HPLC-MSn法鉴定葫芦巴碱及其在大鼠体内的主要代谢产物

目的建立快速灵敏的LC-MSⁿ检测葫芦巴碱及其在大鼠体内代谢物的分析方法。方法以葫芦巴碱对LC-MS²色谱及质谱条件进行优化，分析其电喷雾质谱的一级电离规律和多级质谱裂解规律，以此作为葫芦巴碱大鼠体内代谢物分析鉴定的依据。健康大鼠尾静脉注射8 mg·kg^-1葫芦巴碱，收集0～48 h的尿样，经C₁₈小柱固相萃取分离纯化后，直接采用LC-MSⁿ方法对尿样进行测定。结果根据生物体内药物代谢转化规律及母体药物的色谱-质谱行为规律，在尿样中鉴定出母药及其N-去甲基、N-去甲基环氧化产物，以及母药及其N-去甲基环氧化物的甘氨酸轭合物。结论本方法灵敏、快速、选择性高、专属性好，可用于葫芦巴碱的代谢产物研究。     

人血浆中O-去甲右美沙芬的测定及药代动力学研究

目的建立直接测定人血浆中O-去甲右美沙芬的方法,并应用于药代动力学研究。方法18名健康受试者单剂量po氢溴酸右美沙芬60 mg后,血浆样品经液-液萃取,通过液相色谱-质谱-质谱联用法测定其活性代谢物O-去甲右美沙芬的浓度,用非室模型计算药代动力学参数。结果O-去甲右美沙芬测定的线性范围为0.2～80 μg·L^-1;其主要药代动力学参数T_max为(2.1±0.7) h,C_max为(14±8) μg·L^-1,T_1/2为(3.8±1.8) h,用梯形法计算,AUC_0-t为(60±37) μg·h·L^-1。结论该法灵敏度高,操作简便,可直接测定活性代谢物,适用于右美沙芬的临床药代动力学研究及制剂的生物等效性评价。     

UHPLC-MS/MS法测定依那普利氢氯噻嗪复方制剂中潜在基因毒性杂质

目的 通过合成基因毒性杂质N-亚硝基氢氯噻嗪（NO-HZCT），建立超高效液相色谱-串联质谱法（UHPLC-MS/MS）测定依那普利氢氯噻嗪制剂中N-亚硝基氢氯噻嗪。方法 参考文献方法合成NO-HZCT，采用高分辨质谱对其相对分子量和结构进行确定；采用Agilent EclipsePlus C₁₈ RRHD（3.0 mm×150 mm，1.8 μm）色谱柱，以10 mmol•L^-1甲酸铵-0.1%甲酸的水溶液作为流动相A，以0.1%甲酸的乙腈溶液作为流动相B，梯度洗脱，体积流量0.6 mL•min^-1；采用ESI离子源正离子扫描，多反应监测（MRM）模式下，对NO-HZCT进行定量检测。结果 NO-HZCT质量浓度在0.51~50.67 ng·mL^-1范围内具有良好的线性关系，相关系数（r）为0.999 7；低、中、高3个浓度的加样回收率（n=3）分别为93.10%（RSD 3.7%）、104.30%（RSD 1.0%）和106.48%（RSD 1.8%）；检测限和定量限分别为0.08 ng•mL^-1和0.27 ng•mL^-1，3批样品中均检测出NO-HZCT。结论 该方法灵敏度高、专属性强，可准确地对依那普利氢氯噻嗪制剂中遗传毒性杂质NO-HZCT进行定量检测，可为依那普利氢氯噻嗪制剂的质量控制提供参考，保障药品质量安全。     

