Excretion profile of boldenone in urine of veal calves fed two different milk replacers

The residue profiles of 17alpha-/17beta-boldenone conjugated (17alpha/beta-Bol) and ADD were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male veal calves fed two commercial milk replacers, with different content of cholesterol and phytosterols. The urine samples were collected within 4 h after feeding and further from all the animals. Detectable amounts of 17alpha-Bol conjugated were measured in urine collected from all calves, but the concentrations of 17alpha-Bol were higher in urine from calves receiving the milk replacer with the greater amount of phytosterols. During the whole experiment, 17beta-Bol and ADD were never detected in urine samples collected.     

Development of a method which discriminates between endogenous and exogenous beta-boldenone

One potential explanation for the presence of beta-boldenone in calf urine is contamination of the sample with feces containing beta-boldenone. It has been demonstrated that after oral and intramuscular administration of beta-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6beta-hydroxy-17alpha-boldenone. This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6beta-hydroxy-17alpha/beta-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC-MS) in 2004-2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6beta-hydroxy-17alpha/beta-boldenone and for the presence of coprostanol. Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of alpha-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6beta-hydroxy-17alpha/beta-boldenone. Not finding this metabolite indicates that the origin of alpha-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated beta-boldenone and alpha-boldenone conjugates next to alpha-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated beta-boldenone can be of endogenous origin.     

Fractionation of free and conjugated steroids for the detection of boldenone metabolites in calf urine with ultra-performance liquid chromatography/tandem mass spectrometry

For over a decade there has been an intensive debate on the possible natural origin of boldenone (androst-1,4-diene-17beta-ol-3-one, 17beta-boldenone) in calf urine and several alternative markers to discriminate between endogenously formed boldenone and exogenously administered boldenone have been suggested. The currently approved method for proving illegal administration of beta-boldenone(ester) is the detection of beta-boldenone conjugates. In the presented method the sulphate, glucuronide and free fractions are separated from each other during cleanup on a SAX column to be able to determine the conjugated status of the boldenone metabolites. The sulphate and glucuronide fractions are submitted to hydrolysis and all three fractions are further cleaned up on a combination of C18/NH2 solid-phase extraction (SPE) columns. Chromatographic separation of the boldenone metabolites was achieved with a Waters Acquity UPLC instrument using a Sapphire C18 (1.7 microm; 2x50 mm) column within 5 min. Detection of the analytes was achieved by electrospray ionisation tandem mass spectrometry. The decision limits of this method, validated according to Commission Decision 2002/657/EC, were 0.08 ng mL(-1) for androsta-1,4-diene-3,17-dione, 0.13 ng mL(-1) for androst-4-ene-3,17-dione, 0.11 ng mL(-1) for 17alpha-boldenone, 0.07 ng mL(-1) for 17beta-boldenone, 0.24 ng mL(-1) for 5beta-androst-1-en-17beta-ol-3-one and 0.58 ng mL(-1) for 6beta-hydroxy-17beta-boldenone. Because of the fractionation approach used in this method there is no need for conjugated reference standards which often are not available. The disadvantage of needing three analytical runs to determine the conjugated status of each of the metabolites was overcome by using fast chromatography.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the separation of conjugated and unconjugated 17alpha- and 17beta-boldenone in urine sample

Natural occurrence or illegal treatment of boldenone (BOLD) presence in cattle urine is under debate within the European Union. Separation of conjugated and unconjugated forms of 17alpha-boldenone (alpha-BOLD) and 17beta-boldenone (beta-BOLD) and presence of related molecules as androsta-1,4-diene-3,17-dione (ADD) appear critical points for the decision of an illegal use. The aim of this study is a new analytical approach of BOLD and ADD confirmation in cattle urine. The separation between conjugated and unconjugated forms of BOLD was obtained by a preliminary urine liquid-liquid extraction step with ethyl acetate. In this step the organic phase extracts only unconjugated BOLD and ADD, while BOLD in conjugated form remain in urine phase. Afterwards the urine phase, contains conjugated BOLD, was subjected to an enzymatic deconjugation. Solid-phase extraction (OASIS-HLB Waters) was used for the purification and concentration of analytes in organic and urine phases and liquid chromatography ion electrospray tandem mass spectrometry (LC-MS-MS) was applied for the confirmation of BOLD and ADD, using deuterium-labelled 17beta-boldenone (BOLD-d3) as internal standard. The method was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/CE. The results obtained demonstrate that the developed method show very high specificity, precision, trueness and ruggedness. Decision limits (CCalpha) smaller than 0.5 ng mL(-1) were obtained for each analyte.     

