Possibilities for confirmatory analysis of some β-agonists using two different derivatives simultaneously

A previously described method for the confirmatory analysis of clenbuterol with chemical ionization GC-MS was extended to fit more β-agonists. The method has been used routinely for about one year and a large number of samples have been analysed according to the described procedure. This paper summarizes the results obtained with this method with regard to clenbuterol and furthermore discussion is focussed on applicability to β-agonists other than clenbuterol with respect to the European Community requirements for confirmatory analysis.     

The age of hair matters – the incorporation of cortisol by external contamination is enhanced in distal hair segments of pigs and cattle

Hair cortisol concentration (HCC) is used as an indicator of long-term stress or pathologies in humans and increasingly in animals. Although the main mechanism for the incorporation of cortisol into the hair shaft is by diffusion from blood, cortisol may also be incorporated from external sources by contamination of the hair surface. In farm animals under conventional husbandry conditions and trapped animals, contamination of hair with cortisol-containing body fluids, especially with urine, was shown to be a considerable confounding factor when studying HCCs. We recently found that cattle and pigs exhibit elevated HCCs in distal hair segments and assume that the incorporation of external cortisol is facilitated in these older hair segments. Therefore, the aim of this study was to investigate the effects of urine contamination on HCC in different hair segments of pigs and cattle, and to determine whether different cleaning protocols can prevent contamination effects. In an in vivo experiment in pigs (n = 18) and an in vitro experiment in cattle (n = 12), hairs were repeatedly contaminated with urine of the respective species and then shaved or cut in segments. Cortisol concentrations in hair segments were analysed by enzyme immunoassay after washing with isopropanol and extraction with methanol. Results were compared with HCCs in untreated hairs or hairs treated with water. Moreover, additional bovine hair samples contaminated with urine were subjected to two further cleaning procedures. Contamination with urine generally increased HCCs, and it was demonstrated for the first time that this effect is more pronounced in distal compared to proximal hair segments in both species. The immersion of bovine hair in vitro in water caused a washout of cortisol, which was also more pronounced in distal hair segments. In general, the different cleaning protocols for cattle hair did not prevent contamination effects, so we assume that external cortisol not only adheres but is incorporated into the hair shaft. Structural damage of older, distal hair segments may facilitate permeability of the hair matrix and diffusion of cortisol from and into aqueous solutions. Thus, the validity of HCC as a marker of stress is compromised in animals where soiling of hair with body fluids is a risk factor. Therefore, hair samples should be collected from clean body regions and, if possible, using proximal hair segments.     

Application of a sequential analytical procedure for the detection of the β-agonist brombuterol in bovine urine samples

The multistep analytical procedure routinely applied in our laboratory for the detection of the aryl amine β-agonists clenbuterol, mabuterol and mapenterol in bovine matrices has been extended to the analysis in urine samples of brombuterol, a new clenbuterol-like compound. In the screening steps, the urine samples were first enzyme-linked immunosorbent assay (ELISA)-tested, then the positive samples were analyzed by thin-layer chromatography (TLC) and the β-agonists detected with the Bratton-Marshall color reaction. The TLC spots corresponding to the suspected compounds were scraped off the plates, collected and extracted separately with methanol. The β-agonists in the extracts were detected by HPLC–Vis (limits of detection: 0.5 ng/g). In the confirmatory step the presence and the concentration of the compounds of interest in the samples were established by GC–MS with two different ionization techniques, EI and CI (limits of detection: 1.0 ng/g). The use of this procedure has made possible the detection of brombuterol in officially sampled bovine urine.     

Effect of the myorelaxant clenbuterol on the oxytocin-induced release of prostaglandin F2 alpha in ovariectomized heifers

Clenbuterol, as other sympathomimetic drugs, relaxes the myometrium, thus causing a short-term inhibition of labor and the delay of parturition. This study has examined the influence of clenbuterol on the release of prostaglandin F2 alpha (PGF2 alpha) induced by oxytocin alone or with estradiol-17 beta. Five bilaterally ovariectomized heifers, primed with progesterone for 14 days, were used in two experiments. In the first they received two i.v. injections of oxytocin 6h apart, with and without an i.v. injection of clenbuterol before the second oxytocin injection; the second experiment was similar to the first except that the animals were given estradiol-17 beta 30 min after the first oxytocin injection. Frequent blood samples were taken for the measurement of 13,14-dihydro-15-keto-PGF2 alpha by radioimmunoassay. The data show that clenbuterol does not influence PGF2 alpha release in response to oxytocin alone or with estradiol-17 beta, and it does not inhibit the basal release of PGF2 alpha. This suggests that clenbuterol does not act on the endometrium to alter the secretion of PGF2 alpha in the non-pregnant cow.     