HPLC-MS/MS法测定人血浆中的左西孟旦及其主要代谢物

建立快速、 灵敏、 易操作的LC-MS/MS法测定人血浆中的左西孟旦及其代谢物OR-1855和OR-1896的浓度。根据待测物的不同性质， 采用两套液相色谱系统和电离方式分别测定人血浆中的左西孟旦和代谢物OR-1855、 OR-1896。测定左西孟旦时， 用瑞舒伐他汀为内标， 血浆样品经甲醇沉淀蛋白， 以甲醇-15 mmol·L^-1醋酸铵-甲酸(55∶45∶0.02， v/v/v)为流动相， Capcell MG III C₁₈柱(35 mm×2.0 mm ID， 3 μm)进行分离， 采用电喷雾电离源，以选择反应监测(SRM)方式进行负离子检测。测定代谢物OR-1855和OR-1896时， 用多索茶碱为内标， 血浆样品经乙酸乙酯萃取， 以甲醇-15 mmol·L^-1醋酸铵-甲酸(65∶35∶0.1， v/v/v)为流动相， Zorbax Extend C₁₈柱(150 mm×4.6 mm ID， 5 μm)进行分离， 采用电喷雾电离源， SRM方式进行正离子检测。测定血浆中左西孟旦方法的线性范围为0.10～50.0 ng·mL^-1， 定量下限可达0.10 ng·mL^-1； 测定血浆中代谢物OR-1855和OR-1896方法的线性范围均为0.20～100 ng·mL^-1， 定量下限均可达0.20 ng·mL^-1。本方法专属性好， 准确、 快速， 适用于左西孟旦注射液的临床药代动力学研究。     

高效液相色谱-电喷雾质谱法研究人尿中α-生育酚的主要代谢产物

研究α-生育酚在人尿中的主要代谢物。选择健康受试者5名连续7 d单剂量口服维生素E 250 mg，d 7收集0～6 h尿样，经C₁₈小柱固相萃取(SPE)分离纯化后，直接采用液相色谱-质谱联用方法(LC-MSⁿ)对尿样进行测定。在尿样中鉴定出4个主要代谢物，它们分别为α-生育酸、2,5,7,8-四甲基-2-(2′-羧乙基)-6-硫酸酯苯并二氢吡喃、 γ-生育酸内酯和2,5,7,8-四甲基-2-(4′,8′,12′-三甲基-12′-羧十二烷基)-6-硫酸酯苯并二氢吡喃。该方法灵敏、选择性高，为深入研究α-生育酚在人体内的代谢规律提供了可靠的方法。     

罗红霉素在犬体内的代谢转化罗红霉素在犬体内的代谢转化

目的考察罗红霉素在犬体内的代谢转化及po和iv给药途径对药物代谢的影响。方法采用液相色谱-质谱(LC-MSⁿ)联用技术,检测在iv或po给予单剂量罗红霉素后犬胆汁中的罗红霉素及其代谢物。代谢物经LC/MSⁿ方法分离和分析,并通过与对照品比较质谱和色谱行为确定其结构。结果共检测到13种罗红霉素代谢物,包括N-去甲基和N, N-双去甲基衍生物、肟醚侧链O-去烷醚基衍生物、脱红霉糖衍生物、罗红霉素及其代谢物的Z式几何异构体衍生物。结论罗红霉素在犬体内主要经历4种代谢途径;罗红霉素及其代谢物的几何异构与脱红霉糖代谢在口服和注射两种给药途径间存在显著的差异。     

外周血RANKL/OPG比值在类风湿关节炎合并股骨头坏死患者中的意义

目的 探讨外周血RANKL和OPG水平的变化在类风湿关节炎(RA)患者合并股骨头坏死(ONFH)中的意义。方法 回顾性分析2010年1月至2013年10月住院的644例RA患者各项临床及实验室指标,并选择同期体检的158例健康者为对照组,所有观察对象均行骨盆平片检查。对比分析两组观察对象股骨和腰椎骨密度及外周血RANKL和OPG水平。结果 RA患者中骨质疏松(OP)和ONFH发生率明显高于对照组(χ²=31.92,P<0.001;χ²=8.71,P=0.003);伴ONFH的RA患者外周血RANKL水平[214.50(384.05%) vs 82.67(78.86%)]、RANKL/OPG比值(0.41 vs 0.27)高于无ONFH的RA患者(P<0.05),且病程、健康状况问卷(HAQ)评分和双手关节Sharp评分均明显高于无ONFH的RA患者(P<0.001)。服用糖皮质激素(GC)的RA患者其ONFH发生率明显高于未服用GC组(χ²=20.62,P<0.001)。logistic regression多元回归结果显示,服用GC、总股骨区OP的发生、外周血RANKL/OPG比值为RA发生ONFH的危险因素。结论 RA患者合并ONFH的发生率高于健康人群,且与GC的使用、总股骨区OP的发生、RANKL/OPG比值升高密切相关,也与RA局部骨侵蚀存在一定的相关性。     

