Astroglial CB1 Receptors Determine Synaptic D-Serine Availability to Enable Recognition Memory.

Bidirectional communication between neurons and astrocytes shapes synaptic plasticity and behavior. D-serine is a necessary co-agonist of synaptic N-methyl-D-aspartate receptors (NMDARs), but the physiological factors regulating its impact on memory processes are scantly known. We show that astroglial CB1 receptors are key determinants of object recognition memory by determining the availability of D-serine at hippocampal synapses. Mutant mice lacking CB1 receptors from astroglial cells (GFAP-CB1-KO) displayed impaired object recognition memory and decreased in vivo and in vitro long-term potentiation (LTP) at CA3-CA1 hippocampal synapses. Activation of CB1 receptors increased intracellular astroglial Ca2+ levels and extracellular levels of D-serine in hippocampal slices. Accordingly, GFAP-CB1-KO displayed lower occupancy of the co-agonist binding site of synaptic hippocampal NMDARs. Finally, elevation of D-serine levels fully rescued LTP and memory impairments of GFAP-CB1-KO mice. These data reveal a novel mechanism of in vivo astroglial control of memory and synaptic plasticity via the D-serine-dependent control of NMDARs.

Ionization and collision induced dissociation of steroid bisglucuronides.

Studies on steroid metabolism are of utmost importance to improve the detection capabilities of anabolic androgenic steroids (AASs) misuse in sports drug testing. In humans, glucuronoconjugates are the most abundant phase II metabolites of AAS. Bisglucuronidation is a reaction where two separated functional groups on the same molecule are conjugated with glucuronic acid. These metabolites have not been studied in depth for steroids and could be interesting markers for doping control. The aim of the present work was to study the ionization and collision-induced dissociation of steroid bisglucuronides to be able to develop mass spectrometric analytical strategies for their detection in urine samples after AAS administration. Because steroid bisglucuronides are not commercially available, 19 of them were qualitatively synthesized to study their mass spectrometric behavior. Bisglucuronides ionized as [M+NH4 ]+ in positive mode, and as [M-H]- and [M-2H]2- in negative mode. The most specific product ions of steroid bisglucuronides in positive mode resulted from the neutral losses of 387 and 405 Da (corresponding to [M+NH4 -NH3 -2gluc-H2 O]+ and [M+NH4 -NH3 -2gluc-2H2 O]+ , respectively, being "gluc" a dehydrated glucuronide moiety), and in negative mode, the fragmentation of [M-2H]2- showed ion losses of m/z 175 and 75 (gluc- and HOCH2 CO2- , respectively). On the basis of the common behavior, a selected reaction monitoring method was developed to detect bisglucuronide metabolites in urine samples. As a proof of concept, urines obtained after administration of norandrostenediol were studied, and a bisglucuronide metabolite was detected in those urines. The results demonstrate the usefulness of the analytical strategy to detect bisglucuronide metabolites in urine samples, and the formation of these metabolites after administration of AAS.

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.

Sulfate metabolites as alternative markers for the detection of 4-chlorometandienone misuse in doping control.

Sulfate metabolites have been described as long-term metabolites for some anabolic androgenic steroids (AAS). 4-chlorometandienone (4Cl-MTD) is one of the most frequently detected AAS in sports drug testing and it is commonly detected by monitoring metabolites excreted free or conjugated with glucuronic acid. Sulfation reactions of 4Cl-MTD have not been studied. The aim of this work was to evaluate the sulfate fraction of 4Cl-MTD metabolism by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to establish potential long-term metabolites valuable for doping control purposes. 4Cl-MTD was administered to two healthy male volunteers and urine samples were collected up to 8 days after administration. A theoretical selected reaction monitoring (SRM) method working in negative mode was developed. Ion transitions were based on ionization and fragmentation behaviour of sulfate metabolites as well as specific neutral losses (NL of 15 Da and NL of 36 Da) of compounds with related chemical structure. Six sulfate metabolites were detected after the analysis of excretion study samples. Three of the identified metabolites were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Results showed that five out of the six identified sulfate metabolites were detected in urine up to the last collected samples from both excretion studies. Copyright © 2016 John Wiley & Sons, Ltd.

