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.

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.

On the road of dried blood spot sampling for antidoping tests: Detection of GHRP-2 abuse.

Dried blood spots (DBSs) sampling is gaining support by the antidoping community because of simplicity and cost-effective characteristics, especially in collection, transport, and storage. Nevertheless, DBS applicability demands specific studies for each of the analytes proposed for testing. Here, GHRP-2 has been selected as a representing member of the growth hormone-releasing peptides (GHRPs) family to provide further evidence of DBS suitability for GHRPs abuse detection in sport testing. An analytical procedure to extract GHRP-2 and its main metabolite (AA-3) from DBS and to detect them by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. The method has been validated for the detection of GHRP-2. Specificity and identification capabilities have been assessed in agreement with antidoping guidelines. The low AA-3 levels found in DBS samples prevented its effective application for the determination of this metabolite. The limit of detection (LoD) for GHRP-2 has been established at 50 pg/ml. Long-term stability (>2 years) has been confirmed. The procedure has been successfully applied to actual DBS samples from an administration study with a single intravenous dose of GHRP-2 (100 µg) being detected up to 4 h after drug injection. GHRP-2 concentrations have been higher in venous blood DBS than in capillary blood DBS. Despite the observed differences, a similar detection window has been achieved independently of the type of blood used. In summary, this study provides specific evidence supporting DBS usefulness to detect GHRP-2, and potentially other GHRPs family members, for antidoping tests.

Development and validation of a fast and reliable method for the quantification of glucagon by liquid chromatography and tandem mass spectrometry.

INTRODUCTION: The quantitation of glucagon remains a challenging immunoassay, mainly due to cross-reactivity. A sensitive, rapid and specific intact glucagon method is therefore necessary for quality routine analysis. A tandem mass spectrometry method to fulfill this objective is described in this work. METHODS: Glucagon was extracted from plasma employing a mixed-mode anion exchange solid-phase extraction. Sample stability was assessed in K2-EDTA and P800 tubes at different temperatures. We compared our method to two different immunoassays. FDA and EMA guidelines were followed for validation. An external quality control program served for comparison with other laboratories. RESULTS: Assay imprecision was below 4%. Recoveries were within 95-103%. LoQ was 8.75 pg/mL. Total analytical CV was 2.91%. Samples were found stable at 4 °C for less than 4 h. Diasource® RIA disagreed with our method. Mercodia® ELISA provided a closer agreement, also proven by external quality control samples. CONCLUSIONS: A rapid and specific LC-MS/MS method for glucagon quantitation has been developed, validated and is suitable to routine care. The simplicity and the good performances in terms of time and specificity, could open the possibility to establish a standardized method for glucagon.

Automation of RNA-based biomarker extraction from dried blood spots for the detection of blood doping.

Aim: Transcriptomic biomarkers originating from reticulocytes measured in dried blood spots (DBSs) may be reliable indicators of blood doping. Methods/results: Here, we examined changes in the expression levels of the erythropoiesis-related ALAS2, CA1 and SLC4A1 genes in DBS samples from elite athletes and volunteers of clinical study with recombinant erythropoietin dose. Conclusion: By comparing the mean intraday coefficients of variation for ALAS2L, ALASLC, CA1 and SLC4A1 between manual and automated RNA extractions, an average improvement was observed, whereas the assessment of interday variability provided comparable results for both manual and automated approaches. Our results confirmed that RNA biomarkers on DBS support are efficient to detect blood doping.

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.

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.

If you play with fire, you may get burned.

This article reports an interesting doping case in the late 1990s involving the physical tampering of a urine sample containing a prohibited substance, by adding an alcoholic beverage. The regulations and knowledge available at that time allowed consideration of the case as a sanctioned manipulation and the prohibited substance as an additional element that explained the reasons for it. The development of the case involved some aspects that appeared for the first time in doping control. These include, among others, the participation of an enologist as part of the B sample confirmation, forensic inspection of the sample container, the early development of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS), and the first public hearing by the Court of Arbitration for Sport (CAS), which allowed a wide following of the developments of the case. The information was especially interesting for the general public as the athlete had had, at that time, great Olympic success.

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.

Whole Blood Storage in CPDA1 Blood Bags Alters Erythrocyte Membrane Proteome.

Autologous blood transfusion (ABT) has been frequently abused in endurance sport and is prohibited since the mid-1980s by the International Olympic Committee. Apart from any significant performance-enhancing effects, the ABT may pose a serious health issue due to aging erythrocyte-derived "red cell storage lesions." The current study investigated the effect of blood storage in citrate phosphate dextrose adenine (CPDA1) on the red blood cell (RBC) membrane proteome. One unit of blood was collected in CPDA1 blood bags from 6 healthy female volunteers. RBC membrane protein samples were prepared on days 0, 14, and 35 of storage. Proteins were digested in gel and peptides separated by nanoliquid chromatography coupled to tandem mass spectrometry resulting in the confident identification of 33 proteins that quantitatively change during storage. Comparative proteomics suggested storage-induced translocation of cytoplasmic proteins to the membrane while redox proteomics analysis identified 14 proteins prone to storage-induced oxidation. The affected proteins are implicated in the RBC energy metabolism and membrane vesiculation and could contribute to the adverse posttransfusion outcomes. Spectrin alpha chain, band 3 protein, glyceraldehyde-3-phosphate dehydrogenase, and ankyrin-1 were the main proteins affected by storage. Although potential biomarkers of stored RBCs were identified, the stability and lifetime of these markers posttransfusion remain unknown. In summary, the study demonstrated the importance of studying storage-induced alterations in the erythrocyte membrane proteome and the need to understand the clearance kinetics of transfused erythrocytes and identified protein markers.

