Unveiling a new era with liquid chromatography coupled with mass spectrometry to enhance parathyroid hormone measurement in patients with chronic kidney disease.

Precise determination of circulating parathyroid hormone (PTH) concentration is crucial to diagnose and manage various disease conditions, including the chronic kidney disease-mineral and bone disorder. However, the lack of standardization in PTH assays is challenging for clinicians, potentially leading to medical errors because the different assays do not provide equivalent results and use different reference ranges. Here, we aimed to evaluate the impact of recalibrating PTH immunoassays by means of a recently developed LC-MS/MS method as the reference. Utilizing a large panel of pooled plasma samples with PTH concentrations determined by the LC-MS/MS method calibrated with the World Health Organization (WHO) 95/646 International Standard, five PTH immunoassays were recalibrated. The robustness of this standardization was evaluated over time using different sets of samples. The recalibration successfully reduced inter-assay variability with harmonization of PTH measurements across different assays. By recalibrating the assays based on the WHO 95/646 International Standard, we demonstrated the feasibility for standardizing PTH measurement results and adopting common reference ranges for PTH assays, facilitating a more consistent interpretation of PTH values. The recalibration process aligns PTH results obtained from various immunoassays with the LC-MS/MS method, providing more consistent and reliable measurements. Thus, establishing true standardization across all PTH assays is crucial to ensure consistent interpretation and clinical decision-making.

Concise review on the combined use of immunocapture, mass spectrometry and liquid chromatography for clinical applications.

Immunocapture is now a well-established method for sample preparation prior to quantitation of peptides and proteins in complex matrices. This short review will give an overview of some clinical applications of immunocapture methods, as well as protocols with and without enzymatic digestion in a clinical context. The advantages and limitations of both approaches are discussed in detail. Challenges related to the choice of mass spectrometer are also discussed. Top-down, middle-down, and bottom-up approaches are discussed. Even though immunocapture has its limitations, its main advantage is that it provides an additional dimension of separation and/or isolation when working with peptides and proteins. Overall, this short review demonstrates the potential of such techniques in the field of proteomics-based clinical medicine and paves the way for better personalized medicine.

Validation of an LC-MS/MS Method Using Solid-Phase Extraction for the Quantification of 1-84 Parathyroid Hormone: Toward a Candidate Reference Measurement Procedure.

BACKGROUND: Parathyroid hormone (PTH) measurement is important for patients with disorders of calcium metabolism, including those needing bone-turnover monitoring due to chronic kidney disease-mineral bone disorder. There are currently 2 generations of PTH immunoassays on the market, both having cross-reactivity issues and lacking standardization. Therefore, we developed an LC-MS/MS higher-order method for PTH analysis. METHODS: The method was calibrated against the international standard for 1-84 PTH (WHO 95/646). Antibody-free sample preparation with the addition of an isotope-labeled internal standard was performed by solid-phase extraction. Extracts were analyzed by LC-MS/MS. EDTA-K2 plasma was used throughout the development and validation. Bias and uncertainty sources were tested according to ISO 15193. Clinical Laboratory Standards Institute guidelines and reference measurement procedures were consulted for the design of the validation. Patient samples and external quality controls were compared between LC-MS/MS and 2 third-generation immunoassays. RESULTS: The method was validated for 1-84 PTH from 5.7 to 872.6 pg/mL. The interassay imprecision was between 1.2% and 3.9%, and the accuracy ranged from 96.2% to 103.2%. The measurement uncertainty was <5.6%. The comparison between LC-MS/MS and the immunoassays showed a proportional bias but moderate to substantial correlation between methods. CONCLUSIONS: This LC-MS/MS method, which is independent of antibodies, is suitable for a wide range of PTH concentrations. The obtained analytical performance specifications demonstrate that development of a reference measurement procedure will be possible once a higher order reference standard is available.

Evolution of the slopes of ST2 and galectin-3 during marathon and ultratrail running compared to a control group.