Boldenone, testosterone and 1,4-androstadiene-3,17-dione determination in faeces from horses, untreated and after administration of androsta-1,4-diene-3,17-dione (boldione)

Faeces, which could be a potential alternative medium for doping control, have been used for the detection of 1,4-androstadiene-3,17-dione administration to the horse. Semi-quantitative analyses of 1,4-androstadiene-3,17-dione, testosterone, 17alpha- and 17beta-boldenone have been conducted in pre- and post-administration faeces, and in controls (untreated stallions, geldings and mares). Sample preparation comprised diethyl ether extraction, lipid removal, HPLC purification and derivatisation. 1,4-Androstadiene-3,17-dione, testosterone, 17alpha- and 17beta-boldenone were analysed by GC-EI/MS/MS. Quantitative limits of detection were 0.1 ng/g for 1,4-androstadiene-3,17-dione, and 0.025 ng/g for testosterone, 17alpha- and 17beta-testosterone. In post-administration samples from geldings and mares, peak levels of 1,4-androstadiene-3,17-dione, 17alpha-, 17beta-boldenone and testosterone were attained 24 h after administration. In untreated geldings and mares (in di- or anoestrus), 17alpha- and 17beta-boldenone and testosterone were not detected. Faeces from females in oestrus had detectable levels of boldenone isomers and testosterone. 1,4-Androstadiene-3,17-dione was undetectable in faeces collected from untreated horses, but the presence of this androgen was recently reported in faeces from untreated swine and it would therefore be advisable to check for its possible presence in a larger number of individual faecal samples.     

Analysis of androgenic steroid Girard P hydrazones using multistage tandem mass spectrometry

Seven androgenic steroids have been converted into steroid hydrazones using Girard P hydrazine and analysed by electrospray ionisation multistage tandem mass spectrometry. The cationic derivatives 17alpha-testosterone hydrazone, 17beta-nortestosterone hydrazone, 17beta-bolasterone hydrazone, 17alpha-boldenone hydrazone, 17beta-fluoxymesterone hydrazone, 17alpha-trenbolone hydrazone and 4-chloroandrosten-3,17-dione hydrazone show good response in positive ion mode with enhancements for the method of up to 33 times relative to the native species. Detailed characterisation of fragmentation pathways reveals structurally specific ions formed by fragmentation of the hydrazine moiety. Comparison of structural similarities among the androgenic steroids allows recognition of common ions/fragmentation processes as well as analyte-specific transitions. The suitability of the derivatisation approach in the screening of heifer urine for the presence of administered hormones has been demonstrated using partially purified urine spiked with a mixture of androgenic steroids.     

Determination of hormones in human urine by ultra‐high‐performance liquid chromatography/triple‐quadrupole mass spectrometry

Steroid hormones play a critical role in maintaining the homeostasis of human metabolism. Urine as a noninvasive sample has been extensively used in clinical diagnosis for hormones homeostasis. In this study, the simultaneous characterization of fourteen hormones in urine was performed based on ultra‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPHLC/ESI(+)‐MS/MS) with multiple reaction monitoring in the positive ionization mode. The target hormones were cortisone, cortisol, 11‐deoxycortisol, aldosterone, corticosterone, 11‐deoxycorticosterone, progesterone, 17‐OH‐progesterone, pregnenolone, estrone, estradiol, estriol, testosterone and dehydreopiandrosterone. β‐Glucuronidase/sulfatase deconjugation and liquid–liquid extraction (LLE) were conducted for the determination of urinary hormones (free + conjugated forms). The limits of detection (LODs) ranged from 0.2 ng/mL (11‐deoxycortisol and testosterone) to 1 ng/mL (cortisone). The extraction recovery of the targeted compounds ranged from 87% to 127%, indicating sufficient extraction efficiency for the LLE process. Intraday precision was below 10% and the accuracy ranged from 84% to 122%. The profiling analysis of hormones in urine samples helps to understand the metabolic state of biological systems and can be employed as a diagnostic tool in diseases developed by endocrine‐disrupted systems.     

