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- ItemInvestigation of possible interference of a metabolite of etoricoxib (Arcoxia®) in the detection of 16a-hydroxyprednisolone by LC-MS/MS(2014) Saardpun N; Panan S; Wilairat P; Chaikum N; Mahidol University. National Doping Control CentreEtoricoxib (Arcoxia®) is a recent drug for the effective treatment of pain, inflammation and fever. It is available without prescription in Thailand but may require a physician’s prescription in other countries. This investigation was initiated by the presence of a large peak in about 10% of the MRM chromatograms of our screening procedure for corticosteroids by LC-MS/MS. This peak was in the time window of the MRM transition for 16a-hydroxyprednisolone. Fortunately for some of these samples the data sheet for previous use of medication stated the use of Arcoxia®. To confirm that the interfering peak was due to a metabolite of the drug etoricoxib (Arcoxia®) an excretion study of a male volunteer who had taken a 30-mg single oral dose of etoricoxib was performed. Metabolites of the drug were identified by comparison of the observed mass spectra (both full scan and product ion scan) with that reported in the literature. It was shown that the interference peak was 6’-hydroxymethyl-etoricoxib. The mass spectrum information of this metabolite was then used to modify the MRM transition for 16a-hydroxyprednisolone in the screening method.
- ItemDetection of Glycerol in Human Urine Using the Screening Procedure for Hydroxyethyl Starch and Dextran(2014) Tikum N; Kaewklum S; Wilairat P; Chaikum N; Mahidol University. National Doping Control CentreSince 2010 glycerol has been included in the WADA list of prohibited substances in the class S5 (Diuretics and Other Masking Agents). Starting in 2013 it has been designated as a Threshold Substance, with a threshold value of 1 mg/mL. The procedure for the detection of glycerol is very similar to that for the plasma expanders, hydroxyethyl starch (HES) and dextran. Namely it involves the aliquoting of a small volume (20 µL) of urine sample, acid digestion and drying. All three compounds are then derivatized with MSTFA/NH4 I/2-mercaptoethanol (1000:2:6, v/w/v) and separated by GC-MS. The inclusion of glycerol in the screening procedure for HES and dextran requires a modification of the GC temperature program. The effect of the acid digestion step was checked to show that it had no effect on the stability of glycerol. Combining all three compounds into one procedure leads to a saving of analysis time and equipment. This method has been applied to urine samples receieved by the Centre during the period April to June 2013.
- ItemDetection of deacetylmetipranolol arising from storage of standard solution of metipranolol(2015) Inthong T; Matayatsuk Phechkrajang C; Wilairat P; Chaikum N; Mahidol University. National Doping Control CentreMetipranolol is a beta-blocker in the WADA list of substances prohibited in particular sports. It is converted to deacetylmetipranolol by the first pass metabolism and the parent compound is not excreted. A standard methanolic solution of metipranolol can be converted to deacetylmetipranolol during prolonged storage at -20°C. Spiking blank urine with this aged standard can lead to misinterpretation of the form of metipranolol in a suspected urine sample since the normal GC-MS screening procedure for derivatized beta-blockers cannot separate this metabolite from the parent compound. A specific Ge-temperature program is required. However a LC-MS/MS method can separate metipranolol and its metabolite.
- ItemIncidence of Mitragynine from urine sample at NDCC Bangkok(2016) Kaewklum M; Kaewklum S; Pootrakronchai R; Kusamran T; Wilairat P; Chatsiricharoenkul S; Kongpatanakul S; Mahidol University. National Doping Control CentreMitragynine is one of the main alkaloids found in leaves of Mitragyna speciosa Korth (Thai name "Kratom"), a tropical medicinal plant in Southeast Asia. Kratom is illegal as a drug of abuse because of its stimulating and narcotic effect, but increasingly found misused as a recreational drug, especially in the southern region of Thailand and the northern states of Malaysia. These drugs are widespread used and easily purchased. In contrast to many EU countries such as Denmark or Sweden, mitragynine and its derivative 7-hydroxymitragynine are psychoactive substances with legality. In the U.S., kratom is on "drug of concern" list. Since 2014, WADA has included mitragynine in its list of in-competition monitoring substances, due to potential misuse in sports. At NDCC Bangkok, in previous analysis, we found mitragynine in athletes urine. Mitragynine desmethyl ester has been observed as the main metabolite of mitragynine and identified by comparison with pure compound by GC/MS. Currently we are cooperating with WADA as volunteer laboratory. From our routine screening, mitragynine and mitragynine desmethyl ester were detected in 11 samples from 1,400 urine samples in various sport disciplines using LC-MS/MS.
- ItemMethod validation for routine screening of 145 WADA prohibited substances by LC-MS/MS(2016) Pootrakronchai R; Inthong T; Kaewklum M; Saardpun N; Panan S; Kusamran T; Kongpatanakul S; Mahidol University. National Doping Control CentreLC-MS/MS has been recently a technique essential for analysis of doping substances and currently implemented in all WADA accredited laboratories. The protocols employed for sample preparation and equipment set up vary among different laboratories. In this study, a method has been developed and implemented for analysis of 145 substances in the WADA prohibited list by using the liquid chromatography/triple quadrupole mass spectrometry and multiple reaction monitoring/ electrospray ionization in positive and negative ion modes. The prohibited substances in urine sample were initially prepared by two consecutive liquid-liquid extractions (LLE) with methyl tert- butyl ether (MTBE)(pH 9 and 3) following enzyme hydrolysis. The extracted samples were separately analyzed on two different reverse-phase columns, C-18 column for diuretics, narcotics, beta-blockers, beta2-agonists, corticosteroids, anabolic steroids, aromatase inhibitors, SERM and C-8 column for stimulants. The method validation demonstrated the limit of detection (LOD 0.1-100 ng/ml), and retention time reproducibility (CV% < 1) were appropriate for routine screening the presence of WADA prohibited substances.