Background Previous studies have suggested that exercising may induce cardiac damage. Galectin-3 (Gal-3) and soluble suppression of tumorigenicity 2 (ST2) are very interesting biomarkers for heart failure and myocardial fibrosis. We aimed to compare the kinetics of emerging fibrosis cardiac biomarkers as Gal-3 and ST-2 in endurance runners, and recreational runners before and after a running event represented by a marathon and an ultratrail event. Methods Blood samples were taken from 19 healthy non-elite marathon runners (42 km), 27 ultratour runners (67 km), and 14 recreational runners who represented the control group (10 km) just before the run (T0), just after (T1) and 3 h after (T2), in order to analyze Gal-3, ST2, hsTnT, NT-proBNP, CKMB and hsCRP. We compared the percentage of evolution and the slopes obtained from T0 to T1 (pT0T1) and from T1 to T2 (pT1T2), between the different groups of runners participating in three different races. Results Plasma cardiac biomarker concentrations increased significantly from baseline to immediately post-exercise and most of the time decreased over the subsequent 3-h period. For pT0T1 and pT1T2, the markers Gal-3 and ST2 showed a significant difference between types of run (p < 0.05 and p < 0.0001, respectively). During the recovery time, Gal-3 returned to the baseline values but not ST2 which continued to increase. Conclusions Gal-3 and ST2 are considered as a reflection of cardiac fibrosis and remodeling. The evolution of both was different, particularly after the recovery time. ST2 values exceeding cutoff values at any time.

The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?

Historically, the determination of low concentration analytes was initially made possible by the development of rapid and easy-to-perform immunoassays (IAs). Unfortunately, typical problems inherent to IA technologies rapidly appeared (e.g. elevated cost, cross-reactivity, lot-to-lot variability, etc.). In turn, liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are sensitive and specific enough for such analyses. Therefore, they would seem to be the most promising candidates to replace IAs. There are two main choices when implementing a new LC-MS/MS method in a clinical laboratory: (1) Developing an in-house method or (2) purchasing ready-to-use kits. In this paper, we discuss some of the respective advantages, disadvantages and mandatory requirements of each choice. Additionally, we also share our experiences when developing an in-house method for cortisol determination and the implementation of an "ready-to-use" (RTU) kit for steroids analysis.

Detection of Stimulated Erythropoiesis by the RNA-Based 5'-Aminolevulinate Synthase 2 Biomarker in Dried Blood Spot Samples.

BACKGROUND: Despite implementation of the Athlete Biological Passport 10 years ago, blood doping remains difficult to detect. Thus, there is a need for new biomarkers to increase the sensitivity of the adaptive model. Transcriptomic biomarkers originating from immature reticulocytes may be reliable indicators of blood manipulations. Furthermore, the use of dried blood spots (DBSs) for antidoping purposes constitutes a complementary approach to venous blood collection. Here, we developed a method of quantifying the RNA-based 5'-aminolevulinate synthase 2 (ALAS2) biomarker in DBS. MATERIALS: The technical, interindividual, and intraindividual variabilities of the method, and the effects of storage conditions on the production levels of ALAS2 RNA were assessed. The method was used to monitor erythropoiesis stimulated endogenously (blood withdrawal) or exogenously (injection of recombinant human erythropoietin). RESULTS: When measured over a 7-week period, the intra- and interindividual variabilities of ALAS2 expression in DBS were 12.5%-42.4% and 49%, respectively. Following withdrawal of 1 unit of blood, the ALAS2 RNA in DBS increased significantly for up to 15 days. Variations in the expression level of this biomarker in DBS samples were more marked than those of the conventional hematological parameters, reticulocyte percentage and immature reticulocyte fraction. After exogenous stimulation of erythropoiesis via recombinant human erythropoietin injection, ALAS2 expression in DBS increased by a mean 8-fold. CONCLUSIONS: Monitoring of transcriptomic biomarkers in DBS could complement the measurement of hematological parameters in the Athlete Biological Passport and aid the detection of blood manipulations.

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 differentiation of 22 kDa and 20 kDa Growth Hormone proteoforms in human plasma by LC-MS/MS.

Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes' performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20 kDa GH proteoforms that demonstrate the administration of the synthetic hormone. Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20 kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH purification by protein precipitation. Using GH-MSIA we successfully quantified the 22/20 kDa GH ratio in post-exercise capillary plasma extracted from two individuals. This GH-MSIA is applicable to anti-doping and GH-related disease analysis.

Determination of Recent Growth Hormone Abuse Using a Single Dried Blood Spot.

