Direct quantification of ethyl glucuronide in clinical urine samples by liquid chromatography-mass spectrometry

Ethyl glucuronide is a minor metabolite of ethanol, and its presence in urine can be used as a laboratory test to detect recent alcohol intake, even for some time after the ethanol is no longer measurable. A simple analytical procedure was developed based on direct injection of urine diluted with a deuterated internal standard into an electrospray liquid chromatographic-mass spectrometric (LC-MS) system. A novel LC system using a porous graphite column (Hypercarb) enabled an isocratic elution with retention times of 5-6 minutes. The intra- and inter-assay coefficients of variation were 2-12%, and the measuring range was 0.1-1,500 mg/L (0.45-6,750 micromol/L). Ethyl glucuronide was found to be stable in urine for more than 4 days at room temperature, and no artifactual formation was observed on storage of urine samples fortified with 1% ethanol. Ethyl glucuronide was not detected in urine samples collected after abstinence from alcohol. Intake of a very low amount (7 g) of ethanol produced ethyl glucuronide values up to 8.4 mg/L after 4 hours and was still detectable at 6 hours. When the method was applied for routine screening of 252 clinical urine samples (range, 0-1,240 mg/L), it fulfilled the need for a simple and reliable assay to be used in the evaluation of urinary ethyl glucuronide as a routine test of recent alcohol intake.     

Direct measurement of probenecid and its glucuronide conjugate by means of high pressure liquid chromatography in plasma and urine of humans

Probenecid with its phase-I metabolites, and phase-II glucuronide conjugate can be analysed by a gradient high pressure liquid chromatographic method. Probenecid glucuronide in plasma with pH 7.4 is not stable and declines to 10% of the original value within 6 h (t_1/21 h). Probenecid glucuronide is stable in urine with pH 5.0, moderately unstable at pH 6.0 (t_1/210 h), and unstable at pH 8.0 (t_1/20.5 h). Probenecid glucuronide is stable in water and 0.01 mol/l phosphoric acid in the autosampler of the high pressure liquid chromatograph. The decrease in concentration in water is 5.5% during 9 h and 0% in diluted acid. Probenecid glucuronide and the phase-I metabolites were not detectable in plasma. The main compound in fresh urine is the phase-II conjugate probenecid glucuronide (62% of a 500 mg dose); the phase-I metabolites are present and only a trace of probenecid is present. The percentage of the dose of the phase-I metabolites varies between 5 and 10, while hardly any probenecid is excreted unchanged (0.33%).     

Excretion profiles of ethyl glucuronide in human urine after internal dilution

Ethyl glucuronide (EG) is a useful marker of alcohol consumption because its presence in urine can be detected up to five days. We investigated the impact of diuresis on the urinary excretion of EG, a minor ethanol metabolite. Seven healthy volunteers drank 250 mL of wine (25 g ethanol) in 15 min and, 240 min later, ingested 1 L of water within 15 min. Urine was voided before the drinking started and every 30-60 min for 400-550 min thereafter. Urinary ethyl glucuronide (UEG), creatinine, and ethanol were determined using liquid chromatography-tandem mass spectrometry, Jaffé's method, and the enzymatic ADH method, respectively. The maximum diuresis coincided with the lowest values of the UEG concentrations of 2 mg/L and the lowest creatinine values of 10 mg/dL 250-400 min after drinking. After drinking the wine, the urinary creatinine decreased slowly. After a short period of increasing, it decreased to minimum values caused by the water intake. After the intake of 1 L water, the diuresis increased within 60 min to its maximum. The amount of ethyl glucuronide excreted in urine was 10 mg (SD 5 mg) corresponding to 0.04% (SD 0.02%) of the dose administered. In successive voids during the elimination phase, the UEG and the diuresis were influenced after the subjects drank 1 L of water. Minimum UEG values of 0.5 mg/L could still be measured. Measuring UEG provides a reliable way to monitor recent drinking of alcohol. However, urinary creatinine needs to be measured additionally. Establishing a cutoff value of 25 mg/dL for urinary creatinine in diluted samples, like for the analysis of illicit drugs, is recommended. If the creatinine value is too low, the analyst has to decide about the further procedure.     

Isolation and identification of seven glucuronide conjugates of andrographolide in human urine.

