An UPLC-MS/MS method was developed for the determination of serotonin (5-HT), dopamine (DA), their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfate and glucuronide conjugates in human brain microdialysis samples obtained from two patients with acute brain injuries, ventricular cerebrospinal fluid (CSF) samples obtained from four patients with obstructive hydrocephalus, and a lumbar CSF sample pooled mainly from patients undergoing spinal anesthesia in preparation for orthopedic surgery. The method was validated by determining the limits of detection and quantification, linearity, repeatability and specificity. The direct method enabled the analysis of the intact phase II metabolites of 5-HT and DA, without hydrolysis of the conjugates. The method also enabled the analysis of the regioisomers of the conjugates, and several intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain microdialysis and CSF samples. We were able to show the presence of 5-HIAA sulfate, and that dopamine-3-O-sulfate predominates over dopamine-4-O-sulfate in the human brain. The quantitative results suggest that sulfonation is a more important phase II metabolism pathway than glucuronidation in the human brain.

The interpretation of the steroidal module of the Athlete Biological Passport (ABP) in female athletes is complex due to the large variation of the endogenous urinary steroids. The menstrual cycle seems to be one of the largest confounders of the steroid profile. The duration of the different phases in the menstrual cycle differs between women and is difficult to predict only by counting days after menstruation. Here, we have determined the follicle, ovulation, and luteal phases, by assessing the menstrual hormones in serum samples collected from 17 healthy women with regular menses. Urine samples were collected three times per week during two consecutive cycles to measure the urinary steroid concentrations used in the ABP. The metabolite that was mostly affected by the menstrual phases was epitestosterone (E), where the median concentration was 133% higher in the ovulation phase compared to the follicle phase (p < 0.0001). The women with a large coefficient of variation (CV) in their first cycle also had a large CV in their second cycle and vice versa. The inter‐individual difference was extensive with a range of 11%–230% difference between the lowest and the highest T/E ratio during a cycle. In conclusion, E and ratios with E as denominator are problematic biomarkers for doping in female athletes. The timing of the sample collection in the menstrual cycle will have a large influence on the steroid profile. The results of this study highlight the need to find additional biomarkers for T doping in females.

We present a capillary photoionization (CPI) method for mass spectrometric (MS) analysis of liquid and gaseous samples. CPI utilizes a heated transfer capillary with a vacuum ultraviolet transparent MgF2 window, through which vacuum UV light (10 eV) from an external source enters the capillary. The liquid or gaseous sample, together with dopant, is introduced directly into the heated transfer capillary between the atmosphere and the vacuum of the MS. Since the sample is vaporized and photoionized inside the capillary, ion transmission is maximized, resulting in good overall sensitivity for nonpolar and polar compounds. As in atmospheric pressure photoionization, ionization in CPI occurs either by proton transfer or by charge exchange reactions. The feasibility of CPI was demonstrated with selected nonpolar and polar compounds. A particular advantage of CPI is that it enables the analysis of nonvolatile and nonpolar compounds in liquid samples with high ionization efficiency. This is not possible with existing capillary ionization methods. The performance of CPI as an interface between GC and MS and its applicability for the analysis of steroids in biological samples are also demonstrated. The GC-CPI-MS method shows good chromatographic resolution, linearity (R(2) > 0.993), limits of detection (LOD) in the range of 2-6 pg/mL and repeatability of injection with relative standard deviations of 4-15%.

The objective of the present study was to investigate mesocarb metabolism in humans. Samples obtained after administration of mesocarb to healthy volunteers were studied. The samples were extracted at alkaline pH using ethyl acetate and salting-out effect to recover metabolites excreted free and conjugated with sulfate. A complementary procedure was applied to recover conjugates with glucuronic acid or with sulfate consisting of the extraction of the urines with XAD-2 columns previously conditioned with methanol and deionized water; the columns were then washed with water and finally eluted with methanol. In both cases, the dried extracts were reconstituted and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. Chromatographic separation was carried out using a C(18) column (100 mm x 2.1 mm i.d., 1.7 microm particle size) and a mobile phase consisting of water and acetonitrile with 0.01% formic acid with gradient elution. The chromatographic system was coupled to a mass spectrometer with an electrospray ionization source working in positive mode. Metabolic experiments were performed in multiple-reaction monitoring mode by monitoring one transition for each potential mesocarb metabolite. Mesocarb and 19 metabolites were identified in human urine, including mono-, di-, and trihydroxylated metabolites excreted free as well as conjugated with sulfate or glucuronic acid. All metabolites were detected up to 48 h after administration. The structures of most metabolites were proposed based on data from reference standards available and molecular mass and product ion mass spectra of the peaks detected. The direct detection of mesocarb metabolites conjugated with sulfate and glucuronic acid without previous hydrolysis has been described for the first time. Finally, a screening method to detect the administration of mesocarb in routine antidoping control analyses was proposed and validated based on the detection of the main mesocarb metabolites in human urine (p-hydroxymesocarb and p-hydroxymesocarb sulfate). After analysis of several blank urines, the method demonstrated to be specific. Extraction recoveries of 100.3 +/- 0.8 and 105.9 +/- 10.8 (n = 4), and limits of detection of 0.5 and 0.1 ng mL(-1) were obtained for p-hydroxymesocarb sulfate and p-hydroxymesocarb, respectively. The intra- and inter-assay precisions were estimated at two concentration levels, 50 and 250 ng mL(-1), and relative standard deviations were lower than 15% in all cases (n = 4).