Simultaneous quantification of cholesterol sulfate, androgen sulfates, and progestagen sulfates in human serum by LC-MS/MS

Abstract

Human adrenal glands, and those of some primates, secrete high concentrations of conjugated steroids, particularly 5-androsten-3 ␤ -ol-17-one-3-sulfate [dehydroepiandrosterone sulfate (DHEAS)], which reaches different peripheral tissues through blood ( 1 ). Sulfonation increases the solubility of steroids in water-based biological fl uids such as blood or urine. Sulfated steroids, also known in the literature as steroid sulfates or steroid sulfonates, represent the most abundant fraction of steroids in human blood. Because the physiological activity has been traditionally assigned to the unconjugated steroids, the classical dogma assumes that sulfated steroids are inactive forms of steroids which require sulfate cleavage to be active . In this context, human steroid sulfatase (STS) is the enzyme that desulfates the sulfated steroids to obtain free forms. The complementary reaction, sulfonation, is provided by sulfotransferase enzymes ( Fig. 1 ). This cycle allows for the regulation and excretion of steroids.The concept of steroid sulfates as inactive compounds is under revision. A specifi c transporter for sulfated steroids in the human testis was recently described ( 2 ). Moreover, Abstract Steroids are primarily present in human fl uids in their sulfated forms. Profi ling of these compounds is important from both diagnostic and physiological points of view . Here, we present a novel method for the quantifi cation of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantifi ed for the fi rst time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-␣ -hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 l of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R 2 > 0.99) and recovery for all the compounds, with limits of quantifi cation ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coeffi cient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantifi cation of sulfated steroids in human blood. -S á nchez-Guijo, A., V. Oji, M.

An untargeted metabolomics method for archived newborn dried blood spots in epidemiologic studies

“…For example, 16a-hydroxyDHEAS (C 19 H 28 O 6 S, #17929) has been measured in arterial cord plasma (Mitchell and Shackleton 1969), 5-androstrenetriol (sulfated form, C 19 H 30 O 6 S, #18138) has been measured in amniotic fluid immediately prior to delivery (Schindler and Siiteri 1968), and androsterone sulfate (C 19 H 30 O 5 S, #22415, #22424) and 16a-hydroxyDHEAS (C 19 H 28 O 6 S, #17929) have been measured in infant plasma (Sánchez-Guijo et al 2015). …”

Metabolic profiling putatively identifies plasma biomarkers of male infertility using UPLC-ESI-IT-TOFMS

“…44 Free testosterone, 5a-testosterone, and other steroids transported via the blood system affect target tissues by binding to cell nuclei or cytosolic receptors;…”

Applications and Challenges in Using LC–MS/MS Assays for Quantitative Doping Analysis

“…Wudy's group [55] reported a novel method for quantification of 11 intact steroid sulfates in human serum using LC-MS/MS and the assay offered linearity (R 2 > 0.99) and suitable recovery for all compounds, with LOQs ranging between 1 and future science group Applications & challenges in using LC-MS/MS assays for quantitative doping analysis Review 80 ng/ml. An UHPLC-MS/MS method for direct quantification of two T metabolites (3a-glucuronide-6b-hydroxyandrosterone and 3a-glucuronide-6b-hydroxyetiocholanolone) was developed and validated by Pozo's group [54] and this was accomplished with an UHPLC-MS/MS method because three interfering compounds were noted during GC-MS/MS analysis with the most intense ion transitions. For the free T or its conjugates and other EAAS after hydrolysis, LC-MS/MS methods are available for quantification [57] in urine, plasma and serum [58][59][60][61], hair [62,63] and saliva [64].…”

“…The T/E ratio has been traditionally measured with GC-MS after enzymatic hydrolysis, extraction and derivatization in WADA accredited laboratories. LC-MS/MS is an alternative method, using the negative ionization mode due to the acidity of the glucuronide and sulfate moiety [53,54] or the positive ionization mode because of the proton affinity of the steroid keto functional group (T and ET) [55,56], as shown in Table 5. Different extraction steps have been described for LLE [54], or SPE [53,55,56].…”

“…LC-MS/MS is an alternative method, using the negative ionization mode due to the acidity of the glucuronide and sulfate moiety [53,54] or the positive ionization mode because of the proton affinity of the steroid keto functional group (T and ET) [55,56], as shown in Table 5. Different extraction steps have been described for LLE [54], or SPE [53,55,56]. Badoud's group [53] reported a sensitive and selective method for simultaneous quantification of major urinary metabolites after T intake using ultra high-pressure LC coupled to quadrupole time-of-flight mass spectrometer.…”