Post-administration Samples Research Articles

Mass spectrometric determination of gonadotrophin‐releasing hormone (GnRH) in human urine for doping control purposes by means of LC–ESI‐MS/MS

The decapeptide gonadotrophin-releasing hormone (GnRH) is endogenously produced in the hypothalamus and secreted into the microcirculation between hypothalamus and pituitary gland. Here, the bioactive hormone is responsible for the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the systemic circulation. Because an intermittent application of exogenous GnRH in young males increases the testosterone plasma level by stimulation of the Leydig cells, the potential misuse of the administered substance offers a reasonable relevancy for doping controls and is prohibited in accordance to the list of banned substances of the World Anti-Doping Agency (WADA). The presented method provides a mass spectrometric approach to determine the nondegraded hormone in regular doping control samples by utilizing a sample preparation procedure with solid phase extraction, immunoaffinity purification and a subsequent separation by liquid chromatography with ESI-MS/MS detection. For liquid chromatography/mass spectrometry two alternative instrumental equipments were tested: the first consisted of an Agilent 1100 liquid chromatograph coupled to an Applied Biosystem Q Trap 4000 mass spectrometer, the second equipment was assembled by a Waters Aquity nano-UPLC coupled to a Thermo LTQ Orbitrap high resolution/high accuracy mass spectrometer. In urine specimens provided from healthy volunteers GnRH was not detected in accordance to the recent literature, but in postadministration samples urinary concentrations between 20 to 100 pg/ml of the intact peptide were determined. The method offered good validation results considering the parameter specificity, linearity (5-300 pg/ml), limit of detection (LOD, approx. 5 pg/ml), precision (inter/intraday, < 20%) and accuracy (105%) using Des-pGlu(1)-GnRH as internal standard to control each sample preparation step.

Analysis of iridoids from Harpagophytum and eleutherosides from Eleutherococcus senticosus in horse urine

LC/ESI-MS n methods have been previously set up to detect the administration of (i) Harpagophytum and (ii) preparations containing a plant capable of anti-stress properties: Eleutherococcus senticosus. Harpagoside has been found to be the main indicator of Harpagophytum administration in the horse. These methods have been applied to a large number of horse urine samples of various origins. Regarding the detection of Harpagophytum administration, harpagoside, harpagide and 8-para-coumaroyl harpagide were detected together in only one sample out of 317. Eleutheroside E was found to be the main indicator of Eleutherococcus senticosus administration. It was detected in post-administration samples collected from two horses having received a feed supplement containing Eleutherococcus senticosus for several days. Out of the 382 samples tested, eleutheroside E was found in an unexpected large number of urine samples (39%) of various origins and its presence cannot be only due to the sole use of herbal dietary supplements.

Effects Of Ceftriaxone On Haematological And Biochemical Parameters Of Turkey

Short-term effects of ceftriaxone on haematological and biochemical parameters of Nigerian local turkey poults were studied. The pre-treatment blood and serum samples were collected and the weight of animals taken before the administration of body weight for a period of 4 days. The animals were weighed daiy. The results showed that eosinophilia was significantly increased (P < 0.05) and total bilirubin deceased significantly (P < 005). Furthermore, there was significant decrease in chloride ion (P < 005) and increase in bicarbonate ion (P < 0.05). Oher indices o haematology, liver function test and electrolyte titration were normal (P < 0.05). Ceftriaxone caused eosinophilia in treated samples (2.2 +-0.45a) as compared to pre-treated samples (1.6 +- 0.89b). Total bilirubin in the post-administration samples (135 + 1.05a) was decreased in comparison with pre-administration samples (14.82 +- 0.72b). Chloride ion decreased in the treatedsampes (86.6 +- 8.11a) when compared with untreated samples (98.4 +- 2.88b). Bicarbonate ion increased (24.8 +- 1.79a) in the experimental samples when compared to control (24.4 +- 1.34b).Conclusively, the short term administraton of ceftriaxone may cause eosinophilia, hypobilirubinaemia, hypochloraemia and increased bcarbonate ion which may be positive response to hypochloraemia. Keywords: Haematology, Biochemical Parameters, Ceftriaxone, TurkeyAnimal Research International Vol. 3 (3) 2006 pp. 562-565

Rapid screening of polysaccharide‐based plasma volume expanders dextran and hydroxyethyl starch in human urine by liquid chromatography–tandem mass spectrometry

The increasing number of samples and target substances in doping control requires continuously improved screening methods, combining high-throughput analysis, simplified sample preparation, robustness and reliability. Hence, a rapid screening procedure based on liquid chromatography-electrospray ionization-tandem mass spectrometry with in-source collision-induced dissociation was developed. The detection of the polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch (HES) in human urine was established without further sample preparation. The in-source fragmentation strategy of the approach represented a valuable tool in the analysis of the polysaccharide-based compounds, allowing the use of tandem mass spectrometry. After direct injection of urine specimens, analytes were chromatographically separated on a monolithic reverse-phase column and detected via multiple reaction monitoring of diagnostic ions at detection limits of 10 microg/mL for HES and 30 microg/mL for dextran. Validation was performed regarding the parameters specificity, linearity, precision (8-18%) and accuracy (77-105%) and the method was applied to the investigation of approximately 400 doping control samples and seven dextran and two hydroxyethyl starch post-administration samples. The approach demonstrated its capability as a rapid screening tool for the detection of dextran and hydroxyethyl starch and represents an alternative to existing screening procedures since time consuming hydrolysis or derivatization steps were omitted.