Andrographolide is one of the principal components of a famous traditional Chinese herbal medicine, Andrographis paniculate (Burm) Nees, and has been widely used in the clinic for the treatment of infectious diseases. In this paper, metabolites of andrographolide in the urine of eight healthy volunteers after oral administration were further investigated. Building on previous findings, an additional seven phase II metabolites were isolated by liquid-liquid extraction, open-column chromatography, medium-pressure liquid chromatography, and, finally, preparative high-performance liquid chromatography. Structural elucidation was carried out by mass spectra and NMR spectroscopy including 1H NMR, 13C NMR and two-dimensional NMR (distortionless enhancement by polarization transfer, heteronuclear multiple-quantum correlation, heteronuclear multiple-bond correlation, 1H-1H correlated spectroscopy, and nuclear Overhauser enhancement spectroscopy). All of the metabolites were characterized as glucuronide conjugates, and the structures were determined to be andrographolide-19-O-beta-D-glucuronide (M-1), isoandrographolide-19-O-beta-D-glucuronide (M-2), 14-deoxy-12-hydroxy-andrographolide-19-O-beta-D-glucuronide (M-3), andrographolide-19-O-[6'-methyl-beta-D-glucuronide] (M-4), 14-deoxy-12(13)-en-andrographolide-19-O-beta-D-glucuronide (M-5), 14-deoxyandrographolide-19-O-beta-D-glucuronide (M-6), and 3-oxoandrographolide-19-O-beta-D-glucuronide (M-7), respectively.     

HPLC法同时测定人尿液中霉酚酸及其代谢物葡糖苷酸结合物的浓度

目的用HPLC测定人尿液中霉酚酸(MPA)及其代谢物葡糖苷酸结合物(MPAG)的浓度。方法分析柱为Zorbax Eclipse XDB-C8(150 mm×4．6mm,5μm);流动相为甲醇-0．1％三氟乙酸溶液(55:45,V／V);尿稀释5倍后直接进样。MPAG采用紫外检测,检测波长为250nm;MPA采用柱后添加0．2 mol·L-1 NaOH溶液进行荧光检测,荧光激发波长325 nm,发射波长435 nm。结果MPA和MPAG的线性范围分别为0．1-50 mg·L-1和10-500 mg·L-1,MPA、MPAG日内和日间RSD均<10％。结论此法能简便、灵敏、准确地测定人体尿液中MPA与MPAG的浓度,可用于临床药动学研究和治疗药物浓度监测。     

Stir bar sorptive extraction with in situ de-conjugation and thermal desorption gas chromatography-mass spectrometry for measurement of 4-nonylphenol glucuronide in human urine sample

4-Nonylphenol glucuronide (NP-G) in human urine samples was analyzed using stir bar sorptive extraction (SBSE) with in situ de-conjugation by beta-glucuronidase and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Distilled water (1 ml), 1.0 M ammonium acetate solution (100 microl) and beta-glucuronidase (10,000 units ml(-1), 10 microl) were added to human urine sample (1 ml), and extraction was commenced for 90 min at 37 degrees C while stirring at 250 rpm with a stir bar coated with a 500-microm-thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was subjected to TD-GC-MS in the selected ion monitoring (SIM) mode. The calibration curve was made by SBSE method using 4-nonylphenol (NP) as the standard solution. The method showed good linearity and the correlation coefficients were 0.999 over the concentration range of 5-500 nM. Moreover, to optimize the conditions for SBSE with in situ de-conjugation and the recovery test, NP-G was synthesized by a biochemical technique in our laboratory. The limits of detection (S/N = 3) and quantitation (S/N > 10) for NP were 0.2 ng ml(-1) (1.0 nM) and 1.1 ng ml(-1) (5.0 nM), respectively. The average recoveries in the human urine samples (n = 6) spiked with NP-G at levels of 20 and 100 nM were 104.1 (R.S.D. 7.1%) and 100.6% (R.S.D. 9.2%), respectively, with correction using the added internal standard, 4-(1-methyl) octylphenol-d(5). The method enabled the precise determination of the standard and was applicable to the detection of trace amounts of NP-G in human urine samples.     

