Multiclass screening in urine by comprehensive two-dimensional liquid chromatography time of flight mass spectrometry for residues of sulphonamides, beta-agonists and steroids.

Nowadays routine residue monitoring involves the analysis of many compounds from different classes, mainly in urine. In the past two decades, developments heavily focused on the use of mass spectrometers (MS) and faster and more sensitive MS detectors have reached the market. However, chromatographic separation (CS) was rather ignored and the cognate developments in CS were not in line. As a result, residue analysis did not improve to the extent anticipated. CS by LC x LC is a promising technique and will enable a further increase in the range of compounds and compound classes that can be detected in a single run. In the present study, a self-built LC x LC system, using a 10 port valve, was connected to a single quadrupole MS with electrospray interface. Standards containing a mixture of sulphonamides, ß-agonists and (steroid) hormones, 53 compounds, in total, were analysed. Results demonstrated that these compounds were well separated and could be detected at low levels in urine, i.e. limit of detection (LOD) from 1 µg L-1 for most ß-agonists to 10 µg L-1 for some sulphonamides and most hormones. To enhance the sensitivity, optimisation was performed on an advanced commercial LC x LC system connected to a full scan accurate MS. This ultimately resulted in a fast high throughput untargeted method, including a simple sample clean-up in a 96-well format, for the analysis of urine samples.

Applicability of an innovative steroid-profiling method to determine synthetic growth promoter abuse in cattle.

A robust LC-MS/MS method was developed to quantify a large number of phase I and phase II steroids in urine. The decision limit is for most compounds lower than 1ngml-1 with a measurement uncertainty smaller than 30%. The method is fully validated and was applied to assess the influence of administered synthetic steroids and beta-agonists on the steroidogenesis. From three animal experiments, clenbuterol, diethylstilbestrol and stanozolol, the steroid profiles in urine of bovine animals were compared before and after treatment. It was demonstrated that the steroid profiles were altered due to these treatments. A predictive multivariate model was built to identify deviations from normal population steroid profiles. The abuse of synthetic steroids can be detected in urine samples from bovine animals using this model. The samples from the animal experiments were randomly analysed using this method and predictive model. It was shown that these samples were predicted correctly in the exogenous steroids group.

The effects of bromocriptine, thyrotropin-releasing hormone, and gonadotropin-releasing hormone on hormone secretion by gonadotropin-secreting pituitary adenomas in vivo and in vitro.

The characteristics and dynamics of hormone secretion in vivo and in vitro were investigated in six patients with gonadotropin-secreting pituitary adenomas. All six tumors secreted and contained FSH and different combinations of LH, beta-LH, and alpha-subunit. In addition, immunohistochemical examination of the pituitary tumor tissue showed staining with both LH and FSH in three and either LH or FSH in the other three tumors. TRH and GnRH stimulated hormone secretion in vivo and in vitro, and they also increased the hormone content of the cultured tumor cells. Bromocriptine significantly inhibited hormone release and reduced the hormone content of the tumor cells. In vivo, 2.5 mg bromocriptine significantly suppressed plasma hormone levels; the inhibiting effect on alpha-subunit concentrations was in general more marked than that on LH and FSH. We conclude that hormone release by gonadotropin-secreting pituitary adenomas can be stimulated by TRH and GnRH and inhibited by bromocriptine. Most of these tumors synthesize FSH, but there is a wide variation in the production of LH, beta-LH, and alpha-subunits. The sensitivity of hormone release to bromocriptine suggests that chronic therapy with this drug might have a beneficial effect on pituitary tumor size.

A CO(2)-Flux Mechanism Operating via pH-Polarity in Hydrilla verticillata Leaves With C(3) and C(4) Photosynthesis.