两种测定小鼠体内力达霉素药代动力学方法的比较

目的对测定小鼠血清浓度的总放射性方法和经过高效液相色谱分离后再测定放射性的两种方法进行比较。方法首先对力达霉素进行同位素标记并且分离纯化。给小鼠尾iv ¹²⁵I-力达霉素(100 μg·kg^-1)，采集血样，样品经两种方法测定。对得到的药代动力学参数进行统计学处理。结果两种方法测定得到的药代动力学参数(V_d, t_1/2α, t_1/2β, k₂₁, k₁₀, k₁₂, AUC和CL) 存在显著性的差异 (P<0.05)。结论采用色谱分离后再测定¹²⁵I-力达霉素原形的放射性较为合理和准确。     

HPLC-MS/MS法研究精制蒜氨酸中有关物质

研究精制蒜氨酸样品中的有关物质。采用HPLC-MS/MS一级质谱扫描总离子流谱测定有关物质的准分子离子，并利用他们的色谱保留时间、二级质谱及/或对照品对照进行结构鉴定。全扫描总离子流谱结果表明，精制蒜氨酸中主要含7个有关物质成分，［M+H］⁺离子m/z分别为116，133，147，152，175及两个m/z同为178的蒜氨酸同分异构体。其中m/z为116，133，147，175的［M+H］⁺离子相应的物质分别鉴定为脯氨酸、门冬酰胺、谷氨酰胺、精氨酸，m/z为152的［M+H］⁺离子鉴定为甲基-L-半胱氨酸亚砜，m/z 178的两个蒜氨酸同分异构体分别经二级质谱鉴定为异蒜氨酸和环蒜氨酸。精制蒜氨酸中主要有关物质分别为氨基酸及蒜氨酸的同系物及同分异构体。     

基于Keap1/Nrf2/HO-1通路汉黄芩素7-O-β-D-乙基葡萄糖醛酸苷体外抗氧化应激作用研究

目的 探讨汉黄芩素 7-O-β-D-乙基葡萄糖醛酸苷（WODE）对脂多糖（LPS）诱导的小鼠巨噬细胞（RAW264.7）的抗氧化应激作用及机制。方法 MTS法检测WODE（2.5、5.0、10.0、20.0、40.0、80.0、160.0 μmol·L^-1）对RAW264.7细胞活力的影响；体外培养RAW264.7细胞，WODE（10、20、40 μmol·L^-1）或地塞米松（1 μmol·L^-1，阳性药）预处理1 h，再给予LPS刺激24 h（造模过程不再加药），对照组不加 LPS 和受试物，模型组只给予 LPS 刺激；荧光探针检测胞内活性氧（ROS）水平；Griess反应测定细胞上清液中 NO 生成量；ELISA 检测细胞上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的分泌；实时荧光定量 PCR（qRT-PCR）法检测细胞内诱导型一氧化氮合成酶（iNOS）、环氧化酶-2（COX-2）、白细胞介素-1β（IL-1β）、醌氧化还原酶1（NQO-1）、超氧化物歧化酶-1（SOD-1）的mRNA表达水平；Western blotting法检测细胞核因子E2相关因子2（Nrf2）和血红素加氧酶-（1 HO-1）蛋白表达水平；免疫荧光染色法检测细胞内Kelch ECH相关蛋白（1 Keap1）表达水平。结果 与对照组比较，WODE浓度小于40 μmol·L^-1时，细胞存活率没有明显变化；浓度大于80 μmol·L^-1时，细胞存活率下降，但未见统计学差异。与模型组比较，WODE 10、20、40 μmol·L^-1组 ROS水平显著降低（P＜0.01）；20、40 μmol·L^-1 组 NO 释放显著降低（P＜0.05、0.01）；40 μmol·L^-1 组 iNOS mRNA 表 达 水 平 显 著 降 低（P＜0.01）；10、20、40 μmol·L^-1 组 COX-2 和20、40 μmol·L^-1组IL-1β mRNA表达水平显著降低（P＜0.05、0.01）；10、20、40 μmol·L^-1组TNF-α和IL-6的释放受到显著抑制（P＜0.01）；10、20、40 μmol·L^-1组NQO-1 mRNA表达水平显著升高（P＜0.01），40 μmol·L^-1组SOD-1 mRNA表达水平显著升高（P＜0.01）；10、20、40 μmol·L^-1组Keap1蛋白表达水平显著降低（P＜0.01）；10、20、40 μmol·L^-1组HO-1蛋白和mRNA表达水平显著提高（P＜0.05、0.01）；20、40 μmol·L^-1 组 Nrf2 蛋白和 40 μmol·L^-1 组 Nrf2 mRNA 表达水平显著增加（P＜0.01）。结论 WODE 对 LPS 诱导的RAW264.7细胞的氧化应激具有抑制作用，其作用机制可能与调控Keap1/Nrf2/HO-1信号通路相关。     

