N,N-dimethyl-2-phenylpropan-1-amine quantification in urine: application to excretion study following single oral dietary supplement dose.

N,N-dimethyl-2-phenylpropan-1-amine (NN-DMPPA) is a new designer stimulant prohibited in sport in-competition according to the List of Prohibited Substances and Methods published by the World Anti-Doping Agency (WADA). The first published data on the excretion study of NN-DMPPA to support the knowledge of NN-DMPPA in routine anti-doping control have been presented. The reliable gas chromatography-mass spectrometry quantitative method (GC-MS) has been validated and applied to the excretion study of NN-DMPPA. The validation parameters of the GC-MS method for determination of NN-DMPPA in human urine were the linear calibration range of 100 to 7500 ng/mL, the LOD of 13.9 ng/mL and the LOQ of 42.2 ng/mL. According to the obtained repeatability, intermediate precision, and trueness, the applied GC-MS method was precise and accurate. Urine samples from three volunteers in the excretion study were collected for 5 days after single oral administration of the supplement NOXPUMP containing NN-DMPPA. The obtained results showed the maximum concentration of NN-DMPPA (189-303 ng/mL) in urine samples at a time of 2-3 h post-administration. The NN-DMPPA concentration in urine samples was higher than 50 ng/mL until 22-23 h after the dietary supplement ingestion. This means that according to the WADA rules the use of a supplement containing NN-DMPPA may be related to a positive case when athletes took this supplement in-competition. Moreover, excretion results demonstrate also that NN-DMPPA may be detected in urine samples by the applied GC-MS method till 46 h after supplement administration. Additionally, the excretion study of ß-methylphenethylamine as the second prohibited substance present in the supplement NOXPUMP has been investigated. Graphical Abstract Excretion study of new designer stimulant, N,N-dimethyl-2-phenylpropan-1-amine, and ß-methylphenethylamine following single oral NOXPUMP supplement dose.

Detection of ß-methylphenethylamine, a novel doping substance, by means of UPLC/MS/MS.

Novel substances of expected doping activity are constantly introduced to the market. ß-Methylphenethylamine (BMPEA) is classified as a doping agent by the World Anti-Doping Agency as it is a positional isomer of amphetamine. In this work, the development and application of a simple and rapid analytical procedure that enables discrimination between both isomers is described. The analytes of interest were extracted from urine by a two-step liquid-liquid extraction and then analyzed by UPLC/MS/MS under isocratic conditions. The entire analytical procedure was validated by evaluating its selectivity, discrimination capabilities, carry-over, sensitivity, and influence of matrix effects on its performance. Application of the method resulted in detection of BMPEA in eight anti-doping samples, including the first report of adverse analytical finding regarding its use. Further analysis showed that BMPEA may be eliminated unchanged along with its phase II conjugates, the hydrolysis of which may considerably improve detection capabilities of the method. Omission of the hydrolysis step may therefore, produce false-negative results. Testing laboratories should also carefully examine their LC/MS/MS-based amphetamine and BMPEA findings as both isomers fragment yielding comparable collision-induced dissociation spectra and their insufficient chromatographic separation may result in misidentification. This is of great importance in case of forensic analyses as BMPEA is not controlled by the public law, and its manufacturing, distribution, and use are legal.

Efficacy of photodynamic therapy in vulvar lichen sclerosus treatment based on immunohistochemical analysis of CD34, CD44, myelin basic protein, and Ki67 antibodies.

INTRODUCTION: Lichen sclerosus (LS) is a chronic skin and mucosa inflammatory disease. It affects mainly the female anogenital area especially in postmenopausal period. The main symptoms include pruritus, burning, pain, sometimes urinary problems, or difficulties in defecation. Usually, porcelain-white plaques are seen in the skin and mucosa. The etiology and pathogenesis of LS are still uncertain. There are some research studies on possible genetic predisposition, yet autoimmune, hormonal, or infectious factors are not excluded. The typical treatment of LS is mainly pharmacological, although the alternative treatment method used in LS is photodynamic therapy (PDT), which is noninvasive technique based on selective destruction of lesions. Our study is focused on molecule markers of vascularisation (CD34), nervous cell function (myelin basic protein [MBP]), keratinocyte function (CD44), and proliferation index (Ki67) in cases treated with photodynamic method. MATERIALS AND METHODS: A group of 100 patients treated in our department was included in the study. All 100 women had LS on the basis of clinical and histological criteria. All the subjects underwent PDT. In all cases, skin biopsies were taken before and after treatment, and samples were analyzed with CD34, CD44, MBP, and Ki67 antibodies using immunohistochemical staining. RESULTS: The study shows the high efficacy of PDT in LS treatment including beneficial changes to CD34, CD44, and MBP immunostained molecules. The Ki67 proliferation index did not change significantly. A significant increase of CD34 (microvessel density), MBP, and CD44 expression was confirmed in the histological images and in the partial or full remission of clinical objective and subjective symptoms. CONCLUSIONS: The PDT is a very effective therapeutic method in LS treatment.

