Measurement of progesterone in human serum by isotope dilution liquid chromatography–tandem mass spectrometry and comparison with the commercial chemiluminescence immunoassay

Abstract

Progesterone is one of the steroid hormones. The hormone is especially important in preparing the uterus for the implantation of the blastocyst and in maintaining pregnancy. Its concentration in serum is measured to determine ovarian function and to predict early pregnancy. The progesterone concentration is also important for in-vitro fertilization and embryo-transfer outcomes. We have established isotope dilution liquid chromatography-tandem mass spectrometry as a primary method for the measurement of progesterone in human serum. Progesterone and its isotopic analogue, progesterone-(13)C(2), in serum were monitored at mass transitions of m/z 315.2/109.2 and 317.2/111.2 respectively in multiple-reaction monitoring (MRM) mode with electrospray positive ionization. For validation of the method, progesterone in a National Institute of Standards and Technology standard reference material (NIST SRM) was measured, and the measured results were in good agreement with the reference values within the uncertainty. On the basis of the established method, progesterone certified reference material (CRM) was developed in this work. The certified value was (1.41 ± 0.036) μg kg(-1). The repeatability of 1.1% and reproducibility of 0.14% showed that ID LC-MS-MS is a reliable and reproducible method. The expanded uncertainty for the measurement of progesterone in the CRM was approximately 2.6% within 95% confidence limits. The detection limit of progesterone was approximately 0.6 μg kg(-1). The progesterone CRMs were distributed to representative clinical laboratories in the Republic of Korea for comparison with the chemiluminescence immunoassay (CLIA), which is the most sensitive immunoassay method. The results from the comparison showed quite a large bias among the participating laboratories. This implies that the CRM is a very important material for establishment of traceability to its practical use.

One-pot esterification and amidation of phenolic acids

“…In this study, two types of isotopomers of CAPE, [3,10-13 C2]CAPE (28) and [10- (29) were conveniently synthesized in high yields by using our method at the final condensation step. The isotopomer of CAPE (28) was synthesized as shown in Scheme 1. First, the hydroxyl groups of catechol moiety were protected with methylene acetal to avoid decomposition during the synthetic process.…”

“…The crude product was purified by silica gel column chromatography (ethyl acetate/hexane, 5:1 v/v) to afford 37 (0.72 g, 5.8 mmol, 97% yield) as a clear oil. 1 (28) To a solution of (E)-[3-13 C]caffeic acid (0.18 g, 1.0 mmol) in DCM (1.6 mL) and DMF (0.4 mL) was added 3.6 equiv of TEA (0.5 mL, 3.6 mmol) and 3.6 equiv of iBocCl (0.47 mL, 3.6 mmol) at 15 °C. After stirring for 5 min, 2.0 equiv of phenethyl alcohol (0.24 mL, 2.0 mmol) and 0.1 equiv of DMAP (12 mg, 0.1 mmol) were added, and the mixture was stirred at room temperature After stirring for 3 h, 10.0 equiv of piperidine (1.0 mL, 10.0 mmol) was added, and the reaction mixture was stirred at 0 °C for 1 h. The product was extracted thrice with ethyl acetate and washed with 1.0 M HCl, followed by saturated NaCl aq.…”

“…27 Recently, the accurate mass spectrometric determination of a phenolic compound in vivo has been reported using the corresponding isotopomer as the internal standard. 28 An isotopomer was also used as the labeled compound for the in vivo dynamics analysis. 29 An isotopomer of CAPE is an ideal internal standard and a metabolic tracer.…”

Rapid Quantification of Endogenous Steroids in Human Serum Using Leidenfrost Effect-Assisted Thermal Desorption Atmospheric Pressure Photoionization Orbitrap Mass Spectrometry

“…However, the concentrations of P in human serum evaluated by direct ECLIA (25.2 ng/mL) are nearly 10 times higher than those by LETD-APPI-MS (2.5 ng/ mL) and LC-APCI-MS (2.4 ng/mL) (Figure 3b), and a significant bias was also observed in previous reports. 17,50 When 10 ng/mL P solution was spiked into human serum, the additional concentrations of P evaluated by ECLIA (13.5 ng/ mL) are only slightly higher than those by LETD-APPI-MS (10 ng/mL) and LC-APCI-MS (9.5 ng/mL) (Figure 3b). Direct ECLIA are used for P measurement in most of the clinical laboratories in worldwide.…”

“…The highly sensitive quantification of endogenous steroids in human serum has received increasing attention in clinical diagnosis and pathological research. Because of their high sensitivity, low cost, and commercial availability, immunoassays (IAs), including radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and electrochemiluminescent immunoassay (ECLIA), are the most commonly used methods for the measurements of endogenous steroids in human serum. − However, IAs suffer from a few serious shortcomings, such as poor specificity and false-positive results from antibody cross-reactivity, one single steroid in a single run, and difficulty to identify unknown steroids. − In contrast, mass spectrometry (MS) based methods enable the untargeted identification of a multitude of steroids in a single run with high specificity. MS is normally combined with gas chromatography (GC–MS) or liquid chromatography (LC–MS) for the multiplexed analysis of endogenous steroids in the complex matrix of human serum. − Steroids are a typical kind of weak and nonpolar compounds with high boiling points that are not easy to volatilize.…”