Generic sample preparation combined with high-resolution liquid chromatography-time-of-flight mass spectrometry for unification of urine screening in doping-control laboratories.

A unification of doping-control screening procedures of prohibited small molecule substances--including stimulants, narcotics, steroids, beta2-agonists and diuretics--is highly urgent in order to free resources for new classes such as banned proteins. Conceptually this may be achieved by the use of a combination of one gas chromatography-time-of-flight mass spectrometry method and one liquid chromatography-time-of-flight mass spectrometry method. In this work a quantitative screening method using high-resolution liquid chromatography in combination with accurate-mass time-of-flight mass spectrometry was developed and validated for determination of glucocorticosteroids, beta2-agonists, thiazide diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation of all the compounds of interest and the necessary purification of the resulting extracts, a generic extraction and hydrolysis procedure was combined with a solid-phase extraction modified for these groups of compounds. All 56 compounds are determined using positive electrospray ionisation with the exception of the thiazide diuretics for which the best sensitivity was obtained by using negative electrospray ionisation. The results show that, with the exception of clenhexyl, procaterol, and reproterol, all compounds can be detected below the respective minimum required performance level and the results for linearity, repeatability, within-lab reproducibility, and accuracy show that the method can be used for quantitative screening. If qualitative screening is sufficient the instrumental analysis may be limited to positive ionisation, because all analytes including the thiazides can be detected at the respective minimum required levels in the positive mode. The results show that the application of accurate-mass time-of-flight mass spectrometry in combination with generic extraction and purification procedures is suitable for unification and expansion of the window of screening methods of doping laboratories. Moreover, the full-scan accurate-mass data sets obtained still allow retrospective examination for emerging doping agents, without re-analyzing the samples.

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 CCbeta levels (5-20 microg/kg) after 5-7 weeks. When treatment is repeated two times, the CCbeta 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 17beta-testosterone and 17beta-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.

Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry.

The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.

Comprehensive screening and quantification of veterinary drugs in milk using UPLC-ToF-MS.

Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC-ToF-MS) has been used for screening and quantification of more than 100 veterinary drugs in milk. The veterinary drugs represent different classes including benzimidazoles, macrolides, penicillins, quinolones, sulphonamides, pyrimidines, tetracylines, nitroimidazoles, tranquillizers, ionophores, amphenicols and non-steroidal anti-inflammatory agents (NSAIDs). After protein precipitation, centrifugation and solid-phase extraction (SPE), the extracts were analysed by UPLC-ToF-MS. From the acquired full scan data the drug-specific ions were extracted for construction of the chromatograms and evaluation of the results. The analytical method was validated according to the EU guidelines (2002/657/EC) for a quantitative screening method. At the concentration level of interest (MRL level) the results for repeatability (%RSD < 20% for 86% of the compounds), reproducibility (%RSD < 40% for 96% of the compounds) and the accuracy (80-120% for 88% of the compounds) were satisfactory. Evaluation of the CCbeta values and the linearity results demonstrates that the developed method shows adequate sensitivity and linearity to provide quantitative results. Furthermore, the method is accurate enough to differentiate between suspected and negative samples or drug concentrations below or above the MRL. A set of 100 samples of raw milk were screened for residues. No suspected (positive) results were obtained except for the included blind reference sample containing sulphamethazine (88 microg/l) that tested positive for this compound. UPLC-ToF-MS combines high resolution for both LC and MS with high mass accuracy which is very powerful for the multi-compound analysis of veterinary drugs. The technique seems to be powerful enough for the analysis of not only veterinary drugs but also organic contaminants like pesticides, mycotoxins and plant toxins in one single method.

Analytical strategies for residue analysis of veterinary drugs and growth-promoting agents in food-producing animals--a review.

After a brief introduction into the field of veterinary drugs and growth-promoting agents, the most important EU regulations and directives for the inspection of food-producing animals and animal products regarding the residue control of these substances are presented and discussed. Main attention in the review is on the methods of analysis in use today for the most important classes of veterinary drugs and growth-promoting agents viz. anthelmintics, antibiotics, coccidiostats, hormones, beta-agonists and tranquillizers. Emphasis is given to the potential, and limitations, of state-of-the-art analytical procedures and their performance characteristics. The most obvious conclusion is that, today (reversed-phase) liquid chromatography combined with tandem mass spectrometric detection--either triple-quadrupole or ion-trap multi-stage--is the preferred technique in a large majority of all cases. In the field of sample treatment, the combined use of liquid extraction--i.e., liquid partitioning or liquid-liquid extraction--and liquid-solid extraction--primary on- or off-line solid-phase extraction--is most popular. Finally, while the analytical tools required to meet the demands typically formulated by governments and international organizations today, generally speaking are available, several problems still do exist. To quote three examples, problems are encountered in the area of simultaneously extracting and pre-treating groups of analytes with mutually widely different polarities, with regard to identification-point--based confirmation of analyte identity, and regarding quantification errors caused by ion-suppression effects. Improving the speed of analysis is another aspect that should, and will, receive dedicated interest in the near future.

