Atmospheric Solid Analysis Probe and Modified Desorption Electrospray Ionization Mass Spectrometry for Rapid Screening and Semi-Quantification of Zilpaterol in Urine and Tissues of Sheep.

Ambient ionization mass spectrometric methods including desorption electrospray ionization (DESI) and atmospheric solid analysis probe (ASAP) have great potential for applications requiring real-time screening of target molecules in complex matrixes. Such techniques can also rapidly produce repeatable semiquantitative data, with minimal sample preparation, relative to liquid chromatography-mass spectrometry (LC-MS). In this study, a commercial ASAP probe was used to conduct both ASAP-MS and modified DESI (MDESI) MS analyses. We conducted real-time qualitative and semiquantitative analysis of the leanness-enhancing agent zilpaterol in incurred sheep urine, kidney, muscle, liver, and lung samples using ASAP-MS and MDESI MS. Using ASAP, limits of detection (LOD) and quantitation (LOQ) in urine were 1.1 and 3.7 ng/mL, respectively, while for MDESI MS they were 1.3 and 4.4 ng/mL, respectively. The LODs for tissues were 0.1-0.4 ng/g using ASAP, and 0.2-0.6 ng/g with MDESI MS. The LOQs of the tissues in ASAP were 0.4-1.2 ng/g and 0.5-2.1 ng/g in MDESI MS. Trace levels of zilpaterol were accurately analyzed in urine and tissues of sheep treated with dietary zilpaterol HCl. The correlation coefficient ( R2) between semiquantitative ASAP-MS and MDESI MS results of urine samples was 0.872. The data from ASAP and MDESI MS were validated using LC-MS/MS; urinary zilpaterol concentrations ≥5.0 ng/mL or tissue zilpaterol concentrations ≥1.5 ng/g were detected by ASAP and MDESI MS, respectively, 100% of the time. Forty samples could be analyzed in triplicate, directly from biological matrixes in under an hour.

Urinary Excretion of the ß-Adrenergic Feed Additives Ractopamine and Zilpaterol in Breast and Lung Cancer Patients.

ß2-Adrenergic agonists (ß-agonists) have been legally used in the U.S. for almost two decades to increase lean muscle mass in meat animals. Despite a cardiotoxic effect after high-dose exposure, there has been limited research on human ß-agonist exposures related to meat consumption. We quantified urinary concentrations of ractopamine and zilpaterol, two FDA-approved ß-agonist feed additives, and examined the extent to which the concentrations were associated with estimated usual meat intake levels. Overnight urine samples from 324 newly diagnosed breast cancer patients and spot urine samples from 46 lung cancer patients at the time of diagnosis, prior to treatment, were collected during 2006-2010 and 2014-2015, respectively. Urinary ractopamine and zilpaterol concentrations were measured by LC-MS/MS. Ractopamine and zilpaterol, respectively, were detected in 8.1% and 3.0% of the urine samples collected (n = 370). Only 1.1% (n = 4) of the urine samples had zilpaterol concentrations above the limit of quantification, with the mean value of 0.07 ng/mL in urine. The presence of detectable ractopamine and zilpaterol levels were not associated with meat consumption estimated from a food frequency questionnaire, including total meat (P = 0.13 and 0.74, respectively), total red meat (P = 0.72 and 0.74), unprocessed red meat (P = 0.74 and 0.73), processed red meat (P = 0.72 and 0.15), and poultry intake (P = 0.67 for ractopamine). Our data suggest that the amount of meat-related exposure of ß-agonists was low.

Depletion of urinary zilpaterol residues in horses as measured by ELISA and UPLC-MS/MS.