LC-MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone.

The aim of this study was to evaluate the direct detection of glucuronoconjugated metabolites of metandienone (MTD) and their detection times. Metabolites resistant to enzymatic hydrolysis were also evaluated. Based on the common mass spectrometric behaviour of steroid glucuronides, three liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies were applied for the detection of unpredicted and predicted metabolites: precursor ion scan (PI), neutral loss scan (NL), and theoretical selected reaction monitoring (SRM) methods. Samples from four excretion studies of MTD were analyzed for both the detection of metabolites and the establishment of their detection times. Using PI and NL methods, seven metabolites were observed in post-administration samples. SRM methods allowed for the detection of 13 glucuronide metabolites. The detection times, measured by analysis with an SRM method, were between 1 and 22 days. The metabolite detected for the longest time was 18-nor-17ß-hydroxymethyl-17α-methyl-5ß-androsta-1,4,13-triene-3-one-17-glucuronide. One metabolite was resistant to hydrolysis with ß-glucuronidase; however it was only detected in urine up to four days after administration. The three glucuronide metabolites with the highest retrospectivity were identified by chemical synthesis or mass spectrometric data, and although they were previously reported, this is the first time that analytical data of the intact phase II metabolites are presented for some of them. The LC-MS/MS strategies applied have demonstrated to be useful for detecting glucuronoconjugated metabolites of MTD, including glucuronides resistant to enzymatic hydrolysis which cannot be detected by conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.

Constant Ion Loss Method for the Untargeted Detection of Bis-sulfate Metabolites.

The untargeted detection of phase II metabolites is a key issue for the study of drug metabolism in biological systems. Sensitive and selective mass spectrometric (MS) techniques coupled to ultrahigh performance liquid chromatographic (UHPLC) systems are the most effective for this purpose. In this study, we evaluate different MS approaches with a triple quadrupole instrument for the untargeted detection of bis-sulfate metabolites. Bis-sulfates of 23 steroid metabolites were synthesized and their MS behavior was comprehensively studied. Bis-sulfates ionized preferentially as the dianion ([M - 2H]2-) with a small contribution of the monoanion ([M - H]-). Product ion spectra generated from the [M - 2H]2- precursor ions were dominated by the loss of HSO4- to generate two product ions, that is, the ion at m/z 97 (HSO4-) and the ion corresponding to the remaining monosulfate fragment. Other product ions were found to be specific for some structures. As an example, the loss of [CH3 + SO3]- was found to be important for several compounds with unsaturation adjacent to the sulfate. On the basis of the common behavior of the bis-sulfate metabolites two alternatives were evaluated for the untargeted detection of bis-sulfate metabolites (i) a precursor ion scan method using the ion at m/z 97 and (ii) a constant ion loss (CIL) method using the loss of HSO4-. Both methods allowed for the untargeted detection of the model compounds. Eight steroid bis-sulfates were synthesized in high purity in order to quantitatively evaluate the developed strategies. Lower limits of detection (2-20 ng/mL) were obtained using the CIL method. Additionally, the CIL method was found to be more specific in the detection of urinary bis-sulfates. The applicability of the CIL approach was demonstrated by determining progestogens altered during pregnancy and by detecting the bis-sulfate metabolites of tibolone.

Detection and characterization of clostebol sulfate metabolites in Caucasian population.