Free and conjugated 3-methoxy-4-hydroxyphenylglycol in human urine: Peripheral origin of glucuronide

The aim of our study was to investigate the endogenous origin of the three forms of 3-methoxy-4-hydroxyphenylglycol (MHPG) present in human urine and to further examine the hypothesis of an independent (peripheral or central) origin of glucuronide and sulfate conjugates. The urinary levels of free, sulfate, and glucuronide MHPG were determined in control subjects under normal conditions in relation to age, sex, and diet and in two experimental situations known to alter sympathetic activity. The mean daily excretion of total MHPG in a group of 14 men and 14 women was 1780 +/- 122 micrograms, with the free, sulfate, and the glucuronide representing 8% +/- 0.5%, 40% +/- 1.5%, and 52% +/- 1.6%, respectively. No influence of sex, age, or diet was observed on any form. Strong physical activity and anticipatory stress increased norepinephrine excretion and selectively increased MHPG glucuronide levels without changing the free or the sulfate excretion. We conclude that the total amounts of free, sulfate, and glucuronide MHPG found in urine originate from endogenous body pools with no interference of dietary components. The sympathetic nervous system seems to be the main source of glucuronide and arguments are given supporting the central origin of sulfate.     

Determination of salbutamol and salbutamol glucuronide in human urine by means of liquid chromatography-tandem mass spectrometry

The determination of salbutamol and its glucuronide in human urine following the inhalative and oral administration of therapeutic doses of salbutamol preparations was performed by means of direct urine injection utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) and employing d(3)-salbutamol and d(3)-salbutamol glucuronide as internal standards. Unconjugated salbutamol was detected in all administration study urine samples. Salbutamol concentrations following inhalation were commonly (99%) below 1000 ng/ml whereas values after oral administration frequently (48%) exceeded this threshold. While salbutamol glucuronide was not detected in urine samples collected after inhalation of the drug, 26 out of 82 specimens obtained after oral application contained salbutamol glucuronide with a peak value of 63 ng/ml. The percentage of salbutamol glucuronide compared to unconjugated salbutamol was less than 3%. Authentic doping control urine samples indicating screening results for salbutamol less than 1000 ng/ml, showed salbutamol glucuronide concentrations between 2 and 6 ng/ml, whereas adverse analytical findings resulting from salbutamol levels higher than 1000 ng/ml, had salbutamol glucuronide values between 8 and 15 ng/ml. The approach enabled the rapid determination of salbutamol and its glucuronic acid conjugate in human urine and represents an alternative to existing procedures since time-consuming hydrolysis or derivatization steps were omitted. Moreover, the excretion of salbutamol glucuronide in human urine following the administration of salbutamol was proven.     

Sulphate conjugation in human liver. Direct measurement of N-acetyldopamine-sulphate

The overall three-step sulphate conjugating activity of the human liver was determined by the direct measurement of N-acetyldopamine (NADA)-sulphate from ATP and inorganic sulphate. NADA-sulphate was separated from NADA and quantified by HPLC-ECD (high-pressure liquid chromatography-electrochemical detection). The overall sulphate conjugation of NADA showed at pH optimum of 8.0 with apparent Km values for sodium sulphate and NADA of 103 microM and 4.8 microM. respectively. The optimum concentration of ATP was 5 mM and that for Mg2+ ions was 7 mM. The specific activities of 17 samples of human liver, measured by this HPLC-ECD procedure ranged from 940 to 23,128 pmol NADA-sulphate/hr/mg protein. A comparison of these values with those determined by the radiometric method developed previously showed a direct correlation coefficient, r = 0.89. The rates of overall sulphate conjugation of NADA and dopamine measured in these samples by the radiometric assay procedure were also significantly correlated with r = 0.82.     

Identification in human urine of Δ9-tetrahydrocannabinol-11-oic acid glucuronide: a tetrahydrocannabinol metabolite

A Δ⁹ -THC metabolite has been identified in human urine as an ester linked glucuronide of Δ⁹ -THC-11-oic acid. Its identity was established by a comparison of mass spectra from the metabolite extracted from human urine and synthetically prepared material. Δ⁹ -THC-11-oic acid glucuronide was found to be responsible for the major part of RIA crossreactivity in urine with the Guildhay cannabinoid antiserum used in this study.     