Mercury concentrations in hair exposed in vitro to mercury vapor

Hair is often used as an index of environmental and industrial exposure to different metals. The interpretation of metal levels in hair is difficult because of the risk of external contamination. The aim of this study was to define the degree of external contamination of hair exposed in vitro to mercury vapor. Specimens of hair were exposed to concentration: 0.026, 0.21, and 2.7 mg Hg/m³ for 2–28 d. Mercury levels in hair increased during 28 d of exposure 2, 3 and 13, times, respectively, when compared to initial values. Mercury levels in hair exposed to the first and second (but not third) concentration of mercury vapor attained a steady state on the 21st d of exposure. The contamination of hair with mercury could not be removed by washing with water, solvent, and detergent. Hair may be used as an index of internal uptake of mercury provided that it was not externally exposed to mercury vapor. In cases of occupational exposure to mercury vapor, hair could become a useful tool for monitoring exposures.     

Detection of illegal clenbuterol use in calves using hair analysis: Application in meat quality control

This study describes a real-life situation involving nine calves, 106 days old, which received oral doses of clenbuterol administered through their milk. Powdered skim milk containing 6.7 mg of clenbuterol was given daily for fifteen days under supervision (i.e. 100 mg per calf for the whole study) to seven calves, and two calves did not receive the drug. Hair samples and urine were taken and subjected to analysis by gas chromatography–mass spectrometry. Hairs were pulverized in a ball mill and 100 mg were incubated in a mildly acidic medium. The sample clean-up procedure involved solid-phase extraction on C₁₈ cartridges. Metoprolol was used as the internal standard for quantitation, after formation of methylboronate derivatives. The calibration curve for clenbuterol in hair was linear in the range 20–5000 pg/mg. The limit of detection of clenbuterol was 16 pg/mg in hair and 0.14 ng/ml in urine. Hair testing was effective after 7–10 days of treatment, and concentrations were in the range of 20 to 4372 pg/mg. Urinalysis can detect clenbuterol for up to two weeks after discontinuation of the drug. Conveniently, this is around the time when the hair samples attain greatest sensitivity. Therefore, the combination of the two matrices appears to be the method of choice for testing for the illegal use of drugs in meat-producing animals.     

NGF mediates the neuroprotective effect of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo: evidence from an NGF-antisense study.

Previous studies in our laboratory suggested that neuroprotective effects of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo occurred due to enhanced synthesis of nerve growth factor. The aim of the present study was to evaluate the effects of a phosphothioated NGF oligodeoxynucleotide on neuroprotection by clenbuterol in vitro and in vivo. After clenbuterol treatment (1-100 microM) an increase in nerve growth factor mRNA and protein levels (200-300% of control) was observed in primary cultures of rat cortical astrocytes. Nerve growth factor antisense oligonucleotide (0.3-1 microM for 3 days) reduced the content of nerve growth factor protein in the medium of the astrocytes concentration-dependently to 20% of control level. Nerve growth factor content in the medium of mixed hippocampal cells was reduced to 55% of sister cultures receiving the vehicle or a random control oligonucleotide. In mixed hippocampal cultures pretreated with random oligonucleotide (1 microM, 30 h), clenbuterol (10 microM) reduced the percentage of damaged neurons after glutamate exposure (0.5 mM, 1 h) to 17%. Pretreatment with nerve growth factor antisense oligonucleotide (1 microM) for 30 h before glutamate incubation blocked the protective effect of clenbuterol. In vivo, clenbuterol (0.01-0.1 mg/kg) reduced the infarct volume in a rat model of permanent focal cerebral ischemia dose-dependently. Nerve growth factor antisense oligonucleotides injected into the cortical tissue before ischemia abolished the cerebroprotective effect of clenbuterol. Our results indicate that the nerve growth factor antisense oligonucleotide presented in this study is a useful tool to investigate the effects of nerve growth factor knock down. By using the nerve growth factor antisense oligonucleotide we could demonstrate that nerve growth factor mediated the neuroprotective effects of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo.     