BACKGROUND: Although it is being increasingly applied, blood collection for drug testing in sport presents some logistic issues that complicate full applicability on a large scale. The use of dried blood spots (DBS) could benefit compliant blood testing considerably owing to its simplicity, minimal invasiveness, analyte stability, and reduced costs. The aim of this study was to evaluate the applicability of DBS to the methodology approved by the World Anti-Doping Agency (WADA) for detection of doping by recombinant human growth hormone (rhGH) in serum. METHODS: A protocol for a single DBS analysis using the hGH isoforms differential immunoassays (kit 1 and kit 2) was developed and validated. A clinical study with healthy volunteers injected for 3 consecutive days with a low subcutaneous dose (0.027 mg · kg(-1) · day(-1) · person(-1)) of rhGH was conducted. Finger prick DBS and paired-time serum samples from arm venipuncture were compared. RESULTS: The analysis of the DBS-based protocol indicated that with only a single blood spot it was possible to detect positivity for growth hormone abuse. In spite of the low rhGH dose administered and independently of the kit used, the window of detection for DBS was confirmed in all analyzed samples up to 8 h after rhGH administration and extended up to 12 h in 50% of the cases. Serum positivity was detected in all studied samples for 12 h after administration. CONCLUSIONS: These results support the usefulness of DBS as a biological matrix for testing recent growth hormone abuse.

Surgical Decompression of Exertional Compartment Syndrome of the Forearm in Professional Motorcycling Racers: Comparative Long-term Results of Wide-Open Versus Mini-Open Fasciotomy.

OBJECTIVE: To compare the long-term results of 2 surgical techniques for forearm chronic exertional compartment syndrome (CECS) in professional motorcycling racers and to study a new diagnostic variable for CECS, TRest. DESIGN: Retrospective case series. LEVEL OF EVIDENCE: 4. SETTING: University Hospital. PARTICIPANTS: Thirty-four patients identified from a surgical database who had been operated on for upper-limb CECS. INTERVENTIONS: The purpose of the study was to report and compare the long-term results of 2 surgical techniques using fasciotomies [wide-open fasciotomy (WOF) versus mini-open fasciotomy (MOF)] for forearm CECS in professional motorcycling racers. PATIENT CHARACTERISTICS: Pain [visual analog scale (100-point scale)] and functional scores (Quick-DASH) at 3 months after surgery and at regular intervals during clinical visits. Surgical complications: Level of satisfaction with the outcome. Time to return to full activity after surgery. RESULTS: Thirty-four racers, 22 with bilateral involvement (n = 56), were diagnosed with CECS and were treated either with WOF (n = 24) or MOF (n = 32) depending on the surgeon's indication. Mini-open fasciotomy was usually selected in cases who need a faster recovery because of competition schedule. Visual analog scale and Quick-DASH improved 63 and 73 points, respectively (P < 0.001) with no significant difference between both surgical methods (P = 0.512). Both WOF and MOF were equally effective. Ninety-four percent of the patients were satisfied after 45.35 ± 12 months of follow-up, with no significant difference between surgical groups (P = 0.642). The time to return to full activity was 2.7 ± 1 week, also with no significant difference (P = 0.544). The time between when the stress testing was halted for pain and the return to baseline pressure (TRest) was superior to 15 minutes (defined as the mean minus 2 SDs) in 100% patients. CONCLUSIONS: Surgical open or mini-invasive fasciotomy is equally successful in motorcycling racers with forearm CECS. Although the sensitivity of TRest is quite high in our series, further studies are still needed to validate its diagnostic value. CLINICAL RELEVANCE: Surgical open or mini-invasive fasciotomy is equally successful in motorcycling racers with forearm CECS.

Untargeted metabolomics in doping control: detection of new markers of testosterone misuse by ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry.

The use of untargeted metabolomics for the discovery of markers is a promising and virtually unexplored tool in the doping control field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole Orbitrap (Q Exactive) mass spectrometers, coupled to ultrahigh pressure liquid chromatography, are excellent tools for this purpose. In the present work, QTOF and Q Exactive have been used to look for markers for testosterone cypionate misuse by means of untargeted metabolomics. Two different groups of urine samples were analyzed, collected before and after the intramuscular administration of testosterone cypionate. In order to avoid analyte losses in the sample treatment, samples were just 2-fold diluted with water and directly injected into the chromatographic system. Samples were analyzed in both positive and negative ionization modes. Data from both systems were treated under untargeted metabolomic strategies using XCMS application and multivariate analysis. Results from the two mass spectrometers differed in the number of detected features, but both led to the same potential marker for the particular testosterone ester misuse. The in-depth study of the MS and MS/MS behavior of this marker allowed for the establishment of 1-cyclopentenoylglycine as a feasible structure. The putative structure was confirmed by comparison with synthesized material. This potential marker seems to come from the metabolism of the cypionic acid release after hydrolysis of the administered ester. Its suitability for doping control has been evaluated.