Detection of erythropoiesis-stimulating agents in a single dried blood spot.

The use of dried blood spots (DBS) for anti-doping purposes would facilitate an increase in the number of blood samples because it eliminates the need for specialized personnel and involves minimal invasiveness, reduced costs, stability, and easy transportation and storage. Here, the electrophoretic methodology established by the World Anti-Doping Agency (WADA) to detect erythropoiesis-stimulating agents (ESAs) has been adapted to evaluate their applicability to DBS. A qualitative procedure to detect recombinant erythropoietin (rEPO), novel erythropoiesis-stimulating protein (NESP), and continuous erythropoietin receptor activator (CERA) in a single DBS was optimized and validated. For rEPO and NESP, confirmation was performed in finger-prick DBS from a pilot study and an administration patients study, respectively. For CERA, detection capabilities were evaluated in DBS prepared with modeled-blood spiked with known concentrations of the protein. Main validation parameters concerning DBS sampling such as stability, hematocrit influence, and blood type (capillary vs. venous) described minor variations. Onsite drying appeared not to be essential before transport. Intra- and inter-day variation range was 2.9%-23.5%. Linearity was maintained (r ≥ 0.9) and ESAs were robustly recovered (CV ≤ 20.2%). The validated method permitted the detection of treated subjects after 48 hours and 17 days of rEPO and NESP administration, respectively. The reproduction of a CERA pharmacokinetics showed good possibilities for the method with a detection window that could reach 16 days after its actual administration. Thus, results provided here reinforce the suitability of DBS blood sampling for the analysis of ESA misuse in sports drug testing.

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part II: Intramuscular administration.

In the fight against doping, the introduction of alternative markers to the steroid profile can be considered as an effective approach to improve the screening capabilities for the detection of testosterone (T) misuse. The aim of this study was to evaluate the potential of several T metabolites (cysteinyl conjugated and glucuronoconjugated resistant to enzymatic hydrolysis) to detect both the transdermal and the intramuscular administration of T. In Part I of the study, we studied the potential of these metabolites for the detection of T transdermal administration. Results revealed that resistant glucuronides can be a suitable complement to the current steroid profile. In this, Part II, dedicated to the intramuscular administration, we studied the potential of cysteinyl conjugated, resistant glucuronoconjugated and 1-cyclopentenoylglycine (1-CPG) for the detection of a single intramuscular injection of T cypionate. Possible differences in the excretion profile of all markers were explored between individuals with low basal (n=6) and medium basal (n=6) values of the testosterone/epitestosterone ratio (T/E). The results showed that all tested markers presented low intra-individual stability in basal conditions. Despite this, all glucuronoconjugated markers and 1-CPG, but not the cysteinyl conjugated markers, provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile. Based on the results obtained from the 2 parts of this study and from previously reported data, the potential applicability and the limitations of including these markers in the steroid profile are discussed.

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part I: Transdermal administration.

Although the introduction by the World Anti-Doping Agency (WADA) of the steroid module of the athlete biological passport (ABP) marked an important step forward in the screening of testosterone (T) misuse, it still remains one of the most difficult challenges in doping control analysis. The urinary determination of alternative markers has been recently reported as a promising tool for improving the screening of T oral administration. However, their evaluation for other, commonly used, administration routes is still required. The main goal of this study is the evaluation of the potential of 2 groups of metabolites (cysteinyl conjugated and glucuronoconjugated) after transdermal and intramuscular administration of T. Their suitability was evaluated in individuals with both low basal (L-T/E) and medium basal (M-T/E) values of T/E. In this Part I, we evaluated the urinary excretion profile of these 2 groups of T metabolites after the administration of 3 doses of T gel to 12 volunteers (6 L-T/E and 6 M-T/E) for 3 consecutive days. For this purpose, 9 different concentration ratios (5 cysteinyl conjugated and 4 glucuronoconjugated markers) were studied. Both, the intra-individual variability and the detection windows (DW) obtained by each ratio were evaluated. Cysteinyl conjugates showed a general low intra-individual variability and DWs that were shorter than any other tested marker. Despite the relatively large intra-individual variability, the DWs reached by glucuronoconjugates (2-3 days) were similar to those obtained by markers currently included in the ABP. Overall; this evaluation advises for the introduction of additional glucuronoconjugated markers in the screening of transdermal T administration.