Toxic effects of catecholamines on skin

The purpose of this study was to examine the effects of catecholamines on skin necrosis independent of their vasoactive effects. Rat abdominal or human breast skin was excised, pinned flat, and incubated at 37 degrees C for 6 hours in a buffered salt solution containing catecholamine. At 0.1 and 6 hours the lactate dehydrogenase (LDH) released from the skin and appearing in the buffer was determined spectrophotometrically. All groups showed similar LDH levels at 0.1 hour. Rat skin treated with greater than or equal to 10(-7) M epinephrine (33 times less than the 1:200,000 used clinically) or greater than or equal to 10(-5) M norepinephrine showed a significant increase in the LDH released at 6 hours versus controls (18.75 +/- 1.25 versus 13.75 +/- 1.25 and 29.25 +/- 2.96 versus 22.00 +/- 1.96 IV, respectively). Total tissue LDH levels were not significantly different at 0.1 or 6 hours. The toxic effect of epinephrine was eliminated by the addition of propranolol or selective beta 2 blockade, but not by alpha or beta 1 blockade. Therefore, this effect appears to be mediated largely by beta 2 receptors. Similar toxic effects were seen in human breast skin treated with 1:200,000 epinephrine and were blocked with propranolol. Phenylephrine at 1:20,000 demonstrated toxicity, but angiotensin II and vasopressin did not. These studies indicate that addition of catecholamine to ischemic rat or human skin accelerates skin death within 6 hours, but that the toxicity can be reversed with beta blockade.     

Beta-agonist drugs modulate the proliferation and differentiation of skeletal muscle cells in vitro

Essentially employed for the treatment of airway obstructions in humans, β-agonists are also known to have an anabolic effect in animals’ skeletal muscle. In vivo and in vitro studies have attested the increase in animal body mass and the hypertrophy of muscle cells following the administration of specific β-agonists. However, the contribution of β-agonists to C2C12 myoblasts growth remains obscure. We therefore aimed to investigate the impact of β1-and β2-agonist drugs on the proliferation and differentiation of skeletal muscle cells. Direct observations and cytotoxicity assay showed that clenbuterol, salbutamol, cimaterol and ractopamine enhanced muscle cell growth and viability during the proliferation stage. Structural examinations coupled to Western blot analysis indicated that salbutamol and cimaterol triggered a decrease in myotube formation. A better comprehension of the effect of β-agonists on myogenic regulatory genes in the muscle cells is crucial to establish a specific role of β-agonists in muscle development, growth, and regeneration.     

A dietary regimen alters hepatocyte plasma membrane lipid fluidity and ameliorates ethinyl estradiol cholestasis in the rat

The beta-adrenergic receptor mediating the inhibition of sterol synthesis by catecholamines in freshly isolated human mononuclear leukocytes was defined pharmacologically by using selective beta 1- and beta 2-agonists and -antagonists. Incubation of cells for 6 h in a medium containing lipid-depleted serum resulted in a 3-fold increase in the incorporation of [14C]acetate or tritiated water into sterols. The beta-agonist (-)-isoproterenol was approximately equipotent with (-)-epinephrine and (-)-norepinephrine in suppressing sterol synthesis, yielding a sigmoidal log-dose-effect curve. Accordingly, the effects of the catecholamines were reversed by the beta-antagonist (+/-)-propranolol. The beta 2-agonists terbutaline and salbutamol inhibited sterol synthesis by 42 and 26%, respectively, at a concentration of 0.1 mmol/l. Contrary to that, the beta 1-agonists prenalterol and dobutamine had no effect. In accordance with the influence of the agonists, the beta 2-antagonist butoxamine, but not the beta 1-antagonists atenolol, metoprolol and practolol, reversed the catecholamine action on sterol synthesis. The results provide evidence that catecholamines may regulate sterol synthesis by stimulating beta 2-adrenergic receptors.     