Detection and characterization of triamcinolone acetonide metabolites in human urine by liquid chromatography/tandem mass spectrometry after intramuscular administration.

RATIONALE: Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. intramuscular, IM), whereas they are allowed using other ways of administration. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of triamcinolone acetonide (TA), one of the most used glucocorticosteroids, was studied using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). METHODS: Urine samples obtained after IM administration of TA were analyzed using two sample treatments: (a) hydrolysis with ß-glucuronidase enzymes and liquid-liquid extraction under alkaline conditions, and (b) liquid-liquid extraction under acidic conditions. The extracts were analyzed by LC/MS/MS. RESULTS: TA, commercially available metabolites (6ß-hydroxytriamcinolone acetonide, 6ß-OH-TA, and triamcinolone), and their C20-reduced derivatives showed characteristic fragmentation behavior. Besides common product ions and neutral losses for corticosteroids containing fluorine, additional characteristic neutral losses (58 Da, loss of acetone; 44 Da, loss of acetaldehyde) were observed in positive electrospray ionization. Based on that behavior, two complementary approaches were applied to detect TA metabolites: (a) open detection by precursor ion and neutral loss scan methods and (b) targeted detection by selected reaction monitoring methods (SRM) containing theoretical ion transitions of the potential metabolites. Two main compounds, TA and 6ß-OH-TA, and nine minor potential metabolites, were detected by open screening methods. Using SRM, two additional metabolites were detected. Some of the metabolites were characterized using reference standards and, for the rest of metabolites, feasible structures were proposed based on mass spectrometric data. CONCLUSIONS: Metabolites resulting from hydroxylation in C-6, oxidation of the 11-hydroxyl group, reduction of the Δ(4) double bond and oxidation of the side chain were detected. Some of them have not been previously described. Excretion profiles of the detected metabolites after IM administration are presented.

Time for change: a roadmap to guide the implementation of the World Anti-Doping Code 2015.

A medical and scientific multidisciplinary consensus meeting was held from 29 to 30 November 2013 on Anti-Doping in Sport at the Home of FIFA in Zurich, Switzerland, to create a roadmap for the implementation of the 2015 World Anti-Doping Code. The consensus statement and accompanying papers set out the priorities for the antidoping community in research, science and medicine. The participants achieved consensus on a strategy for the implementation of the 2015 World Anti-Doping Code. Key components of this strategy include: (1) sport-specific risk assessment, (2) prevalence measurement, (3) sport-specific test distribution plans, (4) storage and reanalysis, (5) analytical challenges, (6) forensic intelligence, (7) psychological approach to optimise the most deterrent effect, (8) the Athlete Biological Passport (ABP) and confounding factors, (9) data management system (Anti-Doping Administration & Management System (ADAMS), (10) education, (11) research needs and necessary advances, (12) inadvertent doping and (13) management and ethics: biological data. True implementation of the 2015 World Anti-Doping Code will depend largely on the ability to align thinking around these core concepts and strategies. FIFA, jointly with all other engaged International Federations of sports (Ifs), the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA), are ideally placed to lead transformational change with the unwavering support of the wider antidoping community. The outcome of the consensus meeting was the creation of the ad hoc Working Group charged with the responsibility of moving this agenda forward.

Pyrocarbon interposition (PyroDisk) implant for trapeziometacarpal osteoarthritis: minimum 5-year follow-up.

PURPOSE: To report outcomes after a minimum of 5 years following pyrocarbon interposition (PyroDisk) trapeziometacarpal joint implant for osteoarthritis at a single center. METHODS: We retrospectively reviewed the midterm clinical and radiological outcomes of 19 patients who had a pyrocarbon interposition implant (PyroDisk; Integra Life Sciences, Plainsboro, NJ) arthroplasty. The rate and causes of repeat surgeries, revisions, and complications were examined. RESULTS: The mean follow-up period was 68 months. Patient satisfaction was high. The mobility of the operated thumb was restored to a range of motion comparable with that of the contralateral thumb. Grip strength improved by 26%. Overall function, according to the Quick Disabilities of the Arm, Shoulder, and Hand score, showed an average improvement of 71 to 20. Pain decreased by 78% according to the numerical rating scale. Radiological evaluation using a modification of the system described by Herren revealed progression of the periprosthetic lucency (grade I-II) of the implant after 5 years in 5 of 19 (26%) patients. Progression of lucency did not predict implant loosening or failure at 5 years. Two patients had symptomatic instability that required revision. No dislocations occurred. The 5-year survival of the prosthesis was 90%. CONCLUSIONS: The PyroDisk implant for treating advanced trapeziometacarpal arthritis did not demonstrate superiority over published outcome data of trapeziectomy with or without ligament reconstruction and tendon interposition. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Development and validation of an LC-MS/MS method for the simultaneous quantitation of angiotensin (1-7), (1-8), (1-9) and (1-10) in human plasma.