The aquatic angiosperm Hydrilla verticillata lacks Kranz anatomy, but has an inducible, C(4)-based, CO(2) concentrating mechanism (CCM) that concentrates CO(2) in the chloroplasts. Both C(3) and C(4) Hydrilla leaves showed light-dependent pH polarity that was suppressed by high dissolved inorganic carbon (DIC). At low DIC (0.25 mol m(-3)), pH values in the unstirred water layer on the abaxial and adaxial sides of the leaf were 4.2 and10.3, respectively. Abaxial apoplastic acidification served as a CO(2) flux mechanism (CFM), making HCO (3) (-) available for photosynthesis by conversion to CO(2). DIC at 10 mol m(-3) completely suppressed acidification and alkalization. The data, along with previous results, indicated that inhibition was specific to DIC, and not a buffer effect. Acidification and alkalization did not necessarily show 1:1 stoichiometry; their kinetics for the apolar induction phase differed, and alkalization was less inhibited by 2.5 mol m(-3) DIC. At low irradiance (50 mumol photons m(-2) s(-1)), where CCM activity in C(4) leaves is minimized, both leaf types had similar DIC inhibition of pH polarity. However, as irradiance increased, DIC inhibition of C(3) leaves decreased. In C(4) leaves the CFM and CCM seemed to compete for photosynthetic ATP and/or reducing power. The CFM may require less, as at low irradiance it still operated maximally, if [DIC] was low. Iodoacetamide (IA), which inhibits CO(2) fixation in Hydrilla, also suppressed acidification and alkalization, especially in C(4) leaves. IA does not inhibit the C(4) CCM, which suggests that the CFM and CCM can operate independently. It has been hypothesized that irradiance and DIC regulate pH polarity by altering the chloroplastic [DIC], which effects the chloroplast redox state and subsequently redox regulation of a plasma-membrane H(+)-ATPase. The results lend partial support to a down-regulatory role for high chloroplastic [DIC], but do not exclude other sites of DIC action. IA inhibition of pH polarity seems inconsistent with the chloroplast NADPH/NADP(+) ratio being the redox transducer. The possibility that malate and oxaloacetate shuttling plays a role in CFM regulation requires further investigation.

Comparison of different liquid chromatography methods for the determination of corticosteroids in biological matrices.

Various extraction techniques can be combined with column liquid chromatography (LC) and ultraviolet (UV) or mass spectrometric (MS) detection for the determination of synthetic corticosteroids in biological matrices. Target analysis of low concentrations of 25 microg/kg of dexamethasone in feed can be performed by combining immunoaffinity chromatography (IAC) and LC with UV detection. A straightforward multi-analyte procedure is obtained by tandem solid-phase extraction (SPE) and subsequent LC-UV. However, the limits of detection for feed samples are then relatively poor, viz. 100 microg/kg. A multi-analyte method which meets modern demands of about 5 microg/kg detection limit requires one-step SPE combined with LC-MS analysis. As regards urine corticosteroids can be determined down to a level of 0.5 microg/l by either SPE-LC-MS- MS or SPE(IAC)-LC-MS.

Confirmation of residues of thyreostatic drugs in thyroid glands by multiple mass spectrometry after thin-layer chromatographic screening.

A method is described for the confirmation of high-performance thin layer chromatography (HPTLC) suspect results of residues of thyreostatic drugs in thyroid tissue. The method is based on the infusion of the remainder of the extract used for HPTLC via the electrospray interface into a mass spectrometer operating in the multiple stage mass spectrometry (MSn) mode. The clean-up of the samples was performed with a selective extraction procedure, based on a specific complex formation of the drugs with mercury ions, bound in an affinity column. The thyreostatic drugs were derivatised with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

Repeatability of the KT-1000 arthrometer in a normal population.

Despite its popularity, the MEDmetric KT-1000 arthrometer's reliability remains inadequately documented. We conducted this study to determine the magnitude of trial-to-trial (within installation), installation-to-installation (within day), and day-to-day (between day) variability of anterior/posterior translation measurements in normal knees. We selected six normal subjects, three males and three females, and tested each on 6 consecutive days with three separate installations per day. We recorded the total anterior/posterior translation at +/- 89 and +/- 134 N force at 25 degrees of flexion during three consecutive trials in a single installation. Analysis of variance showed that no significant difference existed between trials (within installation) or between installations (within day) for all parameters. However, we did find a significant difference between days for individual right and left knee translation measurements at 89 and 134 N force. More importantly, no significant difference existed between days for right to left differences at both force levels. The magnitude of the expected measurement variability was expressed by computing 90% confidence limits for total anterior/posterior translation at +/- 89 N force. These were +/- 1.5 mm for the right knees, +/- 1.4 mm for the left knees, and +/- 1.6 mm for the right-left differences. Fischer's protected least significant difference post hoc test revealed that for all parameters, the 1st day measurements were significantly less than those on following days, suggesting that patient and examiner adjust to the testing procedure. We conclude that the standard KT-1000 evaluation should report paired differences rather than individual knee measurements. Additionally, initial evaluation should be supplemented by follow-up examinations for verifying translation values.