- ItemDetection of Ethyl Glucuronide (EtG) in Human Urine Using GC-MS Procedure at NDCC for Plasma Volume Expander(2017) Poohribuncha B; Lohwithee S; Kaewklum W; Joengklinjantana N; Pootrakronchai R; Wilairat P; Kongpatanakul S; Mahidol University. National Doping Control Centre
- ItemPreliminary Study Determination of the Basal concentration of di-(2-ehtylhezyl) phthalates metabolites in urine of thai population by liquid chromatography tandem mass spectrometry(2017) Viriyakhasem N; Kaewklum M; Seetapun S; Wilairat P; Chatsiricharoenkul S; Kongpatanakul S; Mahidol University. National Doping Control Centre
- ItemExperience with LC-MS-MS analysis for routine screening at NDCC(2017) wiriyakosol N; Pootrakronchai R; Inthong T; Panan S; Kusamran T; Wilairat P; Kongpatanakul S; Mahidol University. National Doping Control Centre
- ItemUrinary Luteinizing Hormone (LH) Levels in Male Athletes during 3 – Years Routine Doping Control Analysis at the NDCC Bangkok(2018) Asawesna C.; Nimsoongnern S; Intong T; Seetapun S; Kusamran T; Kongpatanakul S.; Mahidol University. National Doping Control CentreLuteinizing hormone (LH) is a peptide hormone produced by gonadotropic cells from the pituitary gland, and stimulates production of endogenous testosterone by testicular Leydig cells. The LH secretion is suppressed, by negative control mechanism, with high levels of testosterone in the circulating blood. The LH hormone has been included in the World Anti-Doping Agency (WADA) Prohibited List in ClassS.2 as a peptide hormone whose use is prohibited only for male athletes. This study is aimed to provide statistical evaluation of urinary LH levels from male athletes in sport doping control samples determined during January 2014 – December 2016 at the National Doping Control Centre (NDCC), Thailand. A total of 5,699 urine samples were analyzed by IMMULITE® 1000 LH Chemiluminescent Immunometric Assay Kit (Siemens Healthcare, USA) using the Multi-Level Lyphochek® Immunoassay Plus (BIO-RAD, USA)as the assay control. The in-house validation method has a precision of 4.69 %RSD with the limit of detection (LOD) at 0.15 IU/L. The mean LH concentration from the total urine samples analyzed was 10.34 (range 0.06 – 166.67) IU/L from which lower than 1IU/L and higher than 50 IU/L were found in 423 (7.42 %) and 24 (0.52 %) samples, respectively. Furthermore, 15 samples were found to contain LH concentration over 60 IU/L with the numbers declining from 9 (0.36 %) to 5 (0.28 %) and 1 (0.07 %) during 2014, 2015 and 2016 respectively, with no indication of unusual steroid profile. The concentrations of T, E and the T/E ratios of all samples were not significantly out of the WADA guideline normal ranges and showed no correlation with the urinary LH levels. The results provided an additional information for further considering of the ATF findings for the hormone level.
- ItemApplication of LC/MS – IT – TOF for Screening of WADA Prohibited Doping Peptides(2018) Saardpun N; Asawesna C; Wilairat P; Kusamran T; Kongpatanakul S; Mahidol University. National Doping Control CentreGrowth hormone releasing peptides (GHRPs) are synthetic substances which stimulate growth hormone (GH) secretion and are clinically used for the treatment of GH deficiency. GHRPs and other short peptides have been included since March 2016 in World Anti-Doping Agency (WADA) Prohibited List in class S2 as they enhance athletic performance. The purpose of this work was to develop the use of LC/MS-IT-TOF for screening of GHRPs and related substances. Solid phase extraction with a weak-cation exchange (WCX) cartridge was employed for sample preparation of 11 target peptides (GHRP-1, GHRP-2, GHRP-4, GHRP-5, GHRP-6, TB500, Desmopressin, Ipamorelin, Hexarelin, Anamorelin and Alexamorelin). The combined elutions of methanol (Elution 1) and 5% formic acid in methanol (Elution 2) were evaporated under vacuum and the residue reconstituted in water: acetonitrile (90:10, v/v) mixture. Chromatographic separation and mass characterization were performed using a Hypersil Gold C18 column (50 mm, 2.1 mm, 3m) and the Shimadzu LCMS-IT-TOF mass spectrometer. Gradient elution was from 5% to 100% acetonitrile at a flow rate of 0.3 ml/min with total run time of 18 min. Method validation was performed with optimized parameters in the positive mode. The assay limit of detection was 0.2-1.0 ng/mL and the recovery ranged from 9.1 to 53.6%. The instrument precision at 2 ng/mL was 3.2-14.6% RSD. This developed method demonstrates the application of LC/MS-IT-TOF for screening of the WADA prohibited short peptides. Furthermore, modifications of the procedure can be applied for analysis of other prohibited peptides and metabolites.