Simultaneous determination of seven gestagens in kidney fats by Ultra Performance Convergence Chromatography tandem mass spectrometry.

An ultra-performance convergence chromatography (UPC2) system coupled tandem mass spectrometry was successfully utilised to analyse chlormadinone acetate, delmadinone acetate, fluorogestone acetate, medroxyprogesterone acetate, megestrol acetate, melengestrol acetate, chlortestasterone acetate in bovine and porcine kidney fat. This novel approach obtained an improved resolution in comparison to previously reported chromatographic methods combined with MS detector in a shorter analytical time. All the acetylgestagen compounds were well separated on a ACQUITY UPC(2) HSS C18 column (3.0 × 100 mm, 1.7 µm) by applying methanol and carbon dioxide (2/98). The LOQ of delmadinone acetate, melengestrol acetate, medroxyprogesterone acetate and megestrol acetate are 0.5 µg/kg, fluorogestone acetate, chlormadinone acetate and chlortestasterone acetate 1.0 µg/kg. The recoveries of gestagens spiked in kidney fats at a concentration range of 0.5 to 4 µg/kg were above 86.1% with relative standard deviations (RSD) less than 13.1%. These rapid and reliable methods can be used to efficiently separate, characterize and quantify the residues of gestagens in kidney fats with advantages of shorter time, more sensitive and environmental friendly.

Determination of cannabinoids in cannabis products using liquid chromatography-ion trap mass spectrometry.

A method was developed and validated for the simultaneous determination of five cannabinoids, viz. cannabidiol (CBD), cannabidiol acid (CBD-COOH), cannabinol (CBN), delta9-tetrahydrocannabinol (THC), and 3'-carboxy-delta9-all-trans-tetrahydrocannabinol (THC-COOH) in cannabis products. The cannabinoids were extracted from the grinded cannabis samples with a mixture of methanol-chloroform and analysed using liquid chromatography with ion-trap-mass-spectrometry (LC-IT-MSn). For quantification the two most abundant diagnostic MS-MS ions of the analyte in the sample and external standard were monitored. For confirmation purposes the EU criteria as described in Commission Decision 2002/657/EC were followed. Fully satisfactory results were obtained, that is, unequivocal confirmation according to the most stringent EU criteria was possible. The limits of quantification were 0.1 g/kg for CBD, 0.04 g/kg for CBD-COOH, 0.03 g/kg for CBN, 0.28 g/kg for THC and 9.9 g/kg for THC-COOH. The repeatabilities, defined by R.S.D., were 2% for CBN, THC and THC-COOH at the concentration levels of respectively 0.023, 3.3 and 113 g/kg and 5% for CBD-COOH at the level of 0.34 g/kg (n = 6).

Carry-over of veterinary drugs from medicated to non-medicated feeds in commercial feed manufacturing plants.

Different compound feeds have to be manufactured in the same production line. As a consequence, traces of the first produced feed may remain in the production and get mixed with the next feed batches. This "carry-over" is unavoidable, and so non-medicated feed can be contaminated with veterinary drugs like antibiotics added to the previous batch of medicated feed. To monitor the carry-over of antibiotics in the Netherlands, 21 feed mills were visited and 140 samples of flushing feeds were collected and analysed for containing residues of antibiotics. Results show that 87% of all samples contain concentrations of antibiotics in the range of 0.1-154 mg/kg. It is expected that these levels - which are in the same range as previously found for the nowadays banned antimicrobial growth promoters (AMGPs) - have an effect on the occurrence of microbial resistance. Analysis of a second set of samples collected at four different feed mills directly after the production of oxytetracycline-medicated feed demonstrated that the first part of a flushing feed has much higher contamination than the last part of the batch. Furthermore, it was demonstrated that the carry-over percentage shows no correlation with the carry-over determined by one of the standard GMP+ procedures. These observations, unavoidable carry-over, inhomogeneous batches of feed with antibiotics and difficulties to predict the carry-over levels, together with the awareness of the increasing problem of microbial resistance, motivated the NEVEDI, association of Dutch Feed Producers, to announce that they will voluntarily stop the production of medicated feed in 2011. The alternatives for medicated feed are for example water or milk medication or the use of top-dressings at the farm. The consequences and possible new risks of carry-over at the farm are not completely clear yet.

Determination of the stability of antibiotics in matrix and reference solutions using a straightforward procedure applying mass spectrometric detection.