Three horses were dosed with dietary zilpaterol and the urine concentrations measured from withdrawal day 0 to withdrawal day 21. The analyses were carried out using both enzyme-linked immunosorbent assay (ELISA) and an ultraperformance liquid chromatography with triple-quadrupole-tandem mass spectrometric detection (UPLC-MS/MS). The UPLC-MS/MS method was developed to provide rapid analysis with positive analyte identification by following three product ions and computing the two independent ion ratios. When urinary zilpaterol concentrations were between 0.2 and 2 ng/mL, the ELISA had interday recoveries of 114-120% with coefficients of variation (CV) of <22%; intraday recoveries were 79-111% with CVs of <13%. For urinary zilpaterol concentrations of 0.4-40 ng/mL the UPLC-MS/MS method had interday recoveries of 94-104% with CVs of <8%; intraday recoveries were 97-102% with CVs of < or = 7.5%. Correlation analysis demonstrated that the ELISA and UPLC-MS/MS methods returned essentially the same results, especially at urinary zilpaterol concentrations below 2000 ng/mL. Urinary excretion peaked rapidly after dosing between 5300 and 10800 ng/mL (UPLC-MS/MS) or between 5900 and 17900 ng/mL (ELISA) for the different horses, much higher than observed in other species. Urinary zilpaterol concentrations declined rapidly to below 3000 ng/mL within 24 h of study day 1. After about 5 days, zilpaterol elimination slowed markedly, taking nearly 10 days for an order of magnitude decrease. The analytical methods were able to detect zilpaterol in the urine even at withdrawal day 21, demonstrating the sensitivity of each analytical method and the slow rate of zilpaterol depuration from horses.

Analysis of sibutramine metabolites as N-trifluoroacetamide and O-trimethylsilyl derivatives by gas chromatography-mass spectrometry in urine.

A method for identifying the metabolites of sibutramine 1-(4(chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl))cyclobutanemethanamine) in urine, utilizing a double derivatization strategy, with N-methyl-N-(trimethylsilyl)-trifluoroacetamide and N-methyl-bis-(trifluoroacetamide), in gas chromatography/mass spectrometry is proposed. This methodology results in mass spectra with at least three fragments in abundance superior to 20%, attending the World Anti-Doping Agency identification criteria for qualitative assays. The characterization of the derivatives was obtained through two ionization modes: Chemical Ionization and Electron Impact ionization, both in full scan mode. Sibutramine was administered to 5 (five) volunteers and the excretion profile followed for 92h. Routine analytical, hydroxy-cyclobutane-bis-nor-sibutramine which becomes the more abundant metabolite in the first 10h and hydroxy-isopropyl-bis-nor-sibutramine which becomes the most abundant after 40h, were proposed for doping monitoring.

Tissue residues and urinary excretion of zilpaterol in sheep treated for 10 days with dietary zilpaterol.

Zilpaterol is a beta-adrenergic growth promoter approved in Mexico and South Africa for use in cattle. Understanding the rates of zilpaterol depletion from tissues and urine is of interest for the development of strategies to detect the off-label use of zilpaterol. Eight sheep were fed 0.15 mg/kg/day dietary zilpaterol hydrochloride (Zilmax) for 10 consecutive days; two sheep each were slaughtered 0, 2, 5, and 9 days after discontinuation of exposure to the zilpaterol-containing diet. Tissue zilpaterol levels rapidly decreased during the withdrawal period. On the basis of LC-MS/MS-ES (external standard) measurements, liver zilpaterol residues in sheep were 29.3, 1.5, 0.13, and 0.10 ng/g after 0, 2, 5, and 9 day withdrawal periods, respectively; kidney residues were 29.6, 1.10, and 0.09 ng/g and below the detection limit; and muscle residues were 13.3, 0.86, 0.12, and 0.08 ng/g at the same respective withdrawal periods. Between-animal variation in urinary zilpaterol concentrations during the feeding period was considerable, although zilpaterol concentrations converged somewhat as steady state was reached. During the first 3 days of the withdrawal period, zilpaterol elimination followed a first-order excretion pattern, having an average elimination half-life of 15.3 +/- 1.8 h. Urinary zilpaterol concentrations during the withdrawal period were determined using ELISA, HPLC-fluorescence, LC-MS/MS-ES (external standard), and LC-MS/MS-IS (internal standard). Comparison of these methods showed a high correlation with each other. With the exception of LC-MS/MS-IS, the regression coefficients of the linear equations with a zero intercept were between 0.90 and 1.25, indicating the near equivalence of the methods. Because of its simplicity, ELISA is a convenient assay for determining zilpaterol levels in urine giving similar results to HPLC-fluorescence and LC-MS/MS-ES without requiring the extensive cleanup of the latter methods.