Rapid test by liquid chromatography/tandem mass spectrometry to evaluate equine urine reactivity towards 17beta-OH steroids

Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, demonstrating the stereoselectivity of the reaction. A rapid test was performed by spiking one of the four 17beta-OH steroids in samples of diluted equine urine. The steroids were transformed into their respective ketones in the presence of bacterial activity. The test allows direct injection of diluted samples into the LC/MS system, without the need for prior extraction. Results show that the best method of storage is freezing at -18 degrees C. Urine specimens should be analyzed as soon as possible after thawing. This allows bacterial degradation of equine urine to be arrested temporarily, so that the urine can be used for qualitative or quantitative analysis of 17beta-OH steroids.     

Characterisation of steroids in wooden crates of veal calves by accelerated solvent extraction (ASE®) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-QqQ-MS-MS)

Illegal steroid administration to enhance growth performance in veal calves has long been, and still is, a serious issue facing regulatory agencies. Over the last years, stating undisputable markers of illegal treatment has become complex because of the endogenous origin of several anabolic steroids. Knowledge on the origin of an analyte is therefore of paramount importance. The present study shows the presence of steroid analytes in wooden crates used for housing veal calves. For this purpose, an analytical procedure using accelerated solvent extraction (ASE®), solid-phase extraction (SPE) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-MS-MS) is developed for the characterisation of androstadienedione (ADD), boldenone (bBol), androstenedione (AED), β-testosterone (bT), α-testosterone (aT), progesterone (P) and 17α-hydroxy-progesterone (OH-P) in wood samples. In samples of wooden crates used for housing veal calves, ADD, AED, aT and P could be identified. Using the standard addition approach concentrations of these analytes were determined ranging from 20?±?4 ppb to 32?±?4 ppb for ADD, from 19?±?5 ppb to 44?±?17 ppb for AED, from 11?±?6 ppb to 30?±?2 ppb for aT and from 14?±?1 ppb to 42?±?27 ppb for P, depending on the sample type. As exposure of veal calves to steroid hormones in their housing facilities might complicate decision-making on illegal hormone administration, inequitable slaughter of animals remains possible. Therefore, complete prohibition of wooden calf accommodation should be considered.     

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. II. Identification and quantification of 19-norsteroids responsible for 19-norandrosterone and 19-noretiocholanolone excretion in human urine

In previous work (Le Bizec et al., Rapid Commun. Mass Spectrom. 2000; 14: 1058), it was demonstrated that a boar meal intake could lead to possible false accusations of abuse of 17beta-nortestosterone in antidoping control. The aim of the present study was to identify and quantify endogenous 19-norsteroids in boar edible tissue at concentrations that can alter the steroid urinary profile in humans, and lead to excretion of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). The samples were analysed in two laboratories. The methodologies used for extraction and detection (GC/MS(EI) and LC/MS/MS(APCI+)) are compared and discussed. 19-Norandrostenedione (NAED), 17beta- and 17alpha-nortestosterone (bNT, aNT), and 17beta- and 17alpha-testosterone (bT, aT) were quantified. The largest concentrations of NAED and bNT were observed in testicles (83 and 172 microg/kg), liver (17 and 63 microg/kg) and kidney (45 and 38 microg/kg). A correlation between the bNT and NAED content of a typical meal prepared with boar parts and the excreted concentrations of 19-NA and 19-NE in human urine was demonstrated.     

