Quantitation of serum pregnanediol-3-glucuronide level in different endocrine statuses by LC-MS/MS

Abstract

Pregnanediol-3-glucuronide (PdG) is the major terminal metabolite of progesterone, playing an important role in physiological processes, such as the female menstrual cycle, pregnancy (supports gestation), embryogenesis and maternal immune response of humans and other species. Hence, accurate measurement of PdG in serum/plasma is needed for the evaluation of progesterone production. However, such high-specificity determination of PdG is lacking in clinical sample detection. In this study, a highly sensitive and accurate LC-MS/MS method was firstly established for subsequent measurement of PdG in serum of three different female groups: thyroid cancer patients (TCs), healthy controls (HCs) and pregnant women. The factors affecting the sample preparation, MS/MS method, gradient elution program, selection of chromatographic column and internal standard (IS) have been optimized in this study. Compared with enzyme immunoassay (EIA) method, we used LC-MS/MS to shorten analysis time, increase sensitivity, raise specificity, simplify sample preparation, and reduce costs. As a result, the linear range of the method was from 0.38 to 100 ng/mL with a limit of quantification (LOD) of 0.01 ng/mL. Precision assays showed that relative standard deviation (RSD) was less than 10.6, accuracy was between 90.6 % and 110.4 %, and mean recovery was 103.4 %. In addition, the serum PdG/creatinine levels were significantly down-regulated in TCs and up-regulated in pregnant women versus HCs. Receive operating characteristic curve (ROC) analysis enabled to identify TC with a sensitivity of 83.3 %, specificity of 68.0 % and area under curve (AUC) of 0.781 (95 % CI: 0.684 to 0.879), and it enabled to identify pregnant women with a sensitivity of 94.7 %, specificity of 68.5 % and AUC of 0.811 (95 % CI: 0.732 to 0.890). Our results implied that an increase in female serum PdG/creatinine level might be associated with a risk of pregnancy, but serum PdG/creatinine decreasing might be related to a risk of TC.

Keywords: LC–MS/MS; Pregnancy; Pregnanediol-3-glucuronide; Serum; Thyroid cancer.