The stability of an antibiotic is a very important characteristic, especially in the field of antibiotic residue analysis. During method development or validation, the stability of the antibiotic has to be demonstrated no matter if the method is used for screening, confirmation, qualitative or quantitative analysis. A procedure for testing the stability of antibiotics in solutions and food samples using LC-MS/MS is described. The procedure is based on the assumption that the antibiotics are stable when stored at -70 °C. Representative solutions or spiked samples containing the antibiotic were stored at the temperature to be tested (-18 or 4 °C) and at -70 °C. After a selected storing time samples were moved from the chosen storage temperature to -70 °C. At the end of the study, all samples--per class of antibiotic--were analysed in one batch. By applying statistical models, it was finally concluded in which circumstances the antibiotic is stable. The stability of 60 antibiotics belonging to the classes of tetracyclines, sulphonamides, quinolones, penicillins, macrolides and aminoglycosides were tested. The stability of solutions containing tetracyclines and penicillins is only guaranteed for 3 months while stored at -18 °C. Solutions of all other antibiotics tested are stable for at least 6 or 12 months when stored at 4 °C. In muscle tissue stored at -18 °C no severe degradation of the tested antibiotics was observed, with the exception of the penicillins. The stability data reported here are useful as a reference for laboratories carrying out validation studies of analytical methods for antibiotic (residue) detection. The data should save the time needed for long-term stability testing of solutions and samples.

Feasibility of desorption electrospray ionization mass spectrometry for rapid screening of anabolic steroid esters in hair.

Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionization (DESI) mass spectrometry (MS). In this work the feasibility of DESI application for the rapid screening of intact esters of anabolic steroids in bovine hair has been studied. Using a linear ion trap both full scan and data-dependent collision induced dissociation MS(n) spectra were acquired in minutes for testosterone cypionate, testosterone decanoate and estradiol benzoate standard solutions deposited on a glass or PTFE surface. However direct analysis of incurred hair failed due to inefficient desorption ionization and the minute quantities of steroid esters present. Therefore a simplified ultrasonic liquid extraction procedure was developed, allowing rapid DESI analysis of a few microliters of the concentrate and a total analysis time of 2-4h per batch instead of 3 days. The potential of this DESI approach is clearly demonstrated by MS(3) data from hair samples incurred with high levels (300-800 µg kg(-1)) of steroid esters, levels which do occur in samples from controlled- and illegally treated animals. For much lower levels state-of-the-art ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) screening methods remain the method of choice and might benefit from the proposed simplified extraction as well.

Influence of anabolic combinations of an androgen plus an estrogen on biochemical pathways in bovine uterine endometrium and ovary.

The application of anabolic steroids in food producing animals is forbidden in the EU since 1988, but the abuse of such drugs is a potential problem. The existing test systems are based on known compounds and can be eluded by newly emerging substances. The examination of physiological effects of anabolic hormones on different tissues to indirectly detect misuse might overcome this problem. Two studies were conducted with post-pubertal 24-months old Nguni heifers and pre-pubertal female 2-4 weeks old Holstein Friesian calves, respectively. The animals of the accordant treatment groups were administered combinations of estrogenic and androgenic compounds. The measurement of the gene expression pattern was undertaken with RT-qPCR. Target genes of different functional groups (receptors, angiogenesis, steroid synthesis, proliferation, apoptosis, nutrient metabolism and others) have been quantified. Several biochemical pathways were shown to be influenced by anabolic treatment. Both studies identified significant regulations in steroid and growth factor receptors (AR, ERß, LHR, FSHR, Flt-1, PR, IGF-1R, Alk-6), angiogenic and tissue remodeling factors (VEGFs, FGFs, BMPs, ANGPT-2, MMPs, TIMP-2, CTSB), steroid synthesis (S5A1, HSD17, CYP19A1), proliferation (TNFα, IGF-1, IGFBPs, p53, c-fos; CEBPD, c-kit), apoptosis (CASP3, FasL, p53) and others (C7, INHA, STAR). Several genes were regulated to opposite directions in post-pubertal compared to pre-pubertal animals. PCA for Nguni heifers demonstrated a distinct separation between the control and the treatment group. In conclusion, anabolics modify hormone sensitivity and steroid synthesis, and they induce proliferative effects in the whole reproductive tract (uterus and ovary) as well as anti-angiogenic effects in the ovary. However, the extent will depend on the developmental stage of the animals.

Effect of sample pre-treatment on the determination of steroid esters in hair of bovine calves.