Control system for detection of the illegal use of naturally occurring steroids in calves

Within the scope of the National Plan for Hormone Control in The Netherlands, a study was performed to develop a system for control of the illegal use of three naturally occurring hormones [oestradiol-17 beta (E2-17 beta), testosterone (T), progesterone (P)] for fattening purposes in animal production. Using a specific high-performance liquid chromatographic-radioimmunoassay method, reference values were established for concentrations of E2-17 beta, T and P and some of their metabolites in blood plasma and urine from untreated male and female veal calves. E2-17 beta levels of both male and female calves were less than 0.01 microgram/l in blood plasma and less than 0.2 microgram/l in urine. For male veal calves levels of T and epitestosterone (epiT) in blood plasma and urine varied widely. The P levels were less than 0.1-0.3 micrograms/l in blood plasma and less than 0.6-10 micrograms/l in urine from both male and female calves. To investigate the effect of anabolic treatment on the hormone levels in plasma and excreta, male veal calves were injected, subcutaneously into the dewlap, with a solution containing 20 mg of E2-17 beta benzoate and 200 mg of T propionate in 5 ml of arachis oil. Only the levels of E2-17 beta and E2-17 alpha in blood plasma and excreta were elevated until about one week after injection, compared with the untreated control calves and the reference values. T and epiT levels were similar in plasma and excreta from both untreated and treated animals.     

Evaluation of boldenone formation and related steroids transformations in veal faeces by liquid chromatography/tandem mass spectrometry

It is established that bovine urine can result positive for boldenone and androstadienedione in consequence of faecal contamination. The simple transfer of steroids to urine is one minor aspect of faecal contamination. A high de novo production of steroids in faeces after deposition and in faeces-contaminated urine is almost certainly due to microbial activity, although the precursor compounds and transformations leading to the presence of these illegal steroids are unclear. We developed a simple in vitro method - incubation of faecal matter suspended in 0.9% saline - to induce steroid transformations in faeces, and analyzed the products by liquid chromatography/tandem mass spectrometry, without the need for prior extraction. Norethandrolone was the internal standard. The linearity (R(2): 0.987-0.999), sensitivity (LODs: 0.3 to 1.0 ng/mL; LOQs: 1.0 to 3.0 ng/mL), precision (intra-day CVs: 2.6-8.2; inter-day CVs: 4.5-11.5) and accuracy (percentage recovery: 89-120%) were calculated for the studied steroids. Androstenedione, androstadienedione, alpha- and beta-boldenone, testosterone and epitestosterone transformations were investigated. Mutual interconversion of steroids was observed, although 17beta-hydroxy steroids had low stability compared with 17alpha-hydroxy and 17-keto steroids. The results suggest that this simple in vitro system may be an effective way of studying hormone transformations in faeces and, after analogue studies, in faeces-contaminated urine.     

Evaluation of equine urine reactivity towards phase II metabolites of 17‐hydroxy steroids by liquid chromatography/tandem mass spectrometry

Proper storage conditions of biological samples are fundamental to avoid microbiological contamination that can cause chemical modifications of the target analytes. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method through direct injection of diluted samples, without prior extraction, was used to evaluate the stability of phase II metabolites of boldenone and testosterone (glucuronides and sulphates) in intentionally poorly stored equine urine samples. We also considered the stability of some deuterated conjugated steroids generally used as internal standards, such as deuterated testosterone and epitestosterone glucuronides, and deuterated boldenone and testosterone sulphates. The urines were kept for 1 day at room temperature, to mimic poor storage conditions, then spiked with the above steroids and kept at different temperatures (?18°C, 4°C, room temperature). It has been possible to confirm the instability of glucuronide compounds when added to poorly stored equine urine samples. In particular, both 17β‐ and 17α‐glucuronide steroids were exposed to hydrolysis leading to non‐conjugated steroids. Only 17β‐hydroxy steroids were exposed to oxidation to their keto derivatives whereas the 17α‐hydroxy steroids were highly stable. The sulphate compounds were completely stable. The deuterated compounds underwent the same behaviour as the unlabelled compounds. The transformations were observed in urine samples kept at room temperature and at a temperature of 4°C (at a slower rate). No modifications were observed in frozen urine samples. In the light of the latter results, the immediate freezing at ?18°C of the collected samples and their instant analysis after thawing is the proposed procedure for preventing the transformations that occur in urine, usually due to microbiological contamination. Copyright © 2008 John Wiley & Sons, Ltd.     

