High-throughput screening for various classes of doping agents using a new 'dilute-and-shoot' liquid chromatography-tandem mass spectrometry multi-target approach.

A new multi-target approach based on liquid chromatography--electrospray ionization tandem mass spectrometry (LC-(ESI)-MS/MS) is presented to screen for various classes of prohibited substances using direct injection of urine specimens. With a highly sensitive new generation hybrid mass spectrometer classic groups of drugs--for example, diuretics, beta2-agonists--stimulants and narcotics are detectable at concentration levels far below the required limits. Additionally, more challenging and various new target compounds could be implemented. Model compounds of stimulant conjugates were studied to investigate a possible screening without complex sample preparation. As a main achievement, the integration of the plasma volume expanders dextran and hydroxyethyl starch (HES), commonly analyzed in time-consuming, stand-alone procedures, is accomplished. To screen for relatively new prohibited compounds, a common metabolite of the selective androgen receptor modulator (SARMs) andarine, a metabolite of growth hormone releasing peptide (GHRP-2), and 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) are analyzed. Following a completely new approach, conjugates of di(2-ethylhexyl) phthalate (DEHP) metabolites are monitored to detect abnormally high levels of plasticizers indicating for illicit blood transfusion. The assay was fully validated for qualitative purposes considering the parameters specificity, intra- (3.2-16.6%) and inter-day precision (0.4-19.9%) at low, medium and high concentration, robustness, limit of detection (1-70 ng/ml, dextran: 30 µg/ml, HES: 10 µg/ml) and ion suppression/enhancement effects. The analyses of post-administration and routine doping control samples demonstrates the applicability of the method for sports drug testing. This straightforward and reliable approach accomplishes the combination of different screening procedures resulting in a high-throughput method that increases the efficiency of the labs daily work.

Di(2-ethylhexyl) phthalate metabolites as markers for blood transfusion in doping control: intra-individual variability of urinary concentrations.

To indicate homologous or autologous blood transfusion in sports drug testing, quantification of increased urinary concentrations of di(2-ethylhexyl) phthalate (DEHP) metabolites presents a promising approach; however, the possible intra-individual variation of the metabolite concentrations over time has not been well characterized. The aim of this study was to explore the intra-individual variability of urinary DEHP metabolites among seven volunteers without special occupational exposure to DEHP during one week (n = 253) in order to investigate the possibility of increased urinary concentrations of the metabolites caused by, for example, residential, dietary, or environmental exposure. Quantification of three DEHP metabolites--mono(2-ethylhexyl) phthalate, mono(2-ethyl-5-oxohexyl) phthalate, and mono(2-ethyl-5-hydroxyhexyl) phthalate--was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography-tandem mass spectrometry. Although urinary concentrations of DEHP metabolites showed considerable intra-individual variation, no increased values were observed comparable to the concentrations measured in urine specimens collected after blood transfusion.

Determination of Vasopressin and Desmopressin in urine by means of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry for doping control purposes.

The anti-diuretic neurohypophysial hormone Vasopressin (Vp) and its synthetic analogue Desmopressin (Dp, 1-desamino-vasopressin) have received considerable attention from doping control authorities due to their impact on physiological blood parameters. Accordingly, the illicit use of Desmopressin in elite sport is sanctioned by the World Anti-Doping Agency (WADA) and the drug is classified as masking agent. Vp and Dp are small (8-9 amino acids) peptides administered orally as well as intranasally. Within the present study a method to determine Dp and Vp in urinary doping control samples by means of liquid chromatography coupled to quadrupole high resolution time-of-flight mass spectrometry was developed. After addition of Lys-Vasopressin as internal standard and efficient sample clean up with a mixed mode solid phase extraction (weak cation exchange), the samples were directly injected into the LC-MS system. The method was validated considering the parameters specificity, linearity, recovery (80-100%), accuracy, robustness, limit of detection/quantification (20/50 pg mL(-1)), precision (inter/intra-day<10%), ion suppression and stability. The analysis of administration study urine samples collected after a single intranasal or oral application of Dp yielded in detection windows for the unchanged target analyte for up to 20 h at concentrations between 50 and 600 pg mL(-1). Endogenous Vp was detected in concentrations of approximately 20-200 pg mL(-1) in spontaneous urine samples obtained from healthy volunteers. The general requirements of the developed method provide the characteristics for an easy transfer to other anti-doping laboratories and support closing another potential gap for cheating athletes.

Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing.

Methods of blood doping such as autologous and homologous blood transfusion are one of the main challenging doping practices in competitive sport. Whereas homologous blood transfusion is detectable via minor blood antigens, the detection of autologous blood transfusion is still not feasible. A promising approach to indicate homologous or autologous blood transfusion is the quantification of increased urinary levels of di(2-ethylhexyl) phthalate (DEHP) metabolites found after blood transfusion. The commonly used plasticizer for flexible PVC products, such as blood bags, is DEHP which is known to diffuse into the stored blood. Therefore, a straight forward, rapid and reliable assay is presented for the quantification of the main metabolites mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethylhexyl) phthalate that can easily be implemented into existing multi-target methods used for sports drug testing. Quantification of the DEHP metabolites was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography/tandem mass spectrometry. The method was fully validated for quantitative purposes considering the parameters specificity, linearity (1-250 ng/mL), inter- (2.4%-4.3%) and intra-day precision (0.7%-6.1%), accuracy (85%-105%), limit of detection (0.2-0.3 ng/mL), limit of quantification (1 ng/mL), stability and ion suppression effects. Urinary DEHP metabolites were measured in a control group without special exposure to DEHP (n = 100), in hospitalized patients receiving blood transfusion (n = 10), and in athletes (n = 468) being subject of routine doping controls. The investigation demonstrates that significantly increased levels of secondary DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion.

Comparison of extraction methods to monitor pesticide residues in surface water.

A regular monitoring program to study the pesticide concentration in surface waters has been carried out since 1976 in Hungary by the National Plant Protection Organization of the Ministry of Agriculture and Regional Development jointly with the Regional Water Authorities. At the beginning of this program a liquid-liquid partition method is used to extract the pesticides from water samples. After checking the pH value, one sample aliquot is extracted to analyze the basic and neutral compounds. Another aliquot is acidified to pH 2 and extracted to analyze acidic compounds. Disadvantages of this method are high solvent consumption and the need to apply solvents (methylene chloride and diethyl ether) that are harmful to human health. Therefore, the solid-phase extraction method has been introduced. This method has another advantage in that by using the vacuum manifold a number of samples can be extracted simultaneously depending on the capacity (number of ports) of the manifold. Three types of cartridges (LiChrolut EN, ISOLUTE ENV+, and Carbograph) are tested. The suitability and reproducibility of the extraction on various cartridges is studied and compared through recovery experiments. Recoveries are done for 22 active ingredients at spiking levels of 1-5 times the limit of determination (in the range of 0.05-2.5 microg/L) with each extraction method. Individual recovery values as well as average recoveries for all methods are between 70% and 100%, with the relative standard deviation generally below 20%. Carbograph is the only cartridge among those studied that can be used to extract both neutral and acidic compounds in one sample loading step using two different consecutive elution steps.