A pilot wastewater‐based epidemiology assessment of anabolic steroid use in Queensland,Australia

Anabolic‐androgenic steroids are synthetic compounds prohibited due to their performance‐enhancing characteristics. The use of these substances is known to cause health‐related issues, which highlights the importance of being able to evaluate the scale of consumption by the general population. However, most available research on the analysis of anabolic steroids is focused on animals and athletes in connection with doping. The potential of wastewater‐based epidemiology as an intelligence tool for the assessment of community level use of anabolic steroids is presented herein. A liquid chromatography tandem mass spectrometry method was developed for the analysis of 10 anabolic‐androgenic steroids and 14 endogenous hormones in influent wastewater. The validated method was applied to sixteen 24‐hour composite wastewater influent samples that were collected over a period of five years from two wastewater treatment plants in Queensland, Australia. Nine investigated compounds were found to be present at concentrations between 14 and 611 ng L^?1 which translated into 3–104 mg excreted per 1000 individuals per day. It was concluded that the developed analytical method is suitable for the analysis of AAS in wastewater matrix. Additionally, both the inclusion of metabolites and further investigation into deconjugation by enzymatic hydrolysis would aid in understanding and evaluating community anabolic steroid use. For the first time, this study presents the application of wastewater‐based epidemiology on anabolic‐androgenic steroids in Australia.     

Over-the-counter delta5 anabolic steroids 5-androsen-3,17-dione; 5-androsten-3beta, 17beta-diol; dehydroepiandrosterone; and 19-nor-5-androsten-3,17-dione: excretion studies in men

Studies of urinary steroids were performed in males after oral administration of 5-androsten-3,17-dione; 5-androsten-3beta,17beta-diol; dehydroepiandrosterone; and 19-nor-5-androsten-3,17-dione. 5-Androsten-3,17-dione; 5-androsten-3beta,17beta-diol; and dehydroepiandrosterone amplify most endogenous steroids, but to a lesser extent than their delta4 analogues do. Especially affected are androsterone, etiocholanolone, dehydroandrosterone, dehydroepiandrosterone, and isomeric 5-androstendiols. 5-Androsten-3,17-dione; 5-androsten-3beta,17beta-diol; and dehydroepiandrosterone elevate the urinary testosterone to epitestosterone (T/E) ratio by a factor of 2-3 a few hours after administration. This may cause a positive T/E test (> 6) for individuals with normal T/E ratios higher than 2. Most of the steroids return to their original concentrations in less than 24 h. Etiocholanolone and 5beta-androstan-3alpha,17beta-diol remain elevated for several days. A reduced androsterone to etiocholanolone (A/E) ratio may be an indication of delta5 steroids abuse. 19-Nor-5-androsten-3,17-dione has a similar effect, except that all metabolites in urine are 19-nor exogenous steroids. Identification criteria for 19-nor-5-androsten-3,17-dione may be the same as nandrolone, that is, detection of 19-norandrosterone and 19-noretiocholanolone. Specific abundant metabolites of 19-nor-5-androsten-3,17-dione are 19-nordehydroandrosterone and 19-nordehydroepiandrosterone. In the later stages of excretion, higher concentration of 1 9-noreticholanolone relative to 19-norandrosterone specifically indicates administration of 19-nor delta5 steroids.     