Development of a monoclonal antibody-based enzyme-linked immuosorbent assay for the beta-adrenergic agonist zilpaterol.

Zilpaterol is a beta-adrenergic agonist approved for use as a growth promoter in cattle in South Africa and Mexico but not in the European Union, United States, or Asia. Here, we report the development of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for zilpaterol. Mice immunized with zilpaterol-butyrate-keyhole limpet hemocyanin were utilized for monoclonal antibody generation whereas zilpaterol-butyrate-bovine serum albumin was used as a coating antigen for ELISA. Thirteen clones were isolated, and after the initial sensitivity and isotyping experiments, three clones were selected for further ELISA optimization. Studies indicated that the optimum pH was near 7.4. Clone 3H5 had the highest sensitivity to zilpaterol and some interaction with clenbuterol and terbutaline at high concentrations but not other N-alkyl [bamethane, (-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (fenoterol, isoxsuprine, ractopamine, or salmeterol) beta-agonists tested. However, clone 3H5 was not functional at high salt concentrations, which precluded further development for urine analysis. Clone 2E10 showed increased sensitivity as salt concentrations were increased and did not cross-react with any of the structural analogues tested. However, its sensitivity to salt and urine concentration changes could cause high variability. Clone 7A8 showed good sensitivity and only a modest change with the salt concentration changes. Clone 7A8 also demonstrated smaller changes in IC(50) and B(0) with increasing sheep urine or cattle urine concentrations as compared to clones 2E10 or 3H5 and, thus, was selected for further development. The IC(50) for all of the antibodies showed exponential increases with increasing organic solvents concentrations, making it desirable to minimize solvent levels. In conclusion, a sensitive, specific zilpaterol monoclonal antibody-based ELISA has been developed that can serve as a rapid screening assay.

[Sensitivity of GC-MS in the detection of benzodiazepines in the urine in the form of trimethylsilyl derivatives]. / Citlivý GC-MS prukaz benzodiazepinu v moci ve forme jejich trimethylsilylderivátu.

A sensitive evidence of trace concentrations of benzodiazepines and their metabolites in urine can be enabled after special sample preparation including enzymatic hydrolysis, special solid phase extraction, silylation and following analysis by gas chromatography with mass spectrometry in electron impact mode. After optimalization of the procedure the extraction recovery values in the range 50-85% are achieved. The scale of retention times with basic mass spectral data are presented for the spectrum of silylated benzodiazepines which can overlap some gaps in the standard toxicological literature up to now available.

Confirmation of benzodiazepines in urine as trimethylsilyl derivatives using gas chromatography-mass spectrometry.

A confirmation procedure for the identification and quantitation of various benzodiazepines in urine is presented. The urine sample is first hydrolyzed enzymatically because of the glucuronide conjugation of some benzodiazepine metabolites, then extracted using bonded-phase columns. After elution into an organic solvent, the samples are evaporated, converted to the trimethylsilyl ether derivatives and analyzed by electron ionization GC-MS. Quantitation was performed using selected-ion monitoring for each benzodiazepine using prazepam as the internal standard. The method provides excellent linearity and sensitivity for the trimethylsilyl derivatives.

Determination of an acetylcholinesterase inhibitor, MDL 73745, in dog plasma and urine by combined gas chromatography/negative ion chemical ionization mass spectrometry.

