DNA typing for personal identification of urine after long-term preservation for testing in doping control.

When the tampering of a urine sample is suspected in doping control, personal identification of the sample needs to be determined by short tandem repeat (STR) analysis using DNA. We established a method for extracting DNA from urine samples stored at -20 °C without using any additives or procedures, which is consistent with how samples are required to be managed for doping control. The method, using the Puregene® Blood Core kit followed by NucleoSpin® gDNA Clean-up or NucleoSpin® gDNA Clean-up XS kit, does not need any special instrument and can provide a purified extract with high-quality DNA from up to 40 mL of urine suitable for STR analysis using an AmpFlSTR® Identifiler® PCR amplification kit. Storing urine at -20 °C is detrimental to the stability of DNA. The DNA concentration of preserved urine could not be predicted by specific gravity or creatinine level at the time of urine collection. The DNA concentration of a purified extract (10 µL) was required to be >0.06 ng/µL to ensure a successful STR analysis. Thus, the required extraction volumes of urine preserved for 3-7 years at -20 °C were estimated to be 30 mL and 20 mL to succeed in at least 86% of men and 91% of women, respectively. Considering the long half-life of DNA during long-term preservation, our extraction method is applicable to urine samples stored even for 10 years, which is currently the storage duration allowed (increased from 8 years) before re-examination in doping control. Copyright © 2016 John Wiley & Sons, Ltd.

20K-GH and its use in detecting GH abuse.

Following the successful production of recombinant 20K-GH, several studies investigating the physiology of this GH isoform have been undertaken. In this report, we review studies of its biological effect, measurement and secretion. To use serum 20K-GH level in detecting GH abuse, new method has been established and serum 22K-GH, 20K-GH were measured in normal subjects and athletes, and no abnormal results were found among athletes. Another study confirmed that serum 22K-GH increased remarkably and 20K-GH decreased following the exogenous administration of 22K-GH. The duration was relatively short, approximately 24-36h in our and other studies. The increase of the ratio, 22K-GH/20K-GH was the most suitable indicator of GH abuse. Studies supported by the WADA were undertaken in collaboration with an Australian Group. A new approach for the GH isoform assay by beads assay platform is being developed. It is concluded that the direct measurement of 20K-GH is a valid scientific approach, for detecting GH abuse, although the duration of the positive results is short. Our method will be useful in combination with the marker method, an out-of-competition test or test for target cases. Furthermore, its application for the doping test passport is considered to be a possible future strategy.

Newly identified steroid hormone in urine of patients with Cushing's syndrome: 3alpha,11 beta-dihydroxy-4-androsten-17-one.

We have previously reported that the urine of patients with Cushing's syndrome, including pituitary adenoma cases and adrenal adenoma cases, consistently show a conspicuous peak in the chromatographical analysis of 17-ketosteroid fraction but not in the urine of control subjects. The substance emerges just before 11beta-hydroxy-androsterone (11beta-OH-A) in capillary gas chromatography. In the present study, we have identified an "unknown peak substance" observed in the urine of Cushing's syndrome patients using gas chromatography-mass spectrometry (GC/MS). Trimethylsilylether (TMS)-derivative of the substance was found to have a molecular weight (MW) of 448, which is similar to that of 11-OH-A (MW: 450). From these findings, we hypothesized that the substance had the structure of a C-19 steroid with two hydroxyl groups at positions C-3 and C-11, one keto-group at C-17 and a double bond between C-4 and C-5 of the A ring. We hypothesized that the unknown peak substance was 3alpha,11beta-dihydroxy-4-androsten-17-one (3alpha,11beta-DH-A). To confirm this speculation we synthesized 3alpha,11beta-DH-A and compared the elution pattern of it with that of the "unknown peak substance" using GC and GC/MS. We found that both substances were indistinguishable by GC and GC/MS analysis. These results suggest that the unknown substance observed in the urine of patients with Cushing's syndrome is 3alpha,11beta-DH-A.

[Drug of abuse].

Recent development of various dietary supplements after enforcement of "Dietary Supplement Health and Education Act of 1994 (DSHEA)" in the USA enabled better availability of the products through the Internet in Japan as well. Because of differences in the definitions of the term "dietary supplement" and drug control laws between the USA and Japan, health risks due to uncontrolled use of a drug-based foreign dietary supplement without a medical doctor's advice, and side effects due to co-administration of any problematic supplements with prescription drugs has become a problem in Japan. Classes of typical dietary supplements, the method of distribution, and known problems during use or overuse of these products with prescription drugs are discussed. Several recent positive cases are known to be due to the use of contaminated food supplements, which were sold not only to athletes but also to the general public as memory enhancing or anti-aging drugs. These phenomena indicate that trends in drug use in sports and in society becoming increasingly similar.

Automated headspace solid-phase microextraction and in-matrix derivatization for the determination of amphetamine-related drugs in human urine by gas chromatography-mass spectrometry.

An automated extraction and determination method for the gas chromatography (GC)-mass spectrometry (MS) analysis of amphetamine-related drugs in human urine is developed using headspace solid-phase microextraction (SPME) and in-matrix derivatization. A urine sample (0.5 mL, potassium carbonate (5 M, 1.0 mL), sodium chloride (0.5 g), and ethylchloroformate (20 microL) are put in a sample vial. Amphetamine-related drugs are converted to ethylformate derivatives (carbamates) in the vial because amphetamine-related drugs in urine are quickly reacted with ethylchloroformate. An SPME fiber is then exposed at 80 degrees C for 15 min in the headspace of the vial. The extracted derivatives to the fiber are desorbed by exposing the fiber in the injection port of a GC-MS. The calibration curves show linearity in the range of 1.0 to 1000 ng/mL for methamphetamine, fenfluramine, and methylenedioxymethamphetamine; 2.0 to 1000 ng/mL for amphetamine and phentermine; 5.0 to 1000 ng/mL for methylenedioxyamphetamine; 10 to 1000 ng/mL for phenethylamine; and 50 to 1000 ng/mL for 4-bromo-2,5-dimethoxyphenethylamine in urine. No interferences are found, and the time for analysis is 30 min for one sample. Furthermore, this proposed method is applied to some clinical and medico-legal cases by taking methamphetamine. Methamphetamine and its metabolite amphetamine are detected in the urine samples collected from the patients involved in the clinical cases. Methamphetamine, amphetamine, and phenethylamine are detected in the urine sample collected from the victim of a medico-legal case.