[The presence of nortestosterone in edible parts from non-castrated male pigs]. / Het voorkomen van nortestosteron in eetbare delen van niet gecastreerde mannelijke varkens.

Nortestosterone is a major growth-promoting agent in Europe which is often used illegally in various species of meat animal. Recent studies showed that this compound was also present in the urine of young male pigs (boars) to which nortestosterone had not been administered. To determine to which extent nortestosterone may also be present in liver and muscle tissues, samples of the urine, bile, liver and muscle of twenty five boars were analysed. The mean and highest concentrations, detected respectively in muscle were 1.1 and 13 micrograms/kg and were 23 and 200 micrograms/kg in liver. The corresponding concentrations in urine were 55 and 132 micrograms/l and 88 and 212 micrograms/l in bile.

A review of analytical strategies for the detection of 'endogenous' steroid abuse in food production.

Detection of the abuse of synthetic steroids in food production is nowadays relatively straightforward using modern techniques such as gas or liquid chromatography coupled to mass spectrometry (GC-MS/MS or LC-MS/MS, respectively). However, proving the abuse of 'endogenous' (or naturally occurring) steroids is more difficult. Despite these difficulties, significant progress in this area has recently been made and a number of methods are now available. The aim of the current review was to systematically review the available analytical approaches, which include threshold concentrations, qualitative 'marker' metabolites, intact steroid esters, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), longitudinal testing and 'omics' biomarker profiling. The advantages/disadvantages of these methods are considered in detail, but the choice of which to adopt is dictated by a number of practical, political, and economic factors, which vary in different parts of the world. These include the steroid/species combination requiring analysis, the matrix tested, whether samples are collected from live or slaughtered animals, available analytical instrumentation, sample throughput/cost, and the relevant legal/regulatory frameworks. Furthermore, these approaches could be combined in a range of different parallel and/or sequential screening/confirmatory testing streams, with the final choice being determined by the aforementioned considerations. Despite these advances, more work is required to refine the different techniques and to respond to the ever increasing list of compounds classified as 'endogenous'. At this advanced stage, however, it is now more important than ever for scientists and regulators from across the world to communicate and collaborate in order to harmonize and streamline research efforts.

Presence and metabolism of endogenous androgenic-anabolic steroid hormones in meat-producing animals: a review.

The presence and metabolism of endogenous steroid hormones in meat-producing animals has been the subject of much research over the past 40 years. While significant data are available, no comprehensive review has yet been performed. Species considered in this review are bovine, porcine, ovine, equine, caprine and cervine, while steroid hormones include the androgenic-anabolic steroids testosterone, nandrolone and boldenone, as well as their precursors and metabolites. Information on endogenous steroid hormone concentrations is primarily useful in two ways: (1) in relation to pathological versus 'normal' physiology and (2) in relation to the detection of the illegal abuse of these hormones in residue surveillance programmes. Since the major focus of this review is on the detection of steroids abuse in animal production, the information gathered to date is used to guide future research. A major deficiency in much of the existing published literature is the lack of standardization and formal validation of experimental approach. Key articles are cited that highlight the huge variation in reported steroid concentrations that can result when samples are analysed by different laboratories under different conditions. These deficiencies are in most cases so fundamental that it is difficult to make reliable comparisons between data sets and hence it is currently impossible to recommend definitive detection strategies. Standardization of the experimental approach would need to involve common experimental protocols and collaboratively validated analytical methods. In particular, standardization would need to cover everything from the demographic of the animal population studied, the method of sample collection and storage (especially the need to sample live versus slaughter sampling since the two methods of surveillance have very different requirements, particularly temporally), sample preparation technique (including mode of extraction, hydrolysis and derivatization), the end-point analytical detection technique, validation protocols, and the statistical methods applied to the resulting data. Although efforts are already underway (at HFL and LABERCA) to produce more definitive data and promote communication among the scientific community on this issue, the convening of a formal European Union working party is recommended.

Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS.

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.

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.

Determination of resorcylic acid lactones in biological samples by GC-MS. Discrimination between illegal use and contamination with fusarium toxins.