Chlamydia trachomatis infection in women with lichen sclerosus vulvae and vulvar cancer.

OBJECTIVE: Chronic infections in the urogenital area often precede or coexist with vulvar cancer. A strong connection between some tumours and the-appearance of Chlamydia trachomatis infection has been observed, but there is little information concerning a connection of that infection with vulvar cancer and lichen sclerosus vulvae (LS). The aim of this study was the analysis of frequency of antigens appearance and antibodies of IgM and IgG Chlamydia trachomatis in patients with vulvar cancer and LS and we wanted to find the correlation between Chlamydia trachomatis infection and vulvar cancer and LS. METHODS: 80 women treated in the Clinic of Vulva Diseases at the Department and Clinical Ward of Gynaecology, Obstetrics and Oncological Gynaecology in Bytom, in the Silesian Medical University in Katowice were divided into two groups - 30 were treated for vulvar cancer and 50 were treated because of LS. We took bacterial smears vagina and cervical smears for presence of Chlamydia trachomatis antigens and peripheral blood to mark antibodies of IgM and IgG Chlamydia trachomastis. RESULTS: Chlamydia trachomatis antigen was found in 20% women with vulvar cancer and in 12% women with LS (p>0.05). In 13,3% cases with vulvar cancer we observed IgM Chlamydia trachomatis antibodies. In the group with LS IgM antibodies appeared in 16% women (p>0.05). In 50% patients with vulvar cancer in blood serum we observed IgG Chlamydia trachomatis antibodies, and in 16% women with LS (p<0.001). CONCLUSIONS: Previous Chlamydia trachomatis infection can lead to vulvar carcinogenesis.

Effectiveness of photodynamic therapy in the treatment of lichen sclerosus: cell changes in immunohistochemistry.

BACKGROUND: Vulvar lichen sclerosus (LS) affects primarily women at postmenopausal age and its background remains unknown. One of the treatment modalities is photodynamic therapy (PDT). The aim was to investigate the efficacy of PDT in women with LS and the analysis of protein expression before and after PDT. MATERIAL AND METHODS: From 04.2006-01.2008 28 women, with LS underwent photodynamic diagnosis and next PDT: six-courses every second week with using 5-aminolevulinic acid (ALA) as a photosensitizer. Punch biopsies were taken before and after treatment and immunohistochemistry was done with Ki67,CD44,CD34 and CD3. RESULTS: Before PDT all patients suffered from pruritus and after in 89.3% the relief was noted. The histological examination showed that 35.7% patients hadn't LS after therapy completion. Anti-CD44 staining intensities was scored qualitatively - there were no statistical difference at the expression of protein CD44 in the epidermis (p>0.05) before and after therapy. Microvessel density was assessed at the hot spots, marked with anti-CD34. Statistical difference in AVD before and after therapy: (p<0.05). The staining intensity of Ki-67 didn't differ before and after PDT (p>0.05). The expression of CD3 on T lymphocytes showed statistical difference of the lymphocytic infiltration before and after PDT ( p<0.05). CONCLUSION: The immunohistochemical staining in vulvar LS showed increasing microvessel density and decreasing lymphocytic infiltration. There were a clinical, and less histological improvement in patients with LS. We suggest that the photodynamic therapy is an effective, alternative treatment in some but not all patients with LS. Therefore, further studies are needed.

Simultaneous determination of ibuprofen and its metabolites in complex equine urine matrices by GC-EI-MS in excretion study in view of doping control.

A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 µg mL-1, 0.62 µg mL-1, 4.15 µg mL-1, 0.58 µg mL-1 and 4.04 µg mL-1 for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports.

Determination of designer doping agent--2-ethylamino-1-phenylbutane--in dietary supplements and excretion study following single oral supplement dose.