A highly sensitive and specific assay has been developed for the determination of MDL 73745 [2,2,2-trifluoro-1-(3-trimethylsilyl-phenyl) ethanone] (I) and the internal standard (MDL 74398) at the nanomolar level in dog plasma and urine by gas chromatography/mass spectrometry. After a single-step extraction process, an aliquot was directly injected onto the gas chromatograph column. The mass spectrometer was run in the negative ion chemical ionization mode with ammonia as reagent gas, and was set to monitor the abundant M-. ion at m/z 246 of both compounds. The method yielded a linear response over the concentration range 0.1-10 pmol 100 microliters -1 plasma or urine. Within-day reproducibility at a concentration of 0.25, 1 and 5 pmol 100 microliters -1 plasma was 8.6%, 1.0% and 1.0%, respectively. The method was applied to the determination of I in plasma and urine after administration of 1 mg kg-1 i.v. and 10 mg kg-1 p.o. to dogs.

Separation of fluoride from fluoroelastomers by diffusion in test tubes.

The conventional procedure for separation of fluoride as trimethylfluorosilane in Conway diffusion cells involves the use of grease for sealing the cell and also for closing the hole in the lid drilled for introduction of hexamethyldisiloxane. We have developed a simpler procedure in which diffusion is carried out in 5-mL test tubes without the use of grease. Results of analysis of fluoride following diffusion from water, urine, and bone samples are in excellent agreement with those obtained by other procedures not involving diffusion. Separation of fluoride from partly and fully cured fluoroelastomers is achieved by first grinding the samples in a liquid nitrogen mill and then using methyl ethyl ketone as an adjuvant to perchloric acid employed in the diffusion procedure.

Studies on anabolic steroids--4. Identification of new urinary metabolites of methenolone acetate (Primobolan) in human by gas chromatography/mass spectrometry.

The metabolism of methenolone acetate (17 beta-acetoxy-1-methyl-5 alpha-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. After oral administration of a 50 mg dose of the steroid to two male volunteers, twelve metabolites were detected in urine either in the glucuronide, sulfate or free steroid fractions. Methenolone, the parent steroid was detected in urine until 90 h after administration. Its cumulative urinary excretion accounted for 1.63% of the ingested dose. With the exception of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major biotransformation product of methonolone acetate, metabolites were excreted in urine at lower levels, through minor metabolic routes. Most of methenolone acetate metabolites were isolated from the glucuronic acid fraction, namely methenolone, 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, 3 alpha-hydroxy-1 alpha-methyl-5 alpha-androstan-17-one, 17-epimethenolone, 3 alpha,6 beta-dihydroxy-1-methylen-5 alpha-androstan-17-one, 2 xi-hydroxy-1-methylen-5 alpha-androstan-3,17-dione, 6 beta-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione, 16 alpha-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione and 3 alpha,16 alpha-dihydroxy-1-methyl-5 alpha-androst-1-en-17-one. Interestingly, the metabolites detected in the sulfate fraction were isomeric steroids bearing a 16 alpha- or a 16 beta-hydroxyl group, whereas 1-methyl-5 alpha-androst-1-en-3,17-dione was the sole metabolite isolated from the free steroid fraction. Steroids identity was assigned on the basis of the mass spectral features of their TMS ether, TMS enol-TMS ether, MO-TMS, and d9-TMS ether derivatives and by comparison with reference and structurally related steroids. The data indicated that methenolone acetate was metabolized into several compounds resulting from oxidation of the 17-hydroxyl group and reduction of A-ring substituents, with or without concomitant hydroxylation at the C6 and C16 positions.

Studies on anabolic steroids. II--Gas chromatographic/mass spectrometric characterization of oxandrolone urinary metabolites in man.