An EU project, FAIR5-CT-1997-3443, has been undertaken to distinguish illegal use of zeranol from consumption of food contaminated with Fusarium spp. toxin. One of the tasks was development of screening and confirmatory methods of analysis. This paper describes a new method based on two-step clean-up and GC-MS analysis. The first clean-up step is matrix-dependant; the second is applicable to both urine and meat. The MS is operated in negative chemical ionisation mode. The method is quantitative for zeranol and taleranol, alpha- and beta-zearalenol, and zearalenone and qualitative for zearalanone. Validation was performed according to the latest EU performance criteria (Commission Decision 2002/657). For analysis of urine CC(alpha) and CC(beta) for the method (microg L(-1)) were 0.06-0.11 for zeranol, 0.07-0.12 for taleranol, 0.07-0.11 for alpha-zearalenol, 0.21-0.36 for beta-zearalenol, 0.35-0.60 for zearalenone, and 0.19-0.33 zearalanone. Within-laboratory reproducibility was 16.2, 11.2, 31.9, 30.1, 26.6, and 54.2% for zeranol, taleranol, alpha-zearalenol, beta-zearalenol, zearalenone, and zearalanone, respectively. It was found that all the compounds are stable in urine at -20 degrees C for at least a year. Part of the validation program was organisation of a small proficiency study (ringtest) and a correlation study with an LC-MS-MS method developed by the Veterinary Science Division (VSD; Belfast, UK-NI). This study showed there was good correlation between results from both laboratories. The method can be used for quantitative analysis discriminating illegal use of zeranol from consumption of zearalenone-contaminated food.

Immunoaffinity chromatography, its applicability and limitations in multi-residue analysis of anabolizing and doping agents.

The use of (multi-)immunoaffinity chromatography in residue analysis is discussed. After an introduction to the immunochemical background an overview of applications is given. A distinction is made between the following methods: (1) single-antibody, single-analyte procedures; (2) single-antibody, multi-analyte procedures; (3) multi-antibody, multi-analyte procedures. It is concluded that immunoaffinity chromatography is superior to most other techniques for sample preparation and extract clean-up. Its advantages in multi-residue procedures are most clear when compared with e.g. high-performance liquid chromatography. In combination with gas chromatography-low-resolution mass spectrometry, very effective multi-residue methods are possible. Most frequently they concern screening procedures which can fulfill the identification criteria for reference methods. It is concluded that the use of (multi-)immunoaffinity chromatography will proliferate further in the 1990s. However, its future viability is highly dependent on the interest of commercial firms and on the involvement of the EC Community Bureau of Reference in manufacturing and supplying the necessary materials.

Heterogeneity of human luteinizing hormone. Detection and identification of alpha- and beta-subunits in international reference preparations.

The charge heterogeneity of three international reference preparations containing biologically active human luteinizing hormone (LH) or its free alpha- or beta-subunits, respectively, was investigated with isoelectric focussing in sucrose density gradients. The immunoreactive profiles throughout the pH-gradient were assessed with radioimmunoassay (RIA) systems specific for intact, i.e. undissociated, LH or free alpha- or beta-subunits. For the preparation of intact LH (MRC 68/40), two peaks were found at pH = 7.69 and 8.05; for the alpha-subunit preparation (NIBSC 78/554), the peak values were 4.76, 5.04, 5.94, 6.70, 6.96, 7.35, 8.02, 8.72 and 9.32; and for the beta-subunit preparation (NIBSC 78/556), the values were 7.60, 8.40, 8.55 and 9.61. These results are in excellent agreement with those obtained for a highly purified LH preparation (NM14) which was prepared by one of us and on which we have reported earlier. In addition, with the above-mentioned techniques, the spontaneous dissociation of MRC 68/40 into subunits upon incubation at 37 degrees C in phosphate buffer was clearly demonstrated by the increased immunoreactivity in the profiles assessed with both RIA-subunit systems. It is concluded that charge heterogeneity of LHi, alpha- and beta-subunits, as observed for different preparations, is confined to a limited population of forms. Differences between preparations are only quantitative. A single preparation, therefore, can be used as a general model for the study of human luteinizing hormone.

Heterogeneity of human luteinizing hormone. Effect of neuraminidase treatment on biologically active hormone and alpha- and beta-subunits.

By preparative isoelectric focussing of a highly purified LH preparation in a sucrose density gradient, four biologically active LH components were isolated. The effect of neuraminidase treatment of each component on the charge heterogeneity was studied by isoelectric focussing followed by in vitro biological and immunochemical techniques. The number of biologically active components with pI-values greater than 7 containing varying amounts of sialic acid is at least six. The pI-value of the most basic (= asialo) LH component was 9.26. The two most basic components were not present in our preparation (NM14) before neuraminidase treatment. It is concluded that the difference between the pI-values of LH components is caused by a difference in sialic acid content. When an intact LH component was incubated with neuraminidase there was detectable dissociation owing to the elevated temperature (37 degrees C) and necessary acidic conditions of the incubation. Under these conditions we found the same subunits as we have described before. The most basic alpha-subunit had a pI-value of 9.29, whereas two beta-subunits with pI greater than 9 were observed at pI 9.26 and pI 9.9. On the other hand, when an LH component was forced to dissociate by incubation at 56 degrees C prior to neuraminidase digestion, two additional alpha-subunits were found. From this it is concluded that in the intact LH molecule, some sialic acid residues are poorer substrates for neuraminidase action than in the free subunits.