Multicriteria optimization methodology in development of HPLC separation of mycophenolic acid and mycophenolic acid glucuronide in human urine and plasma

Multicriteria optimization methodology was applied for development of isocratic reversed-phased HPLC method for simultaneous determination of mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) in human urine and plasma. In the first stage of method development, pH value of the water phase, percentage of acetonitrile, temperature of the column and flow rate of the mobile phase were investigated using fractional factorial design.Afterwards, the optimal conditions were found employing central composite design and Derringer's desirability function. Two goals were considered, the retention factor of the MPAG to be in the range, between 0.8 and 1.118 which allowed well separation of MPAG from the urine and plasma peaks, and the shortest possible total analysis run time. Then, the obtained sigmoid functions were used to transform the optimization criteria into the desirability values.The satisfactory chromatographic conditions were obtained with mobile phase consisted of acetonitrile–phosphate buffer (pH 2.4; 0.04 M KH₂PO₄) (28:72, v/v). The separation was performed on C₁₈ Chromolith column (100 mm × 4.6 mm) with flow rate of 5 mL/min, the temperature of the column was 25 °C and the chosen wavelength for simultaneous determination of MPA and MPAG was 215 nm. The MPAG eluted at 0.552 min and the duration of run was 3.092 min.Afterwards, the method was subjected to validation. Linearity was observed over the concentration range of 1–50 μg/mL for MPA and 1–500 μg/mL for MPAG in urine and 1–60 μg/mL for MPA and 1–70 μg/mL for MPAG in plasma matrix. The method showed intra-day and inter-day precision with relative standard deviation lower then 5% and accuracy as recovery (%) between 100 ± 5%.     

Specific radioimmunoassays for the measurement of stavudine in human plasma and urine

Sensitive and specific radioimmunoassays (RIAs) have been developed and validated for the determination of stavudine, a nucleoside analog possessing anti-human immunodeficiency virus (HIV) activity, in human plasma and urine. The hemisuccinate of stavudine was conjugated with histamine and radioiodinated to yield the radiotracer. Antisera were produced by injecting the immunogen, stavudine-hemisuccinate-bovine thyroglobulin, into rabbits. The antisera exhibited high specificity for stavudine as the structurally related analogs and other anti-HIV agents did not interfere in the assays. The methods could reliably quantitate stavudine in plasma from 2.5–100 ng ml⁻¹ and in urine from 5.0–1000 ng ml⁻¹ (after 2.5-fold dilution) with good accuracy and precision. The lower limits of quantitation were 2.5 ng ml⁻¹ in human plasma and 5.0 ng ml⁻¹ in urine (after 2.5-fold dilution). The RIA methods were applied to the analysis of stavudine in plasma and urine obtained from HIV-infected patients receiving the drug in clinical trials.     

High-performance liquid chromatographic method for the simultaneous quantitation of diflunisal and its glucuronide and sulfate conjugates in human urine

A direct high-performance liquid chromatographic (HPLC) assay was developed to simultaneously quantitate diflunisal and its three known metabolites (i.e., the phenolic and acyl glucuronides and the sulfate conjugate) in human urine. Chromatographically pure standards of the diflunisal conjugates were isolated from urine of volunteers following ingestion of multiple doses of diflunisal (500 mg twice daily). Diflunisal, its three conjugates, and an internal standard (naproxen) were separated on a reversed-phase column using gradient elution. The column eluate was monitored fluorometrically (excitation: 258 nm; emission: 428 nm). Urine samples were diluted with phosphate buffer (pH 5.75) and injected onto the column. The limit of detection was approximately 1 microgram/mL for each conjugate and 0.1 microgram/mL for diflunisal. Due to the presence in most urine samples of significant concentrations of rearrangement products of the biosynthetic 1-O-acyl glucuronide of diflunisal, the acyl glucuronide could not be reliably quantitated by direct injection of diluted urine samples. Instead, diflunisal acyl glucuronide was quantitated indirectly following alkaline hydrolysis of the urine samples. The method has been successfully used to investigate the dose-dependent glucuronidation and sulfation of diflunisal in humans.     

Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites

Confirmation of opioids in urine samples of clinical patients requires liberation of opioids from their glucuronide conjugates. Both acid hydrolysis and enzyme hydrolysis using beta-glucuronidase from various sources have been reported, with the latter approach prevailing in most clinical toxicology laboratories. The goal of this study was to compare the efficiency of acid versus different enzyme hydrolysis methods in recovering morphine and common semisynthetic opioids from glucuronide standards and 78 patient urine samples that were screened positive for opioids as a class. Specimens were analyzed with a validated gas chromatography-mass spectrometry (GC-MS) procedure. With the exception of oxycodone, the results indicated that the majority of opioids tested were extensively glucuronide-conjugated in urine. Significantly, acid hydrolysis liberated > 90% of morphine and hydromorphone from their glucuronide standards but enzyme hydrolysis had lower and variable efficiency, depending on the opiate type and the enzyme source. In patient specimens, much higher concentrations of free codeine, morphine, hydromorphone, and oxymorphone were obtained with acid hydrolysis than with various enzyme methods. Incomplete hydrolysis using beta-glucuronidase could lead to false-negative results for many opioids when urine is tested for drugs of abuse. We conclude that acid hydrolysis is the method of choice for GC-MS confirmation of urine opioids.     