Screening and confirmatory analysis of β-agonists, β-antagonists and their metabolites in horse urine by capillary gas chromatography—mass spectrometry

A method for the screening and confirmatory analysis of β-agonists and -antagonists in equine urine is described. Following initial enzymic hydrolysis, the basic drugs and metabolites are extracted using Clean Screen^® DAU or Bond Elut Certify™ cartridges, and analysed as their trimethylsilyl ether or 2-(dimethyl) silamorpholine derivatives by capillary gas chromatography—mass spectrometry. The method proved to be very sensitive and selective for basic drugs. After administration of therapeutic doses of propranolol, metoprolol, timolol, isoxsuprine and clenbuterol to thoroughbred horses, the parent compound/metabolites could be detected in urine for upto 14–120 h depending on the drug.     

Removing external DNA contamination from arthropod predators destined for molecular gut-content analysis

Ecological research requires large samples for statistical validity, typically hundreds or thousands of individuals, which are most efficiently gathered by mass-collecting techniques. For the study of interspecific interactions, molecular gut-content analysis enables detection of arthropod predation with minimal disruption of community interactions. Field experiments have demonstrated that standard mass-collection methods, such as sweep netting, vacuum sampling and foliage beating, sometimes lead to contamination of predators with nontarget DNA, thereby compromising resultant gut-content data. We deliberately contaminated immature Coleomegilla maculata and Podisus maculiventris that had been fed larvae of Leptinotarsa decemlineata by topically applying homogenate of the alternate prey Leptinotarsa juncta. We then attempted to remove contaminating DNA by washing in ethanol or bleach. A 40-min wash with end-over-end rotation in 80% EtOH did not reliably reduce external DNA contamination. Identical treatment with 2.5% commercial bleach removed most externally contaminating DNA without affecting the detectability of the target prey DNA in the gut. Use of this bleaching protocol, perhaps with minor modifications tailored to different predator-prey systems, should reliably eliminate external DNA contamination, thereby alleviating concerns about this possible source of cross-contamination for mass-collected arthropod predators destined for molecular gut-content analysis.     

Simultaneous quantification of opiates, amphetamines, cocaine and metabolites and diazepam and metabolite in a single hair sample using GC-MS

A method is described for the simultaneous identification and quantification of opiates, amphetamines, cocainics, diazepam and nordiazepam from one hair extract (typically 10-50mg hair). After decontamination by washing with shampoo, dichloromethane, isopropanol and acetone, drugs were extracted using 0.1M HCl followed by SPE clean-up using mixed-mode extraction cartridges. The SPE extracts were submitted to a two-step derivatisation using MBTFA and MSTFA+1% TCMS and analysis was performed by GC-MS using both SIM and scan modes. Four deuterated standards were used to monitor 14 compounds. The limit of quantification was the total drug detected from the sample. This was 5 ng for amphetamines and 10 ng for remaining drugs which is equivalent to 0.1 and 0.2 ng/mg from a 50mg sample. Standard curves for the range 5-400 ng total drug concentration for all drugs had regression coefficients greater than 0.98. An authentic hair sample was used to validate the method and gave R.S.D.s <25% for both inter and intra-day reproducibility. The results of the analysis of hair taken from four patients attending a drug treatment clinic and six hair samples including head hair, pubic hair, axial hair and beard taken at post-mortem are presented.     

Are Metals Accumulated in Human Hair Affected by Naturally Occurring Asbestos Fiber Contamination? A Case Study from a Rural Area of China