Heterogeneity of human luteinizing hormone. Discrimination between acidic and basic preparations.

The charge heterogeneity of an I. H preparation containing relatively acidic components (LH Type 1) was studied. Four biological active components, with pI-values of 5.04, 5.60, 6.06 and 6.57 were detected. A total of four different alpha-subunits, with pI-values of 4.49, 4.79, 5.16 and 6.02 could be detected after incubation at 37 degrees C. With the exception of the most acidic component all these alpha-subunits were also present in earlier studied 1.H Type II preparations. After neuraminidase treatment a strong shift to more basic components was observed, resulting in a population of components similar to the one detected in I.H Type II preparations. The beta-subunits detected were very different from those observed in Type II preparations. All six components detected had pI-values greater than 7.5. Upon incubation at 56 degrees C these subunits appeared to be unstable resulting in a shift to more basic pI-values these pI-values being very similar to those of beta-subunits observed before in Type II preparations. After neuraminidase treatment, the pH values of the population of beta-subunits became identical to those of the population in I.H Type II. From these results it is concluded that the major charge difference between LH Type I and Type II is located in the beta-subunits. This difference cannot be explained completely by differences in sialic acid content, but may also be due to heat labile charged groups such as sulphate.

Heterogeneity of human lutropin. Detection and identification of alpha- and beta-subunits.

A highly purified LH preparation, prepared from human pituitaries (NM14) was studied using immunochemical and in vitro biological techniques. Using isoelectric focusing 5 different biologically active components could be detected, 4 of which were located between pH = 7.0 and 8.6, one was located at pH = 4.9. The biological activity in the acidic part of the pH gradient is probably due to the formation of an artefact during storage in solution. The experiments were performed with special emphasis on the occurrence of LH subunits, for which until now no pI-values have been reported. Using specific radioimmunochemical (RIA) systems at least 7 different alpha-subunit components and 4 different beta-subunit components could be detected. The presence of even more components is likely. The alpha-subunit components, with pI-values of: 4.6, 5.2, 6.0, 7.1, 8.1, 8.8 and 9.7, were located spread over the entire pH-gradient whereas all beta-subunit components were located above pH = 7.5, the pI-values being 7.7, 8.4, 8.5 and 10.4. The identification of these components as alpha- or beta-subunit was based on the relative response in the different RIA systems, the absence of biological activity and the response changes during incubation at 37 degrees C. Refocusing of the above mentioned biologically active components individually resulted each time in a single component with a pI-value identical to its corresponding 'parent'. After incubation at 37 degrees C of these components each time the same variety of subunit components was found with discrete pI-values, identical to those above.

Heterogeneity of human luteinizing hormone. Hydrophobic interaction chromatographic fractionation of some preparations.

The heterogeneity of human luteinizing hormone was investigated with high performance hydrophobic interaction chromatography on a TSK phenyl-5PW column using an ammonium sulphate gradient. Recovery of individual subunits, expressed as immunochemical activity, was in the order of 90%. Recovery of the intact hormone was less, approximately 25%. The technique appeared to be independent of the charge heterogeneity of the individual subunits. Each component as obtained by hydrophobic interaction chromatography showed considerable charge heterogeneity when studied by subsequent isoelectric focusing. Nonetheless, all chromatography fractions derived from either subunit showed the same collection of isoelectric values, quantitatively only differences for the beta-subunits could be detected. Heterogeneity of the individual subunits was clearly demonstrated after incubation of the preparations at 37 or 56 degrees C. The heterogeneity of the beta-subunit observed after incubation at 56 degrees C was different to the heterogeneity after incubation at 37 degrees C. After incubation at 56 degrees C, an additional component, with a longer retention time, developed directly from the intact molecule. The component is unstable and is transformed to one of the components detected also after incubation at 37 degrees C. Based on these results the existence of two populations of intact LH molecules with respect to their thermal stability is hypothesized.