The effect of three sample pre-treatment steps, washing, cutting and grinding on the determination of steroid esters in hair is studied. The study is performed by using hair samples obtained after pour-on application of steroid esters to bovine calves. After sample pre-treatment the hair is treated with a mild reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] to extract the steroid esters. After a solid-phase extraction clean-up step the extracts are analysed by using liquid chromatography combined with triple-quadrupole mass spectrometric detection. For the washing step the use of non-organic washing solvents like (warm) water and a solution of 0.1% sodium dodecyl phosphate and organic solutions containing different percentages of methanol are tested. By using the non-organic solvents and the organic solvents with a percentage of methanol <20% the recovery results are as good as the results obtained without washing the hair. Cutting the hair samples increases the analyte recoveries of incurred steroid esters by 20% compared to the non-cut hair. The analyte recoveries of cut hair samples are about 60-80% that of ground hair samples. The obtained surface expansion of hair samples by grinding proves to be necessary in order to achieve the highest possible analyte yields. Finally the use of pressurised liquid extraction (PLE) for the extraction of steroid esters from plain (no washing, cutting or grinding) hair is investigated. The first results show lower (up to 40%) extraction recoveries in comparison with the classical solvent extraction procedures. If the limit of detection requirement is met, PLE may be an alternative for extracting large numbers of hair samples due to the short sample treatment procedure involved.

Multi-residue screening of veterinary drugs in egg, fish and meat using high-resolution liquid chromatography accurate mass time-of-flight mass spectrometry.

The last 2 years multi-compound methods are gaining ground as screening methods. In this study a high-resolution liquid chromatography combined with time-of-flight mass spectrometry (HRLC-ToF-MS) is tested for the screening of about 100 veterinary drugs in three matrices, meat, fish and egg. While the results are satisfactory for 70-90% of the veterinary drugs, a more efficient sample preparation or extract purification is required for quantitative analysis of all analytes in more difficult matrices like egg. The average mass measurement error of the ToF-MS for the veterinary drugs spiked at concentrations ranging from 4 to 400 microg/kg, is 3.0 ppm (median 2.5 ppm) with little difference between the three matrices, but slightly decreases with increasing concentration. The SigmaFit value, a new feature for isotope pattern matching, also decreases with increasing concentration and, in addition, shows an increase with increasing matrix complexity. While the average SigmaFit value is 0.04, the median is 0.01 indicating some high individual deviations. As with the mass measurement error, the highest deviations are found in those regions of the chromatogram where most compounds elute from the column, be it analytes or matrix compounds. The median repeatability of the method ranges from 8% to 15%, decreasing with increasing concentration, while the median reproducibility ranges from 15% to 20% with little difference between matrices and concentrations. The median accuracy is in between 70% and 100% with a few compounds showing higher values due to matrix interference. The squared regression coefficient is >0.99 for 92% of the compounds showing a good overall linearity for most compounds. The detection capability, CCbeta, is within 2 times the associated validation level for >90% of the compounds studied. By changing a few conditions in the analyses protocol and analysing a number of blank samples, it was determined that the method is robust as well as specific. Finally, an alternative validation strategy is proposed and tested for screening methods. While the results calculated for repeatability, within-lab reproducibility and CCbeta show a good comparison for the matrices meat and fish, and a reasonable comparison for the matrix egg, only 27 analyses are required to obtain these results versus 63 analysis in the traditional, 2002/657/EC, approach. This alternative is suggested as a cost-effective validation procedure for screening methods.

Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis.

The applicability of ultra-performance liquid chromatography (UPLC) combined with full-scan accurate mass time-of-flight (TOF) and Orbitrap mass spectrometry (MS) to the analysis of hormone and veterinary drug residues was evaluated. Extracts from blank bovine hair were fortified with 14 steroid esters. UPLC-Orbitrap MS performed at a resolving power of 60,000 (FWHM) enabled the detection and accurate mass measurement (<3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background. A 5 ppm mass tolerance window proved to be essential to generate highly selective reconstructed ion chromatograms (RICs) having reduced background from the hair matrix. UPLC-Orbitrap MS at a lower resolving power of 7500 and UPLC-TOFMS at mass resolving power 10,000 failed both to detect all of the steroid esters in hair extracts owing to the inability to mass resolve analyte ions from co-eluting isobaric matrix compounds. In a second application, animal feed extracts were fortified with coccidiostats drugs at levels ranging from 240 to 1900 ng/g. UPLC-Orbitrap MS conducted at a resolving power of 7500 and 60,000 and UPLC-TOFMS detected all of the analytes at the lowest investigated level. Thanks to the higher analyte-to-matrix background ratio, the utilization of very narrow mass tolerance windows in the RIC was not required. This study demonstrates that even when the targeted sample preparation from conventional LC-MS/MS is applied to UPLC with full-scan accurate mass MS, false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions. The current trend towards more generic and less selective sample preparation is expected to aggravate this issue further.