RATIONALE: Urine contains free and conjugated steroids. Total and free steroids were comprehensively quantified; UPLC-MS/MS based on Li adduction allowed for detecting thirteen 3-OH-containing steroids, two of which were detected in human urine for the first time. METHODS: Free urinary steroids were isolated by solid-phase extraction (SPE) with 80% acetonitrile. The total steroids were prepared by enzymatic treatment of urine with a cocktail of sulfatase and glucronidase, protein precipitation, and separation with the above SPE. The free and total steroids were separately analyzed by UPLC-MS/MS with and without introduction of Li + solution. The steroids were quantified by two standard curves created using product ion transitions derived from MH + and [M+Li] +. RESULTS: Two groups of human urine, male and female urine, were analyzed. The absolute amount of each steroid was determined based on creatinine levels. The differences between the male and female groups are clearly attributable to sex steroids. 7-OH P5 and 7-OH DHEA were, for the first time, quantified in the total steroids of female urine, and the latter was identified in both female and male urine. CONCLUSIONS: By combining UPLC-MS/MS based on lithium ion incorporation with conventional UPLC-MS/MS, a total of 29 steroids were identified in human urine containing two newly found steroids.

RATIONALE: The thiosuccinimide linker is widely used in the synthesis of bioconjugates. However, it is susceptible to hydrolysis and is transformed into its hydrolyzed and/or the isobaric thiazine forms, the latter of which is a fairly common product in a conjugate that contains a cysteinyl peptide. MALDI-MS and MS/MS are useful for differentiating these isobaric species. METHODS: Four cross-linked peptides with thiosuccinimide linkers were synthesized. Analogs with the linker that were transformed into thiazine and/or the hydrolyzed thiosuccinimide linkers were then generated by incubating the samples at neutral or basic pH. All of the cross-linked peptides were purified by rp-HPLC and differentiated by MALDI-MS, -MS/MS and UVPD. RESULTS: A cysteinyl peptide-containing conjugate, the thiosuccinimide form, was largely transformed into the hydrolyzed or thiazine forms after incubation at neutral or basic pH. MALDI-MS allowed the three forms to be differentiated: the thiosuccinimide and its hydrolysis product gave two constituent peptides after reductive cleavage between the Cys and succinimide moieties; no fragment ions were produced from the thiazine form. In addition, MALDI-MS/MS of the thiosuccinimide form yielded two pairs of complementary fragment ions via 1,4-elimination: Cys-SH and maleimide, and dehydro-alanine and thiosuccinimide, which are different from those produced via reductive cleavage in MALDI-MS. The thiazine form gave fragment ions resulting from the cleavage of the newly formed amide bond in the linker that arose from thiazine formation. CONCLUSIONS: The thiosuccinimide (but not thiazine) form of the cross-linked peptide yielded individual constituent peptides in MALDI-MS; MALDI-MS/MS showing specific 1,4-elimination for the thiosuccinimide form and cleavage at the newly formed peptide bond via transcyclisation for the thiazine form.