Derivatization‐based quadruplex LC/ESI–MS/MS method for high throughput quantification of serum dehydroepiandrosterone sulfate

Abstract

The quantification of the circulating dehydroepiandrosterone sulfate (DHEAS) might be of diagnostic help for several diseases. For the DHEAS quantification, LC/ESI–MS/MS has the advantage of a high specificity compared with immunoassay, whereas LC/ESI–MS/MS has room to improve the analysis throughput. One of the promising solutions to enhance the analysis throughput is sample‐multiplexing in the same injection, which can reduce the total LC/ESI–MS/MS run time. In this study, a quadruplex LC/ESI–MS/MS method was developed to quantify DHEAS in four different serum samples in a single run. After the four samples were separately deproteinized and derivatized with one of four Girard reagents (Girard reagent T, P and their isotopologs), the resulting samples were mixed, then injected into the LC/ESI–MS/MS. The applicability and advantage of the developed method were evaluated based on the analysis of nine batches of serum samples from healthy subjects (total 36 samples). The limit of quantitation was 0.050 μg/ml, which was sensitive enough for clinical laboratory use. The method was precise (intra‐ and inter‐assay RSDs ≤ 3.6%), accurate (94.4–108.1%) and robust for the matrix effects. The analysis time was also shortened by about 60% for 36 samples by the introduced method compared with the conventional method.

An LC/MS/MS method for quantifying testosterone and dehydroepiandrosterone sulfate in four different serum samples during a single run

“…It is well known that the 3-oxo-4-ene steroids, such as TS, are readily derivatized with the Girard reagents. 17,23 Although the keto group of DHEAS (at the 17-position) is less reactive than that of TS (at the 3-position), our previous study verified that the derivatization using any Girard reagents almost quantitatively proceeded to DHEAS under the stated conditions 19 (Fig. 1).…”

“…DHEAS and 2 H4-DHEAS (IS for DHEAS) were synthesized in our laboratories and the same as those used in a previous study. 19 Stock solutions were prepared by dissolving a precisely-weighed quantity of the steroids with ethanol.…”

“…dGT and dGP were synthesized in our laboratories. 19,21 An Oasis ® HLB solid-phase extraction (SPE) cartridge (30 mg adsorbent; Waters, Milford, MA) was used to purify the serum samples. All other reagents and solvents were of analytical grade or LC/MS grade.…”

“…This strategy was also applied to the steroid quantification. [17][18][19][20] A set of structurally analogous derivatization reagents with different masses is indispensable for the sample-multiplexing strategy; the resulting derivatives closely elute from the LC, show similar ESI-MS/MS behaviors, and are separately quantified by different selected reaction monitoring (SRM) transitions. Both TS and DHEAS have a carbonyl group, therefore, Girard reagents can be used to derivatize these androgens.…”

“…We have reported the sample-quadruplex LC/ESI-MS/MS method for the high throughput quantification of serum DHEAS using the Girard reagents. 19 It is of significance to expand this strategy to the quantification of the most clinically-important androgen, TS, for the assessment of androgen status.…”