A fast and simple method for simultaneous measurements of 25(OH)D, 24,25(OH)2D and the Vitamin D Metabolite Ratio (VMR) in serum samples by LC-MS/MS.

BACKGROUND: Rapid, easy and reliable measurement of the major vitamin D metabolites is required in order to fulfill the needs of a clinical routine laboratory. To overcome these challenges, we have developed and validated a LC-MS/MS method for the quantification of 25-hydroxyvitamin D2 and D3, epi-25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. METHODS: Sample preparation was based on precipitation and centrifugation of 100µL of patient serum, followed by injection into the LC-MS/MS system. Samples from Vitamin D Standardization Program (n=80) and patient samples (n=281) have been compared with a reference LC-MS/MS method. For the analytical validation NIST and Labquality quality control materials were used. RESULTS: Mean intra-assay and inter-assay imprecision were <6.0 and 6.4% and mean recoveries were within 95-104%. LOQ's were 0.5µg/L for 24,25(OH)2D3, 1.1µg/L for 25(OH)D3 and epi-25(OH)D3 and 1.7µg/L for 25(OH)D2. A 3% bias obtained between the proposed and the reference method satisfies Vitamin D Standardization Program recommendations. CONCLUSIONS: We present a rapid, easy, reliable and cost-effective method completely adequate for routine testing, which permits the measurement of the ratio of 24,25-dihydroxyvitamin D to 25-hydroxyvitamin D, Vitamin D Metabolite Ratio (VMR), in serum samples.

Quantifying endogenous androgens, estrogens, pregnenolone and progesterone metabolites in human urine by gas chromatography tandem mass spectrometry.

A method for the quantitation of 22 urinary steroids (androgens, estrogens and the main pregnenolone and progesterone metabolites) by means of gas chromatography tandem mass spectrometry using a triple quadrupole analyzer has been developed. Two different enzymatic hydrolysis protocols were investigated; one capable of releasing steroids present as both sulfates and glucuronides (total fraction), and another with ß-glucuronidase activity only. After selecting adequate internal standards and choosing the optimal instrumental parameters, i.e. chromatographic separation and ion transition conditions, the method was fully validated using both hydrolysis protocols. The method was shown to be linear (r >0.99) in the range of endogenous concentrations for all studied steroids with extraction recoveries higher than 80%. The use of labeled internal standards allowed for both a correct quantification and the evaluation of the rate of deconjugation for sulfates and glucuronides in every sample. In general, the sensitivity of the method was suitable for the detection of the endogenous levels, with limits of quantification ranging from 0.1 to 20ng/mL. Accuracies ranging from 80% to 120%, and relative standard deviations below 25% in intra- and inter- assay experiments were found for most of the analytes. The applicability of the validated method was tested by quantifying twenty-two metabolites in 24-h urine samples collected from healthy individuals. The ranges for the excretion of steroids in the total and glucuronide fractions obtained with the new method were compared with those available in the literature. By comparing the figures in both fractions, an estimation of the percentage that the sulfation represents for each steroid was also calculated. The presence of side enzymatic activities and the utility of the method for clinical studies as well as for doping control analysis is discussed.

Evaluation of two glucuronides resistant to enzymatic hydrolysis as markers of testosterone oral administration.

Testosterone (T) has traditionally been the most commonly reported doping agent by doping control laboratories. The screening of T misuse is performed by the quantification of six endogenous androgenic steroids and the ratio T/E included in the Athlete Biological Passport (ABP). The inclusion of additional metabolites can improve the screening capabilities of ABP. In this study, the potential of 3α-glucuronide-6ß-hydroxyandrosterone (6OH-Andros3G) and 3α-glucuronide-6ß-hydroxyetiocholanolone (6OH-Etio3G) as markers of T oral administration was evaluated. These glucuronides have been shown to be resistant to enzymatic hydrolysis and their quantification by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was reported as the only way to obtain feasible results. Urine samples were collected from five volunteers before and after the oral administration of 40mg of T undecanoate and were analyzed by a LC-MS/MS method recently developed. Concentration of 6OH-Andros3G and 6OH-Etio3G compounds and those of the glucuronides of T (TG), epitestosterone (EG), androsterone and etiocholanolone were established and different concentration ratios were calculated. The detection windows (DWs) for the T administration obtained by each selected ratio were compared to the one of TG/EG. The results showed that four out of the nine tested markers presented DWs much larger for all volunteers than those obtained by the World Anti-Doping Agency established T/E marker or other alternative markers. The 6OH-Andros3G/EG, 6OH-Etio3G/EG, 6OH-Andros3G/TG and 6OH-Etio3G/TG markers were able to identify the T abuse up to 96h after the administration, extending our detection capability for the misuse up to 84h more than the classic marker. The importance of these markers was also highlighted by their prolonged capacity to detect the T misuse in the case of one volunteer whose TG/EG barely exceeded his individual threshold. As a consequence, the four markers presented in this study seem to have an exceptional potential as biomarkers of T oral administration.

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.