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Conventionally, the concentration of steroidal sulfates was estimated by indirect or immuno-based assays before the use of liquid-chromatography tandem mass spectrometry (LC-MS/MS). In the present study, a validated LC-MS/MS method is described for the simultaneous quantification of dehydroepiandrosterone sulfate (DHEA-S), estrone sulfate (E-1-S), androsterone sulfate (ADT-S), pregnenolone sulfate (Preg-S) and allopregnanolone sulfate (Allopreg-S). E-1-S binding to serum proteins was observed, especially for the high concentration quality control serum samples, leading to -10 to -15% bias using a polymer-based SPE. This protein binding can be efficiently eliminated using a Waters Oasis (TM) WAX following the same extraction procedure. Most likely, the E-1-S binding elimination on Oasis (TM) WAX can be attributed to its different sorbent structure, where the benzeno group of E-1-S can interact with the benzene of the backbone of Oasis (TM) WAX. With this improvement, the method has been fully validated according to the FDA guidelines. The low quantification limits (LLOQs) are 40 ng/mL, 40 pg/mL, 5 ng/mL, 1.5 ng/mL and 0.25 ng/mL for DHEA-S, E-1-S, ADT-S, Preg-S and Allopreg-S, respectively. A good linearity is obtained with R>0.99 for all compounds within the appropriate calibration range. Accuracies of all levels of QCs are within the range of 10% for DHEA-S, E-1-S, ADT-S and Preg-S while for Allopreg-S, the accuracy is within the 15% range. The interday coefficient variance is 5.5-9.5% for the low limits of quantification of all five compounds while values of 1.3-9.9% are found for higher levels of QCs of all five compounds. Recovery of the five compounds in stripped serum is equivalent to that in unstripped serum. The average recovery difference is less than 5% between stripped and unstripped serum for each compound. All results of other test parameters such as matrix, hemolysis and lipemic effects as well as stabilities meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. (C) 2015 Elsevier Ltd. All rights reserved.