The experience in the development and validation of method for testosterone measurement in blood serum of premenopausal women using HPLC-MS/MS

Abstract

Testosterone assessment is essential for detecting biochemical hyperandrogenism, one of the important diagnostic criteria of polycystic ovary syndrome (PCOS) both in clinical practice and in epidemiological studies. Currently, tandem liquid chromatography-mass spectrometry (LC-MS/MS) is the most preferred technique to measure testosterone level in women. Its validation is important to reproducibility of androgen tests results for clinical practice and for epidemiological studies of the prevalence PCOS.The aim of the study. To develop and validate a method for determining total testosterone in blood serum using highly efficient LC-MS/MS to assess androgenemia in the epidemiological study of the prevalence of PCOS and its phenotypes in Eastern Siberia (ESPEP STUDY).Materials and methods. We determined a total testosterone level in serum blood using triple quadrupole mass spectrometer LCMS-8060 (Shimadzu, Japan). The protocol of technique was developed using self-prepared purified human testosteronefree serum with a known concentration of analyzed compound. We used the serum samples of women of reproductive age to test the developed method.Results. Optimum chromatographic conditions were obtained with a Kromasil 100-2.5-C18 column (2.1 mm × 100 mm; AkzoNobel, Netherlands), and an isocratic elution mode using a mobile phase consisting of acetonitrile and 0.1 % aqueous solution of formic acid. The total flow rate was 0.35 ml/min. The lower limit of quantification was 5 ng/dl with an average accuracy of 100.2 %. During the approbation of the method in a test population sample of 1138 premenopausal women (mean age – 34.3 ± 6.3 years), the median testosterone concentration was 26.9 ng/dl.Conclusion. It was found that the proposed method for determining testosterone in blood serum has acceptable linearity and reproducibility and meets the requirements for bioanalytical methods under the regulatory documentation. This method can be used for clinical practice and epidemiological study of the prevalence of PCOS.