tetralol, which was isolated as methyl (1,2,3,4-tetrahydro-1-naphthyl tri-O-acetyl-beta-d-glucosid)uronate; the major metabolite of beta-tetralol and beta-tetralone is the glucuronide of beta-tetralol, which was characterized as methyl (1,2,3,4-tetrahydro-2-naphthyl tri-O-acetyl-beta-d-glucosid)uronate. 5-Hydroxytetralin is conjugated with glucuronic acid, and was characterized as methyl (5,6,7,8-tetrahydro-1-naphthyl tri-O-acetyl-beta-d-glucosid)uronate. 6-Hydroxytetralin is conjugated with glucuronic acid, and was characterized as methyl (5,6,7,8-tetrahydro-2-naphthyl tri-O-acetyl-beta-d-glucosid)uronate. 6. A metabolic sequence accounting for the observed biological transformation products is proposed.     

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.     

Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.     

Direct analysis of salicylic acid,salicyl acyl glucuronide,salicyluric acid and gentisic acid in human plasma and urine by high-performance liquid chromatography

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C₁₈ column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3–4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2–20 μg/ml and linearity was observed from 0.1–200 μg/ml and 5–2000 μg/ml for SA in plasma and urine, respectively. The method was validated to 0.2 μg/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.     

Immunochemical tests of potential fertility.

There are many methods of determining the physiological basis of cyclical periods of potential fertility: calculations, sensations, or physical or chemical biosensors. This article reviews the development of self-tests of potential fertility using immunoassay of hormone metabolites in urine. Indices of potential fertility in urine are the concentration of principal metabolites of steroid hormones and luteinizing hormone (LF). The ovaries produce an estimated 50-800 mcg of estradiol/day, the most active estrogen which is concentrated in the peripheral circulation during the preovulatory period of rapid follicular growth. Measurements may be taken from the blood, saliva, or cervico-vaginal fluid, and urine as estrogen-3 or 17 beta-glucuronide. The metabolism of progesterone shows changes at carbon atoms 3, 11, 17, and 20. Urine contains lower concentrations of 5 beta-pregnanediol-3 alpha-glucuronide (P3G), followed by 5 beta-pregnanediol glucuronide and 5 alpha-pregnanediol glucuronide. Methods of measurement are presented, including the mean changes in the urinary concentrations of estrogen 3-glucuronide (E3G) and P3G and their concentration ratio in relation to day 1 of menses, the day of luteinizing hormone LH peak, and the time limits for ovulation during menstrual cycle. A laboratory test of E3G in early morning urine (EMU) from 38 subjects showed that delineating a defined fertile period (day of maximum follicular diameter minus 3 to day plus 2) was possible in 89% of cases. New methods with immunotubes or immunostrips, novel antibodies, and idiometric assay attempt to improve the signal/background ratio; each method is described. Immunotubes for measuring steroid glucuronide may be light absorbing, light emitting (fluorescence polarization), and light emitting with time resolved fluorescence. Each procedure is described. The present technology demonstrates that immunoassays can be performed as self-tests, and what remains to be done is ascertaining the most appropriate one for determining potential fertility.     

Reversed-phase chromatography of urinary metabolites of paracetamol using ion suppression and ion pairing

High-performance liquid chromatography (HPLC) has proven particularly useful for the study of paracetamol metabolism. Two alternative methods were developed using reversed-phase C₁₈ columns. A rapid ion suppression technique was used for the analysis of free paracetamol, paracetamol mercapturic acid and cysteine conjugate in urine samples obtained from isolated perfused rat kidney preparations, which has conveniently demonstrated the oxidative metabolic capacity of the kidney towards paracetamol. A somewhat longer, but higher resolution, ion-pair HPLC procedure was developed for the analysis of paracetamol metabolites in urine samples from experimental animals. The ion-pairing solvent was composed of tetrabutylammonium hydroxide, Tris and EDTA buffered to pH 7.2 with phosphoric acid. Gradient programming was further used to enhance resolution. Using this system two new metabolites, the sulphate and glucuronide conjugates of 3-thiomethyl-paracetamol were detected and routinely determined along with other known paracetamol metabolites, viz. free paracetamol, paracetamol sulphate, glucuronide, mercapturic acid, and cysteine conjugates, 3-methoxyparacetamol glucuronide and sulphate, p-aminophenol and its O-glucuronide and O-sulphate conjugates. Phenolic O-substituted glucuronide and sulphate conjugates of N-hydroxyparacetamol were also separated.     

Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 μg/ml for both MPA and MPAG in plasma and from 1 to 50 μg/ml of MPA and 5 to 2000 μg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 μg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.     

Determination of urinary 5-hydroxytryptophol glucuronide by liquid chromatography-mass spectrometry

5-Hydroxytryptophol glucuronide (GTOL) is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin metabolite under normal conditions. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. This study describes the development and evaluation of a liquid chromatography-electrospray ionization mass spectrometry (LC-MS) procedure for direct quantification of GTOL in human urine. Deuterium labelled GTOL (GTOL-(2)H(4)) was used as internal standard. GTOL was isolated from urine by solid-phase extraction on a C(18) cartridge prior to injection onto a gradient eluted Hypurity C(18) reversed-phase HPLC column. The detection limit of the method was 2.0 nmol/L and the measuring range 6-8500 nmol/L. The intra- and inter-assay coefficients of variation were <3.5% (n=10) and <6.0% (n=9), respectively. The new LC-MS method was highly correlated with an established GC-MS method for urinary 5-HTOL (r(2)=0.99, n=70; mean 5-HTOL/GTOL ratio=1.10). This is the first direct assay for quantification of GTOL in urine. The method is suitable for routine application.