Anabolic androgenic steroids (AAS) are synthetic testosterone derivatives which undergo extensive metabolism in man. Differences in the excretion of phase II metabolites are strongly associated with inter-individual and inter-ethnic variations. Sulfate metabolites have been described as long-term metabolites for some AAS. Clostebol is the 4-chloro derivative of testosterone and the aim of the present study was the evaluation of clostebol sulfate metabolites in Caucasian population by LC-MS/MS technology. Clostebol was orally administered to four healthy Caucasian male volunteers, and excretion study urines were collected up to 31 days. Several analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) were applied to detect sulfate metabolites in post-administration samples. Sixteen sulfate metabolites were detected, five of them having detectability times above 10 days (S1a, S2a, S3b, S3g and S4b). Interestingly, metabolite S1a could be detected up to the last collected sample of all excretion studies and it was characterized by LC-MS/MS and GC-MS as 4ξ-chloro-5α-androst-3ß-ol-17-one 3ß-sulfate. Thus, monitoring of S1a improves the detection time of clostebol misuse with respect to the commonly monitored metabolites, excreted in the glucuronide fraction. Importantly, this new metabolite can be incorporated into recently developed LC-MS/MS screening methods base on the direct detection of phase II metabolites.

Screening for anabolic steroids in sports: analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry.

In order to improve the detection capabilities of anabolic androgenic steroids (AAS) in sports, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for the simultaneous detection of AAS phase I and phase II intact urinary metabolites (glucuronides and sulfates) was developed. A total of 36 metabolites (7 unconjugated; 19 glucuronides and 10 sulfates) corresponding to 15 of the most reported AAS were included. Analytes were extracted from urine using C18 cartridges. LC and MS conditions were studied in-depth to determine the most sensitive and selective conditions for each analyte. A selected reaction monitoring method was set up. The optimization of the experimental parameters for 13 metabolites not available as standards was performed using excretion study urines. Extraction recoveries were above 77% for all 23 validated analytes. Intra-day precision was lower than 21%, and LODs were in the range 0.25-4ng/mL for 18 of the 23 analytes. Matrix effect was evaluated using post column infusion and ranged from 92 to 147%. The method was successfully applied to excretion study urines of different exogenous AAS. The suitability of the strategy was demonstrated with methyltestosterone and stanozolol excretion study urines by achieving detection times of 22 and 21 days, respectively. The method is compliant with the World Antidoping Agency requirements for most of the studied compounds. It represents a cost-effective approach that improves the detection capabilities of AAS by increasing the sensitivity for some metabolites and by including recently described phase II long-term metabolites not detectable using the current screening strategy.

Determination of five di-(2-ethylhexyl)phthalate metabolites in urine by UPLC-MS/MS, markers of blood transfusion misuse in sports.

Di-(2-ethylhexyl)phthalate (DEHP) is the most commonly used plasticizer for polyvinyl chloride, which is found in a large variety of products, including most of the bags used for blood storage because of its protective role on erythrocytes survival. DEHP metabolites have been recently proposed as markers of the misuse of blood transfusion in athletes. In this study, a method to quantify the main five DEHP metabolites in urine has been developed: mono-(2-ethylhexyl)phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP), and mono-(2-carboxymethylhexyl)phthalate (2cx-MMHP). The method involved an enzymatic hydrolysis with ß-glucuronidase from Escherichia coli followed by an acidic extraction with ethyl acetate. The hydrolysed extracts were analysed by ultraperformance liquid chromatography tandem mass spectrometry. Isotope labelled MEHP, MEOHP and 5cx-MEPP were used as internal standards. Analysis of all the metabolites was achieved in a total run time of 10min, using a C(18) column and a mobile phase containing deionized water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6mLmin(-1). Detection of the compounds was performed by multiple reaction monitoring, using electrospray ionization in positive and negative ion modes. The method was validated for quantitative purposes. Extraction recoveries were greater than 90% and the limits of quantitation ranged from 1.2 to 2.6ngmL(-1). Intra-day precisions were better than 8% for all metabolites while inter-assay precisions were better than 12%. Concentrations of DEHP metabolites were measured in a control group (n=30, subjects reflecting the common environmental DEHP exposure), and in sportsmen (n=464), to evaluate population distribution exposure to DEHP. Additionally, threshold concentrations indicating outliers of common exposure for DEHP metabolites are proposed.