Cardiovascular diseases have cast a significant negative impact on the lives of millions worldwide. Over the years, extensive efforts have been dedicated to enhancing diagnostic and prognostic tools for these diseases. A growing body of evidence indicates that the angiotensin convertase enzyme (ACE) and the angiotensin convertase enzyme 2 (ACE2), and angiotensin peptide levels could hold a pivotal role in assisting clinicians with the management of cardiovascular conditions, notably hypertension and heart failure. However, despite the considerable body of knowledge in this domain, a void remains in the field of analytical methodologies for these molecules. In this study, we present a fully validated LC-MS/MS method for the precise quantitation of plasma angiotensin (1-7), (1-8), (1-9), and (1-10), following the guidelines set by the Clinical and Laboratory Standards Institute (CLSI). Our method not only enables the accurate quantification of angiotensin peptides but also provides a means to assess ACE and ACE2 activity. Remarkably, our method achieved a Lower Limit of Measurement Interval (LLMI) as low as 5 pg/mL. This has enabled the detection of angiotensin (1-7), (1-8), (1-9) and (1-10) and the accurate quantitation of angiotensin (1-7), (1-8) and (1-10) in all analyzed groups, including healthy controls, patients with high blood pressure, and patients with chronic kidney disease. To our knowledge, our method represents the most sensitive approach allowing for simultaneous quantitation of these four angiotensin peptides. A distinct advantage of our method, when compared to immunoassays, is its high sensitivity combined with comprehensive chromatographic separation of all currently known angiotensin peptides. This combination translates to an exceptional level of selectivity, underscoring the value and potential of our methodology in advancing cardiovascular disease research.

Use of LC-MS/MS for the open detection of steroid metabolites conjugated with glucuronic acid.

In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied by using ß-glucuronidase enzymes to release the phase I metabolites. It is well-known that hydrolysis with ß-glucuronidase presents some limitations that may result in the underestimation of some conjugates. The aim of the present work was to develop and to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for the open detection of steroid glucuronides in urine samples. The mass spectrometric behavior of thirteen representative steroid glucuronides, used as model compounds, was studied. Characteristic ionization and collision induced dissociation behaviors were observed depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI of m/z 141, 159, and 177, in positive mode and m/z 75, 85, and 113, in negative mode) scan methods were evaluated. The NL scan method was chosen for the open detection of glucuronoconjugated steroids due to its sensitivity and the structural information provided by this method. The application of the NL scan method to urine samples collected after testosterone (T) undecanoate administration revealed the presence of two T metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the urine. 3α,6ß-Dihydroxy-5α-androstan-17-one (6ß-hydroxyandrosterone) glucuronide and 3α,6ß-dihydroxy-5ß-androstan-17-one (6ß-hydroxyetiocholanolone) glucuronide were established as the structures for these metabolites, by comparing the structure of the steroids released after chemical hydrolysis with reference materials. An increase of 50-300-fold of these metabolites after oral administration of T undecanoate was observed, proving that their determination can be useful in the doping control field. Moreover, these results exemplify that significant information might be missed, unless direct methods for the determination of steroid glucuronides are employed.

Discrimination of prohibited oral use from authorized inhaled treatment of budesonide in sports.