The metabolism of 17 alpha-methyl-17 beta-hydroxy-2-oxa-5 alpha-androstan-3-one (oxandrolone) in man has been investigated by gas chromatography/mass spectrometry. After oral administration of a 10 mg dose to man, five metabolites were detected in the free fraction of the urinary samples. Oxandrolone, the major compound excreted in urine, was detected within 72 h after administration. During this period 35.8 and 8.4% of the administered dose was excreted as unchanged oxandrolone and 17-epioxandrolone, respectively. In addition, minute amounts of 16 alpha- and 16 beta-hydroxyoxandrolone and a delta-hydroxy acid resulting from the hydrolysis of the lactone group of oxandrolone were detected in the urine samples 8-60 h after administration. Furthermore, the susceptibility of oxandrolone to hydrolysis was investigated under several pH conditions. Extraction and fractionation of steroidal metabolites was achieved by using C18 and silica Sep Pak chromatography. The mass spectra of the metabolites are presented and major fragmentation pathways discussed.

Increased urinary excretion of aromatic amino acid catabolites by Microtus montanus chronically infected with Trypanosoma brucei gambiense.

Microtus montanus infected with Trypanosoma brucei gambiense for 16 and 21 days excreted significantly greater quantities of several aromatic amino acid catabolites when compared to uninfected control animals. Very large quantities of three aromatic alpha-keto acids (alpha-oxocarboxylic acids), phenylpyruvic acid, 4- hydroxyphenylpyruvic acid and indole-3-pyruvic acid, were excreted by infected animals. Increased excretion of indole-3-lactic acid and indole-3-acetic acid was also detected. Gas chromatographic-mass spectral analysis of the trimethylsilyl derivatives of phenylpyruvic acid, 4- hydroxyphenylpyruvic acid and indole-3-pyruvic acid confirms the identity of the aromatic alpha-keto acids elevated during infection. The marked alpha-keto aciduria indicates that a large disturbance exists in aromatic amino acid metabolism in this chronic animal model of African trypanosomiasis. The disturbance may contribute to the pathogenesis of the disease. The increased catabolite concentrations may also prove to be useful diagnostically and prognostically.

Procedure for the gas chromatographic-mass spectrometric confirmation of some exogenous growth-promoting compounds in the urine of cattle.

As gas chromatography-mass spectrometry is the most conclusive confirmation technique available today for the detection of ppb levels of anabolics in the urine of cattle, the following procedure was used. The urine is hydrolysed with Helix pomatia intestinal juice, extracted, and the extract cleaned by gel-permeation chromatography or with Extrelut. In one fraction are eluted diethylstilboestrol, dienoestrol, hexoestrol, methyltestosterone, ethinyloestradiol, zeranol and trenbolone. This fraction is injected into a two-dimensional high-performance liquid chromatography system. From the first column, the fraction containing the above-mentioned compounds is transferred to the second column, where a separation into two fractions is obtained. The first fraction contains zeranol and trenbolone, the second fraction the stilbenes, methyltestosterone and ethinyloestradiol. In general, the compounds are derivatized with an N,O-bis (trimethylsilyl)trifluoroacetamide-trimethylchlorosilane mixture to a trimethylsilyl derivative. With stilbene, confirmatory derivatization into heptafluorobutyryl derivatives is necessary. In combination with a Finnigan 4000/INCOS system, a CP-Sil-5 CB and a CP-Sil-19 CB capillary are used for final confirmation. Two capillaries with different polarities are necessary to overcome problems with possible interferences from compounds extracted from the urine. Recoveries at the ppb level are better than 80%.

Studies on drug metabolism by use of isotopes. XXIV-Determination of 3-phenylpropyl carbamate metabolites using stable isotope labelling with deuterium or carbon-13.

Metabolism of 3-phenylproply carbamate was investigated by using a stable isotope tracer technique. 3-Phenylpropanol, 3-hydroxy-3-phenylpropanol, 3-hydroxy-3-phenylpropyl carbamate, 2,3-dihydroxy-3-phenylproply carbamate, benzoic acid and hippuric acid were identified as the rat urinary metabolites. Using the dilution analysis, the amounts of metabolites in urine and faeces in rat and man were determined. In rats, 2,3-dihydroxy-3-phenylproply carbamate and 3-phenylpropanol glucuronide were excreted into the urine as the major metabolites of this drug. On the other hand, in man, the major metabolite was hippuric acid and about 30% of the administered dose was excreted as hippuric acid in the 24 h urine. The tracer technique using a singly labelled drug with carbon-13 employed in the present study provided a reliable methods for the analysis of drug metabolites and was comparable with the tracer technique using a multilabelled drug with deuterium.