The quantitative analysis of a new designer doping agent, 2-ethylamino-1-phenylbutane (EAPB) and its metabolite, 2-amino-1-phenylbutane (APB) in urine samples, and the determination of EAPB in dietary supplement samples, have been presented. The main purpose of the present study was to develop simple and reliable gas chromatography-mass spectrometry method (GC-MS) for excretion study following a single oral administration of dietary supplements containing EAPB. Three analytical methods for the determination of EAPB in urine and supplement samples, and APB in urine samples using the GC-MS system, have been validated. The method of the determination of EAPB in supplement samples was applied to analyze seventeen dietary supplements, CRAZE and DETONATE. Two other methods were used to determine the urinary excretion profile of EAPB and APB in the case of three healthy volunteers and, on further investigation, it was applied to the anti-doping control in sport. Quantification was obtained on the basis of the ions at m/z 86, 58 and 169, monitored for EAPB, APB and diphenylamine (used as an internal standard), respectively. The limits of detection and quantification were 2.4 and 7.3µg/g for EAPB in the case of supplement analysis, 2.9 and 8.8ng/mL for EAPB in the case of urine analysis, and 3.2 and 9.7ng/mL for APB. The other validation parameters as linearity, precision and trueness have been also investigated with the acceptable results. The extraction yield of all presented methods was above 69%. EAPB was detected in fourteen analyzed supplements (not included EAPB in their labels) and its content varied between 1.8 and 16.1mg/g. Following oral administration of three supplements with EAPB to one male and two female volunteers, the parent compound of EAPB and its metabolite were monitored and the excretion parameters as the maximum concentration of the analyte in urine (2.2-4.2µg/mL for EAPB; 1.1-5.1µg/mL for APB) and the time for the maximum height of the excretion peak (2-8h and 22h in one case for EAPB; 20-22h and 4h in one case for APB) have been indicated. EAPB and APB were detected at the level above 50ng/mL (50% of the minimum required performance level for stimulants in the anti-doping control in-competition in sport) in the urine up to 46-106h and 58-120h, respectively. Additionally, the result of the anti-doping control during swimming competition of one athlete, whose urine sample was analyzed for stimulants and narcotics, has been presented. The qualitative and quantitative analyses of new designer agents in urine samples and the excretion studies of these substances are of a great importance in the anti-doping control in sport. Moreover, the presentation of detection examples of these agents in supplements that haven't got included an information about them in the labeling, make athletes (and other supplement customers) more and more aware of the risk of the supplement use and possible health and doping consequences.

N,N-dimethyl-2-phenylpropan-1-amine - new designer agent found in athlete urine and nutritional supplement.

Reports of new designer agents banned in sport being detected in supplements widely available for athletes are constantly emerging. The task of anti-doping laboratories is to control athletes for the presence of substances listed by the World Anti-Doping Agency (WADA) and those that are structurally/biologically similar to them. Recently, a new designer stimulant, N,N-dimethyl-2-phenylpropan-1-amine (NN-DMPPA), was detected by the WADA accredited anti-doping laboratory in Warsaw during routine anti-doping control. The urine samples from four athletes were analyzed in the screening method for stimulants and narcotics and the presence of NN-DMPPA was detected. The identity of NN-DMPPA was confirmed by gas chromatography-mass spectrometry using a synthesized reference standard. The measured concentrations of NN-DMPPA were between 0.51 and 6.51 µg/mL. The presence of the NN-DMPPA compound has been detected in the 'nutritional supplement' NOXPUMP that had been purchased in a store in Poland. NN-DMPPA at 121.7 µg/g was indicated in the investigated supplement together with another banned stimulant ß-methylphenethylamine. The presence of this new stimulant was not indicated on the labelling of the supplement, a situation which is not unusual within this market. Thus, it is important to make athletes aware of the risk related to the use of supplements. Moreover, specific legistation dealing with the commercialization of drugs banned for sport should be undertaken.

The prevalence of trimetazidine use in athletes in Poland: excretion study after oral drug administration.

Stimulants, together with anabolic androgenic steroids, are regarded as one of the most popular doping substances in sport. Owing to a great variety of these substances and new designer drugs being introduced to the market, each year the World Anti-Doping Agency (WADA) updates the list of substances and methods prohibited in sport. On 1 January 2014, a new doping agent - trimetazidine (TMZ) - was added to the WADA Prohibited List. TMZ, a substance prohibited in competition, is classified in the S6b Specified Stimulant Group. TMZ is used as a well-known cardiologic drug with confirmed biochemical and clinical activity. According to knowledge of the pharmacology and mechanism of TMZ action, TMZ can be used by athletes to improve physical efficiency, especially in the case of endurance sports. This study presents the phenomena of TMZ use by Polish athletes involved in anti-doping control in the WADA-accredited laboratory in Warsaw (Poland) between 2008 and 2013. Samples were taken from the athletes of such disciplines as cycling, athletics, and triathlon. Moreover, the elimination study of TMZ has been conducted to establish the change of TMZ concentration in urine sample after oral administration of a single or double (during the long-term therapy) dose. TMZ was monitored in urine samples by gas chromatography-mass spectrometry-nitrogen phosphorus detection (GC-MS-NPD).

In memory of Alfons Bukowski on the centenary of anti-doping research.

Alfons Bukowski (1858-1921) is commonly regarded as the pioneer of anti-doping research. In 1910, he developed a method to detect alkaloids in horse saliva. One hundred years later, this is a good moment to remember Bukowski, an outstanding Polish pharmacist, often mistakenly represented in world literature as a Russian chemist. It is also an occasion to mention that the real driving forces in the history of doping were events related to horse rivalry.