Assay for trenbolone and its metabolite 17 alpha-trenbolone in bovine urine based on immunoaffinity chromatographic clean-up and off-line high-performance liquid chromatography-thin-layer chromatography

An high-performance liquid chromatography (HPLC)-thin-layer chromatography (TLC) method was developed to detect the illegal use of the xenobiotic growth promotor Trenbolone acetate (TBA). Very effective clean-up of bovine urine was achieved by immunoaffinity chromatography (IAC). The active form of TBA, the steroid 17 beta-Trenbolone (17 beta-TB), as well as its major metabolite 17 alpha-Trenbolone (17 alpha-TB), were assayed simultaneously with HPLC and on-line UV detection. The fraction containing 17 alpha-TB and 17 beta-TB (TB-fraction) was collected, and for confirmation 17 beta- and 17 alpha-TB were subsequently separated and identified by TLC. The limit of detection by on-line HPLC-UV (350 nm) was 1-2 micrograms TB/l. Off-line TLC detection was even more sensitive, 0.5 microgram 17 beta- or 17 alpha-TB/1. The assay was validated by investigating urine samples from veal calves implanted with TBA. The presence of 17 beta- and 17 alpha-TB was clearly demonstrated. A survey of the illegal use of TBA in cattle was performed by applying the assay to urine obtained at slaughter. No residues of TBA or its metabolites were found in any of the 144 random samples from the Dutch public health surveillance programme.     

Multi-residue screening of bovine urine on xenobiotic oestrogens with an oestrogen radioreceptor assay

An improved radioreceptor assay (RRA) is used for the screening of urine samples from cattle for the presence of exogenous oestrogenic anabolic compounds, e.g., stilbenes and zeranol. The method includes extraction of the hormones from urine samples with simultaneous purification using reversed-phase C18 cartridges. High-performance liquid chromatography is used to isolate the anabolics from the naturally occurring oestrogens. Fractions containing the stilbenes and zeranol are collected and subsequently analysed using the RRA with the oestrogen receptor, isolated from immature calf uteri, as a binder and tritiated 17 beta-oestradiol as a tracer. Urine samples from untreated calves and cows and samples from calves treated with zeranol-trenbolon acetate, dienoestrol or hexoestrol or samples containing diethylstilboestrol were analysed with this RRA method. Sensitivity, specificity and predictive values were calculated at different classification levels (0.4, 0.5, 0.6 and 1.0 ng/ml 'apparent' oestradiol in urine). An optimum sensitivity (89%) with a maximum specificity (95%) was reached at a classification level of 0.6 ng/ml. At this level the detection limits in urine samples are 0.5 ng/ml for hexoestrol, 0.6 ng/ml for diethylstilboestrol, 0.9 ng/ml for dienoestrol and 5.0 ng/ml for zeranol.     

Determination of beta-19-nortestosterone and its metabolite alpha-19-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid chromatography with on-line immunoaffinity sample pretreatment

An immunoaffinity precolumn (immuno precolumn) packed with Sepharose-immobilized polyclonal antibodies against the anabolic hormone 17 beta-19-nortestosterone (beta-19-NT) was used for the selective on-line pretreatment of raw extracts of urine, bile and tissue samples by high-performance liquid chromatography. Using UV detection (247 nm), beta-19-NT and its metabolite 17 alpha-19-nortestosterone (alpha-19-NT) can be determined in biological samples with a detection limit of 0.05 microgram/kg. Owing to the high clean-up efficiency of the immuno precolumn and the large sample volumes used, confirmation by gas chromatography-mass spectrometry is possible at this level. In urine samples from a calf treated with 19-nortestosterone 17 beta-laurate, the maximum concentrations of beta-19-NT (1.3 micrograms/l) and alpha-19-NT (3.1 micrograms/l) were found seven days after intramuscular administration. In a bile sample from this calf only alpha-19-NT (55 microgram/l) was detected. In meat samples from three treated calves, the concentration of beta-19-NT varied from 0.1 to 1.6 micrograms/kg and no alpha-19-NT could be detected. In liver samples from these calves, the concentrations of beta-19-NT and alpha-19-NT were less than 0.05-0.1 and 0.5-0.9 micrograms/kg, respectively. In the corresponding kidney samples, the concentrations of beta-19-NT and alpha-19-NT were 0.4-0.5 and 0.5-1.6 micrograms/kg, respectively. The application of the same immuno precolumn to the determination of 17 beta- and 17 alpha-trenbolone, two structurally related steroids, is also demonstrated.     

Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10–12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCβ levels (5–20 μg/kg) after 5–7 weeks. When treatment is repeated two times, the CCβ levels are reached after 9–11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but—in contrast with the pour-on application—after i.m. injection, significant increase of 17β-testosterone and 17β-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood.     

Comparison of multiple API techniques for the simultaneous detection of microconstituents in water by on‐line SPE‐LC‐MS/MS

This study described a fully automated method using on‐line solid phase extraction of large volume injections coupled with high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) to simultaneously detect a group of recalcitrant microconstituents (pharmaceuticals and personal care products, steroid hormones and sterols) in aqueous matrices. Samples (1 mL to 20 mL) were loaded to the preconcentration column at 1 mL/min, and the column was washed with 1000 μL of 25% methanol in LC/MS water to remove polar and ionic interferences before LC‐MS/MS analysis. Three different atmospheric pressure ionization (API) techniques, including photoionization (APPI) with four different dopants (acetone, anisole, chlorobenzene and toluene), heated electrospray ionization (HESI) and atmospheric pressure chemical ionization (APCI), were evaluated on the basis of method detection limits (MDLs) and recoveries from different aqueous matrixes. Results indicated that APPI with toluene as dopant was the most sensitive ionization method for the majority of the analytes. When using 5 mL of sample, MDLs for pharmaceuticals and personal care products, including carbamazepine, DEET, caffeine, naproxen, acetaminophen and primidone, were between 0.3 ng/L and 15 ng/L. MDLs of hormones, including testosterone, equilenin, progesterone, equilin, 17β‐estradiol, 17α‐ethynylestradiol, estrone, androsterone, mestranol and estriol, were between 1.2 ng/L and 37 ng/L. The combination of APPI with dopant allowed the detection of two difficult to ionize fecal related sterols, such as coprostan‐3‐ol and coprostan‐3‐one with MDLs of 5.4 ng/L and 11 ng/L, respectively. Calculated MDLs are more than adequate for analysis of wastewater using 1 to 5 mL sample size and for surface waters using up to 20 mL sample size. Copyright © 2012 John Wiley & Sons, Ltd.     

Unambiguous identification of thiouracil residue in urine collected in non-treated bovine by tandem and high-resolution mass spectrometry

Thyrostats are banned compounds in Europe since 1981 (directive 81/602/EC) because of their carcinogenic and teratogenic properties. However, the occurrence of thiouracil (TU) in bovine urines from national monitoring plans with quantifications in the range 1-10 microg . L(-1) occasionally raises the question of its origin which might either be the consequence of an illegal administration or the result of 'endogenous' production. In order to definitively and unambiguously identify the so-called thiouracil signal in non-treated bovine urines, independent mass spectrometry (MS) approaches have been used. Different reagents (3-IBBr, 3-BrBBr and PFBBr) were used to derivatise and to extract TU from urine samples and characterisation of the residues was performed by means of different MS approaches [LC/(ESI-)MS/MS, GC/(EI+)MS/MS and HRMS (EI and NCI)]. These combined strategies allowed for an independent and confident identification of TU in bovine urine samples collected from animals never treated with any thyrostatic drugs. This result is of prime importance for laboratories and risk managers involved in the field of forbidden growth promoters control: detection of TU residue in bovine urine will have to be carefully considered as a non-systematic proof of illegal administration.     

Identification of 17-hydroxyprogesterone and other steroid hormones in saliva from a normal child and patients with congenital adrenal hyperplasia by plasmaspray liquid chromatography/mass spectrometry

A very sensitive, selective and simultaneous method for the analysis of salivary steroid hormones was examined by discharge-assisted thermospray liquid chromatography mass spectrometry (plasmaspray LC/MS). Plasmaspray LC/MS gave [M + H]+ or [MH - H2O]+ as a predominant ion in most of the steroids in this work and in some cases fragment ions were also observed. Eight salivary steroid hormones, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, aldosterone, and testosterone were identified within 10 minutes using the selected ion monitoring technique in conjunction with plasmaspray LC/MS. Progesterone, 17-hydroxyprogesterone, cortisol, aldosterone and testosterone were detected in 1 mL of saliva from a normal child and two patients with congenital adrenal hyperplasia.