Metabolism of biphenyl in the rat.

The metabolism of biphenyl in the rat has been studied by using gas chromatographic and mass spectrometric methods. The free and conjugated urinary metabolites were characterized. Eight new metabolites were isolated: a dihydrodiol and two hydroxydihydrodiols were characteristic for the epoxide--diol pathway. There were two dihydroxybiphenyls, a trihydroxybiphenyl, a trihydroxymethoxybiphenyl and 4,4'-dihydroxy-3-methylthiobiphenyl. The mass spectra of the trimethylsilyl derivatives of the metabolites exhibited characteristic doubly charged and metastable ions.

Quantitative analysis of steroid profiles from urine by capillary gas chromatography. I. Accuracy and reproducibility of the sample preparation.

A method is described for the determination of steroid profiles from urine by means of gas chromatography using high-efficiency glass capillary columns. The accuracy and reproducibility of essential steps in the sample preparation (extraction of steroids and steroid conjugates by means of XAD-2, enzymatic hydrolysis with Helix pomatia juice, solvolysis in acidified ethyl acetate and alkali wash) are established using different endogenously labelled urine samples, obtained from normal subjects to whom labelled steroids had been administered. Preliminary results are given on the reproducibility of the derivatization procedure (formation of methoxime-trimethylsilyl (MO-TMS) ethers), the gas chromatographic analysis and the whole method. Two procedures for the purification of MO-TMS steroid derivatives are compared. Application of the method to urine samples of patients with various endocrine disorders is included.

n-Butoxyacetic acid, a urinary metabolite from inhaled n-butoxyethanol (Butylcellosolve).

Male albino rats were exposed to butylcellosolve (n-butoxyethanol). Collected urine was found to contain a characteristic metabolite, identified as n-butoxyacetic acid by mass spectrometry. The identity of the compound was confirmed by synthesis.

The metabolism of biphenyl. IV. Phenolic metabolites in the guinea pig and the rabbit.

The phenolic metabolites of biphenyl in guinea pigs and rabbits were qualitatively and quantitatively analysed as trimethylsilyl (TMS) ethers by combined gas chromatography/mass spectrometry and gas chromatography, respectively. The parent compound was hydroxylated to monohydroxylated biphenyls and minor amounts of dihydroxylated derivatives, and the main route of body clearance appeared to be by the urine in both species. Thus, in the urine of guinea pigs 32.9% of the dose was detected 96 hrs after dosing, while the major part (29.5%) was eliminated during the first day as conjugates. The main metabolite was 4-hydroxybiphenyl (25.5%). During the first 24 hrs faecal recovery was 20.3% of the dose, and most of this (14.3%) consisted of biphenyl itself. Biliary excretion of the metabolites of biphenyl origin amounted to 3.3% of the dose during the first day, and 4-hydroxybiphenyl was the major metabolite. In the urine of rabbits 49.1% of the dose was recovered 96 hrs after dosing, and most of this (25.4 and 15.9%, respectively) was eliminated during the first two days as conjugates. The major metabolite was 4-hydroxybiphenyl (35.3%). On the first day faecal recovery was 1.6%, of which 1.4% was detected as biphenyl itself. Less than 1% of the dose was found in the 7 hrs rabbit bile, and exclusively as 4-hydroxybiphenyl. The experiments thus show that both qualitative and quantitative differences in the metabolism of biphenyl exist between the guinea pig and the rabbit even though 4-hydroxybiphenyl was the most prominent metabolite of biphenyl in both species.