Sample-multiplexing by derivatization using multiple analogous reagents for enhancing throughput in LC/ESI-MS/MS assay of steroids: Plasma 17α-hydroxyprogesterone as an example

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2020-06, Vol.1146, p.122117-122117, Article 122117
• Main Authors: Kamemura, Miki, Yokota, Mai, Ogawa, Shoujiro, Sugiura, Takahiro, Higashi, Tatsuya
• Format: Article
• Language: English
• Subjects: 17α-Hydroxyprogesterone; Derivatization; Girard-type reagent; LC/ESI-MS/MS; Sample-multiplexing
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2020.122117

[Display omitted] •A sample-multiplexed LC/ESI-MS/MS method for 17OHP was developed.•The method employed the derivatization with 4 Girard-type reagents.•17OHP in 4 different plasma samples was quantified within a single run.•The total LC run time was reduced by about 60% for the 32 samples.•The method was successfully used for the analysis of cord plasma samples. The measurements of steroids in biological fluids are of importance for the diagnosis and treatment of many diseases. Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has a high specificity and accuracy for the steroid analysis, whereas it has a lower analysis throughput, which could become a big issue in clinical practice. One of the promising solutions to this issue is the multiplexing of samples in the same injection. In this study, the utility of the sample-multiplexing by the derivatization using multiple analogous reagents was evaluated for enhancing the throughput of the LC/ESI-MS/MS assays of steroids. The plasma 17α-hydroxyprogesterone (17OHP), which is a diagnostic marker for the 21-hydroxylase deficiency, was chosen as the model analyte. The four plasma samples (20 μL each) were separately derivatized with one of four different analogous Girard-type reagents, combined, then injected together into the LC/ESI-MS/MS. By this method, four plasma samples could be analyzed within a single LC run. The developed method could significantly reduce the total LC run time (about 2/5 for 32 samples, compared with the conventional method) with a satisfactory sensitivity (lower limit of quantification 0.5 ng/mL), precision (intra- and inter-assay RSDs ≤ 4.0% and ≤ 3.5%, respectively) and accuracy (97.6–106.7%), and negligible matrix effect. The developed method had a satisfactory applicability for the quantification of 17OHP in the cord plasma samples.