BACKGROUND: Budesonide (22(R,S)-16α,17α-butylidenedioxy-11ß,21-dihydroxypregna-1,4-diene-3,20-dione) (BUD) is a glucocorticoid widely used for the treatment of asthma and rhinitis. Its use in sport competitions is prohibited when administered by oral, intravenous, intramuscular, or rectal routes, but its use by other routes (eg, inhalation) is allowed. The objective of this study was to evaluate the urinary profiles of different metabolites of BUD after oral and inhaled administrations in order to define a criterion to discriminate between forbidden and authorized administrations of the drug. METHODS: A liquid chromatography-tandem mass spectrometry method was validated to quantify BUD, 16α-hydroxy-prednisolone, 6ß-hydroxy-budesonide, and 6α-hydroxy-budesonide and to qualitatively determine 13 additional BUD metabolites. The method was applied to urine samples collected in clinical studies where BUD was administered to healthy volunteers by the oral route (n = 2) and by inhalation for 3 consecutive days followed by a single oral dose (n = 8). RESULTS: Reporting levels of the different metabolites were evaluated in terms of specificity (no false-positive results after inhalation) and sensitivity (no false-negative results after oral intake). CONCLUSION: Taking into consideration the administered doses, the best compromise to discriminate between authorized inhaled administration and forbidden oral intake of BUD was found using a reporting level of 20 ng/mL of metabolite 6ß-hydroxy-budesonide.

Plasticizers excreted in urine: indication of autologous blood transfusion in sports.

BACKGROUND: Misuse of autologous blood transfusions in sports remains undetectable. The metabolites of the plasticizer di-(2-ethylhexyl)phthalate (DEHP) were recently proposed as markers of blood transfusion, based on high urinary concentrations of these compounds observed in patients subjected to blood transfusion. This study evaluates DEHP metabolites in urine for detecting autologous blood transfusion. STUDY DESIGN AND METHODS: One blood bag was drawn from moderately trained subjects and the red blood cells (RBCs) were reinfused after different storage periods. Group 1 (12 subjects) was reinfused after 14 days, and Group 2 (13 subjects), after 28 days of storage. Urine samples were collected before and after reinfusion for determination of the concentrations of three DEHP metabolites, mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate, and mono-(2-ethyl-5-oxohexyl)phthalate. RESULTS: Concentrations of DEHP metabolites on the days before reinfusion were in agreement with those described after common environmental exposure. A few hours after the reinfusion a significant increase was observed for all metabolites in all volunteers. Concentrations 1 day later were still higher (p < 0.05) than before reinfusion. Variations in urine dilution supported normalization by specific gravity. Concentrations of DEHP metabolites tended to be higher after longer storage times of RBCs. CONCLUSION: Autologous transfusion with RBCs stored in plastic bags provokes an acute increase in the urinary concentrations of DEHP metabolites, allowing the detection of this doping malpractice. The window of detection is approximately 2 days. The method might be applied to urine samples submitted for antidoping testing.

Using complementary mass spectrometric approaches for the determination of methylprednisolone metabolites in human urine.

RATIONALE: The metabolism of methylprednisolone is revisited in order to find new metabolites that could be important for distinguishing between different routes of administration. Recently developed liquid chromatography/tandem mass spectrometry (LC/MS/MS) strategies for the detection of corticosteroid metabolites have been applied to the study of methylprednisolone metabolism. METHODS: The structures of these metabolites were studied using two complementary mass spectrometric techniques: LC/MS/MS in product ion scan mode with electrospray ionization and gas chromatography/mass spectrometry (GC/MS) in full scan mode with electron ionization. Metabolites were also isolated by semipreparative liquid chromatography fractionation. Each fraction was divided into two aliquots; one was studied by LC/MS/MS and the other by GC/MS after methoxyamine-trimethylsilyl derivatization. RESULTS: The combination of all the structural information allowed us to propose a comprehensive picture of methylprednisolone metabolism in humans. Overall, 15 metabolites including five previously unreported compounds have been detected. Specifically, 16ß,17α,21-trihydroxy-6α-methylpregna-1,4-diene-3,11,20-trione, 17α,20ß,21-trihydroxy-6α-methylpregna-1,4-diene-3, 11-dione, 11ß,17α,21-trihydroxy-6α-hydroxymethylpregna-1,4-diene-3,20-dione, 11ß,17α,20ξ,21-tetrahydroxy-6α-hydroxymethylpregna-1,4-diene-3-one, and 17α,21-dihydroxy-6α-hydroxymethylpregna-1,4-diene-3,11,20-trione are proposed as feasible structures for the novel metabolites. In addition to the expected biotransformations: reduction of the C20 carbonyl, oxidation of the C11 hydroxy group, and further 6ß-hydroxylation, we propose that hydroxylation of the 6α-methyl group can also take place. CONCLUSIONS: New metabolites have been identified in urine samples collected after oral administration of 40 mg of methylprednisolone. All identified metabolites were found in all samples collected up to 36 h after oral administration. However, after topical administration of 5 g of methylprednisolone aceponate, neither the parent compound nor any of the metabolites were detected.