Little is known about the link between metals accumulated in human and asbestos fiber contamination in the environment. Therefore, hair samples of 368 subjects (128 males and 240 females) from a rural area contaminated by crocidolite asbestos fibers were collected to investigate the distributions of 17 metals accumulated in human. The results showed that the mean concentrations of As, Al, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Mo, Na, Ni, Pb, Sr, and Zn in hair of the total subjects were 0.23, 23.36, 4.33, 0.11, 0.05, 0.70, 10.53, 29.74, 0.37, 241.57, 3.52, 0.08, 153.21, 0.72, 4.26, 10.96, and 113.35 mg/kg, respectively. Moreover, approximately 86.14, 52.17, 73.91, 85.05, 80.98, 74.46, and 53.80 % of the hair samples of the total subjects contained much higher concentrations of Al, Ba, Fe, Mg, Mn, Na, and Sr compared with the highest reference values, respectively. The mean concentrations of the determined metals (except for As, Co, Cr, Hg, and Mo) significantly varied among different age groups for both male and females. The results of correlation analysis and cluster analysis revealed that strong correlations were found between Al, Fe, Zn, Mg, and Na accumulated in human from the study area. These might suggest that Al, Ba, Fe, Mg, Mn, Na, and Sr were significantly derived from contamination of crocidolite asbestos fibers. Zn, Mg, and Na might also originate from diet. However, Cd, Mo, Co, As, Cr, Hg, Ni, Mn, Pb, and Ba accumulated in human seemed to be mainly derived from soil. It can be concluded that metals accumulated in human hair have a link with asbestos fiber contamination in the environment.     

Hair analysis for opiates: evaluation of washing and incubation procedures

Hair analysis of drugs of abuse has been a subject of interest from a clinical, social and forensic perspective for years because of the broad time detection window after intake in comparison to urine or blood. However, the correct and reliable interpretation of opiates findings in an authentic hair sample requires optimalisation and standardisation of decontamination and incubation procedures. Comparing various published methods, we have found some variability in them and no unequivocal recommended procedure for starting with a method directly. Therefore, various combinations of solvents, of various polarity, as washing solvents were tested for removing opiates from the external surface of real hair samples. The yields of opiates from these washings were compared with the yields from the interior of the hair matrix after digestion with various procedures. The opiates after digestion were cleaned up from resulting solution on extraction columns with mixed solid-phase and analysed by GC–MS in standard EI mode after silylation. The efficiencies of neutral (Söerensen buffer, pH 7.4), acid (0.1 M HCl) and basic (1 M NaOH) digestion of the hair matrix were evaluated and the relative recoveries for morphine, codeine, dihydrocodeine and hydrocodone were compared. As it is very problematic to imitate the reference hair sample with a specific amount of analytes incorporated inside, which can be used for calibration to get a close estimate of the quantities of analytes inside the solid authentic sample, the total digestion of a hair sample in basic medium was considered to be a very important reference basis for quantitative determinations. The ratios of hydrolysis of labile 6-acetylmorphine or acetylcodeine were tested and evaluated in practical routine conditions of acid or neutral digestion of hair. Comparing the three methods of incubation of authentic hair samples, the methods using 1 M NaOH or 0.1 M HCl yielded higher recoveries of total equivalents of morphine or codeine, whereas the incubation in Söerensen buffer allowed the reflection of real ratios of labile metabolites and/or parent compounds in an original sample. This method has been shown to be capable of detecting hydrocodone in hair with other opiates concomitantly and to indicate the drug abuse pattern of a person at various time intervals in the past.     

Ethylene promotes the induction by auxin of the cortical microtubule randomization required for low-pH-induced root hair initiation in lettuce (Lactuca sativa L.) seedlings

Transverse cortical microtubule (CMT) arrays in lettuce root epidermal cells randomize soon after a shift from pH 6.0 to pH 4.0, and this randomization is essential for root hair initiation. We investigated the hormonal regulation of CMT randomization. At pH 4.0, 1 micro M of the auxin competitive inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid (PCIB), 0.1 micro M of the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) or 0.1 micro M of the ethylene action inhibitor Ag(+) suppressed CMT randomization and root hair initiation. At pH 6.0, addition of 0.1 micro M indole-3-acetic acid (IAA) or 1 micro M of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) induced CMT randomization and root hair initiation. Culturing with 0.1 micro M IAA plus 0.1 micro M AVG, or 1 micro M ACC plus 1 micro M PCIB also induced these phenomena. ACC (1 micro M) plus 100 micro M PCIB inhibited CMT randomization and root hair initiation, but 1 micro M AVG with 0.1 micro M Ag(+) and 0.1 micro M IAA induced them. These results suggest that auxin is essential for CMT randomization. As a higher concentration of PCIB was required to suppress CMT randomization when ACC was added, the greater amount of ethylene produced at pH 4.0 may promote the induction by auxin of CMT randomization in hair-forming cells.     