Quantitative assay of lorazepam and its metabolite glucuronide by reverse-phase liquid chromatography-tandem mass spectrometry in human plasma and urine samples

A LC/MS/MS method for the quantitative determination of lorazepam in human plasma and urine samples was developed and validated. The enantioselective assay allowed to separate the enantiomers and to verify the stereochemical instability of lorazepam. The linearity assessed for lorazepam unchanged was 0.2-20 ng of each enantiomer/ml plasma and 0.2-15 ng of each enantiomer/ml urine. The linearity assessed for total lorazepam (after enzymatic hydrolysis) was 1-30 ng of each enantiomer/ml plasma and 10-150 ng of each enantiomer/ml urine. The coefficients of variation obtained for the intra- and interassay precision were less than 15%. The method was applied to the investigation of the kinetic disposition and metabolism of racemic lorazepam administered as a single oral dose of 2 mg to a parturient. The occurrence of racemization required the calculation of the pharmacokinetic parameters as enantiomeric mixtures of lorazepam (t(1/2a) 3.5h; K(a) 0.198 ngh(-1); t(1/2) 11.5h; beta 0.060 h(-1); AUC(0-infinity) 192.1ngh/ml; CLt/f 2.41ml/minkg; Vd/f 173.5l; Fel 0.41%, and Cl(R) 0.0099 ml/minkg) and its metabolite lorazepam-glucuronide (t(1/2f) 1.2h; K(f) 0.578 h(-1); t(1/2) 16.6h; beta 0.042 h(-1); AUC(0-infinity) 207.6 ngh/ml; Fel 51.80%, and Cl(R) 98.32 ml/minkg). However, the determined confidence limits make the method suitable for application to clinical pharmacokinetic studies, even if the quantification of both the enantiomers is required.     

Interference from a glucuronide metabolite in the determination of ramipril and ramiprilat in human plasma and urine by gas chromatography-mass spectrometry

In the course of development and validation of a gas chromatography-mass spectrometry (GC-MS) method for ramipril and its biologically active metabolite ramiprilat, evidence was found for an unknown interfering metabolite. Sample treatment included isolation from plasma or urine by solid-phase extraction, methylation with trimethylsilyldiazomethane and acylation with trifluoroacetic anhydride (TFAA). When liquid chromatography was used to fractionate plasma extracts prior to derivatization, the alkyl, acyl-derivative of ramipril was obtained from two separate LC fractions. Electrospray ionization mass spectral data, together with circumstances for the derivatization, were consistent with the presence of an N-glucuronide of ramipril. Interference from the metabolite was eliminated by including a wash step after extraction/alkylation, prior to acylation. The final assay had a lower limit of quantification at 1.0 nmol/L and a linear range of 1-300 nmol/L. Intra- and inter-batch precision for ramipril and ramiprilat in plasma or urine were better than 10 and 5% at 2 and 80 nmol/L, respectively.     

Establishing biomarkers of oxidative stress: the measurement of hydrogen peroxide in human urine

Hydrogen peroxide (H(2)O(2)) can be detected in freshly-voided human urine from healthy subjects and has been proposed as a "biomarker" of oxidative stress. This paper summarizes our studies to examine the extent to which urinary H(2)O(2) measurement fulfils the criteria for the "ideal biomarker". Levels of H(2)O(2), standardised for creatinine, varied widely between subjects. In most subjects, levels also varied considerably when measurements were made at different times and on different days. A reproducible increase in urinary H(2)O(2) was detected in all subjects examined after drinking coffee, a beverage rich in H(2)O(2). By contrast, green tea decreased urinary H(2)O(2) levels. We conclude that the H(2)O(2) in coffee is not excreted into urine. Instead, hydroxyhydroquinone from coffee is absorbed, excreted and oxidises in urine to produce H(2)O(2). No other confounders of urinary H(2)O(2) have been identified to date. Work is underway to compare H(2)O(2) levels with variations in other biomarkers of oxidative damage, to test the possibility that there are daily or other periodic variations in oxidative damage rates.