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.

Determination of IGF-I in horse plasma by LC electrospray ionisation mass spectrometry

The insulin-like-growth factor (IGF-I) peptide is considered to be the main indirect marker for growth hormone administration (GH) in a horse. Further to a previous investigation on measurement of IGF-I in plasma samples by mass spectrometry, this study focuses on quantitative and qualitative analysis of intact IGF-I in horse plasma. First, protein-transposing software has been developed for IGF-I to facilitate its quantification by HPLC-electrospray-ion-trap mass spectrometry. Second, product-ion scan experiments on IGF-I have been conducted on standard samples, non-fortified equine plasma samples, fortified plasma samples, and equine GH post-administration samples. This "top-down" approach method enables characterisation of fragment ions corresponding to the carboxy terminal end, which can be useful for the confirmation of the presence of IGF-I in plasma samples.

Detection of the Administration of Human Erythropoietin (HuEPO) to Canines

Recombinant human erythropoietin (rHuEPO) doping is prohibited in animal (canine and equine) sport. The effectiveness of a range of immunoassay screening methods for the detection of rHuEPO in canine urine was evaluated. The excretion profiles following rHuEPO administration to dogs were investigated. The presence of rHuEPO in postadministration samples was confirmed using the World Anti-Doping Agency (WADA)-approved isoelectric focusing immunoblotting confirmatory technique. Following the administration study, a screening program involving approximately 6000 greyhound sport (mostly racing) samples was undertaken for rHuEPO. This resulted in the detection of the first rHuEPO positives in the world of canine or equine sport. In an additional case, endogenous HuEPO was detected in a sample submitted as greyhound urine. It was determined that this arose from the submission to control stewards, as greyhound urine, of a substance that was, in fact, human urine. This was a particularly welcome development as definitive confirmatory evidence of such sample switching can be difficult to obtain in the case of greyhounds.

The Effect of Volume Replacement on Serum Protein Concentration During Cardiopulmonary Bypass

Although controversy exists concerning the optimal total protein and colloid osmotic pressure that should be maintained during cardiopulmonary bypass (CPB), the primary volume expanders remain albumin and 6% hetastarch. The purpose of this study was to quantify the effect of adding boluses of volume replacement agents under various conditions to total serum protein values during CPB. A standard CPB circuit was utilized in eight 45-kg swine that had a priming volume (physiologic saline solution) of 2309 ± 245 mL. Volumetric alterations occurred throughout the CPB period by the addition of combinations of physiologic saline solution, 6% hetastarch or 5% swine albumin. Pre- and postadministration samples were assayed for total serum protein, total protein, and albumin throughout the CPB period and at pre- and postvolume administration times. There was a significant decline in total serum protein with the initiation of CPB (6.14 ± 0.49 g/dL vs. 3.40 ± 0.43 g/dL, p &lt;.0001). Addition of 12.5 g of swine albumin (N = 5) to two different swine increased total serum protein significantly when compared to adding 500 mL of 6% hetastarch (N = 6) (swine albumin 12.4 ± 6.3% vs. hetastarch 3.3 ± 2.1%, p &lt;.005). A reduction in total serum protein occurred after hemodilution with varying amount of physiologic saline solution: 250–450 mL (7.4 ± 4.5%), 451–650 mL (9.6 ± 5.6%), and 651–1050 mL (19.4 ± 4.0%). In summary, knowledge of total serum protein concentration and estimated circulating blood volume can be used to guide albumin and hetastarch administration following hemodilution.

Intravascular coagulation phenomena associated with prevalent fall in fibrinogen and plasminogen during L-asparaginase treatment in leukemic children.

12 children affected by acute lymphoblastic leukemia had higher baseline levels of Normotest, Antithrombin III activity (AT III:A) and antigen (AT III:Ag) than those found in the control group, due to increased liver protein synthesis, as shown by the higher values of 305llbumin. Treatment with L-asparaginase (L-Asp) induced a marked decrease in fibrinogen and plasminogen and only a slight but significant reduction in AT III:A, AT III:Ag, protein C and alpha 2 antiplasmin levels. Platelet counts progressively increased. Each single L-Asp administration acutely induced (as recorded in the 2-hour postadministration samples) a significant increase in fibrinopeptide A values and reduction in several factors and inhibitors, due to activation of coagulation and consumption phenomena. It is suggested that these acute changes, but not the marked and selective reduction in fibrinogen and plasminogen, were partly compensated during L-Asp treatment by an overall increase in clotting and nonclotting protein synthesis in the liver.