Detection of dihydrotestosterone gel, oral dehydroepiandrosterone, and testosterone gel misuse through the quantification of testosterone metabolites released after alkaline treatment

The natural occurrence of endogenous anabolic steroids together with their availability in different administration forms makes the detection of their misuse a great challenge for doping control laboratories. Nowadays, the detection of endogenous steroids abuse is performed by the analysis of the steroid profile. Recently, androst-1,4-dien-3,17-dione (1,4-AD), androst-4,6-dien-3,17-dione (4,6-AD), 17β-hydroxy-androst-4,6-dien-3-one (6-T), and androst-15-en-3,17-dione (15-AD) have been described as testosterone (T) metabolites released after basic treatment of the urine. In the present work, the usefulness of these metabolites has been evaluated detecting the use of three different forms of endogenous steroids in a single dose: dihydrotestosterone gel (DHT), oral dehydroepiandrosterone (DHEA), and T gel. After the independent administration of these endogenous steroids, a rise in the value of several of the ratios calculated between the tested metabolites was noticed. For DHT, a small increase was observed for the ratios 1,4-AD/15-AD, 6-T/15-AD and 4,6-AD/15-AD although only for one volunteer. Better results were obtained for oral DHEA and T gel where an increase was observed in all volunteers for several of the tested ratios. The detection time in which the misuse can be detected (DT) has been evaluated using two different approaches: (1) comparison with population based reference limits, and (2) comparison with individual threshold levels. The obtained DTs were compared with the results of previously published markers for the misuse of such substances. When using basic released metabolites, shorter DTs were obtained for DHT, similar DTs for DHEA, and the detectability was substantially improved for T gel.     

Screening of anabolic steroids in horse urine by liquid chromatography-tandem mass spectrometry

Anabolic steroids have the capability of improving athletic performance and are banned substances in the Olympic games as well as in horseracing and equestrian competitions. The control of their abuse in racehorses is traditionally performed by detecting the presence of anabolic steroids and/or their metabolite(s) in urine samples using gas chromatography–mass spectrometry (GC–MS). However, this approach usually requires tedious sample processing and chemical derivatisation steps and could be very insensitive in detecting certain steroids. This paper describes a high performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) method for the detection of anabolic steroids that are poorly covered by GC–MS. Enzyme-treated urine was processed by solid-phase extraction (SPE) using a Bond Elut Certify^® cartridge, followed by a base wash for further cleanup. Separation of the steroids was carried out on a reversed-phase DB-8 column using 0.1% acetic acid and methanol as the mobile phase in a gradient elution programme. The mass spectrometer for the detection of the steroids was operated in the positive electrospray ionisation (ESI) mode with multiple reaction monitoring (MRM). Urine samples fortified with 15 anabolic steroids (namely, androstadienone, 1-androstenedione, bolasterone, boldione, 4-estrenedione, gestrinone, methandrostenolone, methenolone, 17-methyltestosterone, norbolethone, normethandrolone, oxandrolone, stenbolone, trenbolone and turinabol) at low ng/mL levels were consistently detected. No significant matrix interference was observed at the retention times of the targeted ion masses in blank urine samples. The method specificity, sensitivity, precision, recoveries, and the performance of the enzyme hydrolysis step were evaluated. The successful application of the method to analyse methenolone acetate administration urine samples demonstrated that the method could be effective in detecting anabolic steroids and their metabolites in horse urine.     

Long-term detection of methyltestosterone (ab-) use by a yeast transactivation system

The routinely used analytical method for detecting the abuse of anabolic steroids only allows the detection of molecules with known analytical properties. In our supplementary approach to structure-independent detection, substances are identified by their biological activity. In the present study, urines excreted after oral methyltestosterone (MT) administration were analyzed by a yeast androgen screen (YAS). The aim was to trace the excretion of MT or its metabolites in human urine samples and to compare the results with those from the established analytical method. MT and its two major metabolites were tested as pure compounds in the YAS. In a second step, the ability of the YAS to detect MT and its metabolites in urine samples was analyzed. For this purpose, a human volunteer ingested of a single dose of 5 mg methyltestosterone. Urine samples were collected after different time intervals (0–307 h) and were analyzed in the YAS and in parallel by GC/MS. Whereas the YAS was able to trace MT in urine samples at least for 14 days, the detection limits of the GC/MS method allowed follow-up until day six. In conclusion, our results demonstrate that the yeast reporter gene system could detect the activity of anabolic steroids like methyltestosterone with high sensitivity even in urine. Furthermore, the YAS was able to detect MT abuse for a longer period of time than classical GC/MS. Obviously, the system responds to long-lasting metabolites yet unidentified. Therefore, the YAS can be a powerful (pre-) screening tool with the potential that to be used to identify persistent or late screening metabolites of anabolic steroids, which could be used for an enhancement of the sensitivity of GC/MS detection techniques.     