Comparison and evaluation of the specificity and binding capacity of commercial and in house affinity columns used in sample preparation for analysis of growth-promoting drugs

Immunoaffinity chromatography (IAC) and affinity chromatography (AC) are widely used for extraction of drugs from biological samples. Fifteen column types were purchased from five different manufacturers and their ability to bind specific drugs including β-agonists and anabolic steroids over a range of analyte concentrations in fortified bovine urine samples was assessed. The performance data obtained from these columns were compared with columns produced in this laboratory (in house columns). The in house columns gave the highest recoveries, ranging from 92 to 100% at the 1 ng spiking concentration, for five of the seven analytes assessed. Forty percent (11 of 27) of all the commercial column assessments recorded recoveries of less than 50% even when the lowest spiking concentration was applied (1 ng). For one manufacturer, only one of seven different columns purchased delivered extraction efficiencies greater than 50%. The extraction efficiencies of the clenbuterol columns were the highest with all commercially prepared columns showing at least 50% binding of radiolabelled tracer. Recoveries of -nortestosterone were the lowest. The variability of these products with respect to quality control requires constant monitoring.     

Potential role for nonesterified fatty acids in beta-adrenoceptor-induced increases in brain tryptophan

We tested the hypothesis that beta2- and beta3-adrenergic receptor-mediated increases in brain tryptophan are due to the liberation of fatty acids, which in turn displace tryptophan from its albumin-binding site and thus facilitate its entry into the brain. Male CD-1 mice were injected with subtype-selective beta-adrenergic agonists 1h before brain samples were collected for analysis of tryptophan content by HPLC with electrochemical detection, and blood samples were collected for analysis of total and free tryptophan and nonesterified fatty acid (NEFA) concentrations. The beta2-selective agonist, clenbuterol (0.1 mg/kg), increased concentrations of tryptophan in all brain regions studied and decreased plasma total tryptophan, but had no effect on plasma free tryptophan or NEFAs. The beta3-selective agonists, BRL 37344 (0.2 mg/kg) or CL 316243 (0.01 mg/kg), increased brain tryptophan, plasma NEFAs and free tryptophan. Pretreatment with nicotinic acid (500 mg/kg), an inhibitor of lipolysis, almost completely prevented the increase in plasma free tryptophan and NEFAs, and attenuated the increase in brain tryptophan induced by CL 316243. These results suggest that beta2- and beta3-adrenergic agonists increase brain tryptophan by a mechanism other than the liberation of NEFAs. Nonetheless, beta3-adrenergic agonists appear to increase brain tryptophan by a mechanism that may depend partially on elevations of plasma NEFAs.     

Quantitative multi-residue determination of β-agonists in bovine urine using on-line immunoaffinity extraction-coupled column packed capillary liquid chromatography-tandem mass spectrometry

This report demonstrates the potential of on-line immunoaffinity extraction and coupled column packed capillary liquid chromatography-ion spray tandem mass spectrometry for multi-residue determination of five β-agonists, clenbuterol, mabuterol, mapenterol, methylclenbuterol, and tolubuterol, in bovine urine using an automated column switching system. Trace enrichment and preliminary sample cleanup was performed on-line using bovine urine diluted with phosphate-buffered saline. The column switching process involves trapping the target analytes onto a mini-bore immunoaffinity column, whereupon the target analytes are released from the immunoaffinity column onto a trapping column and subsequently eluted onto a packed capillary analytical column. The latter packed capillary column was used to provide the optimum sensitivity for ion spray LC-MS-MS analyses. The three-column system consists of a 2.0 mm I.D. immunoaffinity column, a 1 mm I.D. reversed-phase trapping column and a 320 μm I.D. packed capillary analytical column. Both qualitative and quantitative results are presented for the multi-residue determination of the target β-agonists from the complex urinary matrix. Using tolubuterol as an internal standard, the quantitative data showed good linear response within the concentration ranges studied. Lower levels of quantitation were 50 part per trillion (ppt) for clenbuterol and methylclenbuterol, 20 ppt for mabuterol and 10 ppt for mapenterol. The bovine renal elimination is described using the technique for one of the β-agonists, clenbuterol. The concentration of clenbuterol was detectable 15 days after the cessation of oral administration.