Determination Of Serum Progesterone By Isotope Dilution Gas Chromatography/Mass Spectrometry

Progesterone is a steroid hormone that is involved in the regulation of the menstrual cycle and is especially important in preparing the uterus for the implantation of the blastocyst and in the maintenance of pregnancy. Measurements of serum progesterone are widely used for the detection of ovulation and the investigation of pregnancy. Serum progesterone measurements are routinely performed with immunoassays which are simple and sensitive but susceptible to various sources of interference. Thus there is a strong need to establish a critically evaluated reference measurement procedure for serum progesterone.The aim of our study is to develop a candidate reference measurement procedure for the measurement of serum progesterone. Reference measurement procedures need to be analytically specific and precise and have an uncertainty of measurement commensurate with its use in assessing the accuracy of routine methods and characterizing reference materials. Isotope dilution mass spectrometry(ID-MS) is highly specific and precise and is widely accepted as one of the most reliable analytical principles. In ID-MS, an isotopically labeled analogue of the analyte is used as an internal standard and the analyte is quantified by measuring the ratio of the analyte to the internal standard with mass spectrometry. Since the internal standard has essentially the same properties as the analyte, once equilibrated, it behaves very similarly to the analyte during the analysis and the absolute recovery of the analyte does not affect the accuracy of the analysis. Mass spectrometry is highly specific and is a necessity of isotope dilution analysis. ID-MS was thus selected as the measurement principle in our development of the reference procedure for serum progesterone. In ID-MS analysis, mass spectrometry is normally coupled with gas chromatography(GC/MS) or liquid chromatography. GC/MS was used in the present study.The serum matrix is complex and the concentration of progesterone in serum is low(normally ranging from 0.1 to 20 ng/ml in adult females. Besides, there are plentiful analogue compounds in serum, such as cholesterol and other steroid hormones, which potentially interfere with progesterone analysis. It is necessary to remove these substances before GC/MS analysis. Various approaches for recovering progesterone from serum were investigated and a simple procedure for the extraction and purification of progesterone was established. The procedure involved an extraction of serum with hexane and a treatment of the extracts with 2-hydroxypropyl-β-cyclodextrin(HP-β-CD). The cyclodextrin has an unique structure of a hydrophobic internal cavity and a hydrophilic outer surface and thus selectively isolate progesterone form other serum substances.GCMS analysis of progesterone also requires a pre-column derivatization for improved gas chromatographic efficiency and mass spectrometric detectability of the analyte and the internal standard. Acylation with heptafluorobutyric anhydride was selected as the derivatization reaction and various reaction conditions was investigated. With acetone as the solvent progesterone was converted to the 3-enol heptabutyrate within 60 min at room temperature.Various gas chromatographic and mass spectrometric conditions were also tested and optimized for best chromatographic and mass spectrometric performances.Based on the above investigations, an ID-GC/MS method for serum progesterone was established. Serum samples were mixed with[3,4-¹³C₂] progesterone(the internal standard) and extracted with n-hexane. The hexane extract was treated with HP-β-CD for the isolation of progesterone with other serum substances. The progesterone recovered was converted to 3-enol heptafluorobutyrate and analyzed by GC/MS with selected ion monitoring. The concentration of serum progesterone was calculated by a bracketing calibration.The procedure showed total coefficients of variation(CVs) of 0.69% to 2.12%. The detection limit at a signal-to-noise ratio of three was 1.5 pg of progesterone. The analytical recoveries ranged from 98.3 % to 100.1 %. The accuracy of the measurement was further evaluated by analyzing a certified reference material(ERM-DA347) and the bias was less 1.5%. We also participated with this method in the international comparison CCQM-P77b(determination of serum progesterone) held by the Consultative Committee for Amount of Substance(CCQM). Our results agreed well with that reported by other participating national metrology institutes.In Conclusion, a measurement procedure for progesterone in serum by isotope dilution gas chromatography mass spectrometry has been developed. The method is accurate, precise and sensitive and may be used as a candidate reference method.