Long-term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

The most frequently detected substances prohibited by the World Anti-Doping Agency (WADA) belong to the anabolic steroids class. The most challenging compounds among this class are the endogenous anabolic steroids, which are detected by quantitative measurement of testosterone (T) and its metabolites with a so-called “steroid profiling” method. The current steroid profile is based on the concentrations and ratios of the sum of free and glucuronidated steroids. Recently, our group developed a steroid profiling method for the detection of three free steroids and 14 intact steroid conjugates, including both the glucuronic acid conjugated and sulfated fraction. The study aimed at evaluating the long-term stability of steroid conjugate concentrations and ratios, and the influence of different endogenous steroids on this extended steroid profile. A single dose of oral T undecanoate (TU), topical T gel, topical dihydrotestosterone (DHT) gel, and oral dehydroepiandrosterone (DHEA) was administered to six healthy male volunteers. One additional volunteer with a homozygote deletion of the UGT2B17 gene (del/del genotype) received a single topical dose of T gel. An intramuscular dose of TU was administered to another volunteer. To avoid fluctuation of steroid concentrations caused by variations in urinary flow rates, steroid ratios were calculated and evaluated as possible biomarkers for the detection of endogenous steroid abuse with low doses. Overall, sulfates do not have substantial additional value in prolonging detection times for the investigated endogenous steroids and administration doses. The already monitored glucuronides were overall the best markers and were sufficient to detect the administered steroids.     

Selective androgen receptor modulators: comparative excretion study of bicalutamide in bovine urine and faeces

Besides their development for therapeutic purposes, non‐steroidal selective androgen receptor modulators (non‐steroidal SARMs) are also known to impact growth‐associated pathways as ligands of androgenic receptors (AR). They present a potential for abuse in sports and food‐producing animals as an interesting alternative to anabolic androgenic steroids (AAS). These compounds are easily available and could therefore be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies should be developed for specific and sensitive detection of these compounds in biological matrices. The present study focused on Bicalutamide, a non‐steroidal SARM used in human treatment of non‐metastatic prostate cancer because of its anti‐androgenic activity exhibiting no anti‐anabolic effects. To select the most appropriate matrix to be used for control purposes, different animal matrices (urine and faeces) have been investigated and SARM metabolism studied to highlight relevant metabolites of such treatments and establish associated detection time windows. The aim of this work was thus to compare the urinary and faecal eliminations of bicalutamide in a calf, and investigate phase I and II metabolites. The results in both matrices showed that bicalutamide was very rapidly and mainly excreted under its free form. The concentration levels were observed as higher in faeces (ppm) than urine (ppb); although both matrices were assessed as suitable for residue control. The metabolites found were consistent with hydroxylation (phase I reaction) combined or not with glucuronidation and sulfation (phase II reactions). Copyright © 2016 John Wiley & Sons, Ltd.     

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17‐epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC‐MS/MS to establish potential long‐term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M‐I to M‐XI) were detected and characterized by LC‐MS/MS. This paper provides valuable data on the ionization and fragmentation of O‐ sulfates and N‐ sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long‐term metabolite (epistanozolol‐N ‐glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Urinary detection of conjugated and unconjugated anabolic steroids by dilute‐and‐shoot liquid chromatography‐high resolution mass spectrometry

Anabolic androgenic steroids (AAS) are an important class of doping agents. The metabolism of these substances is generally very extensive and includes phase‐I and phase‐II pathways. In this work, a comprehensive detection of these metabolites is described using a 2‐fold dilution of urine and subsequent analysis by liquid chromatography‐high resolution mass spectrometry (LC‐HRMS). The method was applied to study 32 different metabolites, excreted free or conjugated (glucuronide or sulfate), which permit the detection of misuse of at least 21 anabolic steroids. The method has been fully validated for 21 target compounds (8 glucuronide, 1 sulfate and 12 free steroids) and 18 out of 21 compounds had detection limits in the range of 1–10 ng mL^?1 in urine. For the conjugated compounds, for which no reference standards are available, metabolites were synthesized in vitro or excretion studies were investigated. The detection limits for these compounds ranged between 0.5 and 18 ng mL^?1 in urine. The simple and straightforward methodology complements the traditional methods based on hydrolysis, liquid‐liquid extraction, derivatization and analysis by gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography‐mass spectrometry (LC‐MS). Copyright © 2014 John Wiley & Sons, Ltd.