Isotopic characterisation of in-house purified progesterone for 13C/12C isotope ratios by multicollector ICP-MS

Abstract

In this paper, a multicollector inductively coupled mass spectrometer (MC-ICP-MS) has been used for the first time to characterise a steroid sample for carbon isotope ratio traceable to SI. The progesterone sample was purified in house using semi-preparative high performance liquid chromatography (HPLC) and characterised using a multi-method approach. Carbon ¹³C/¹²C isotope amount ratios have been measured for the progesterone standard and NIST RM materials have been used as QCs to test instrumental performance and linearity over the δ¹³C range from −46 to +37‰. Internal normalisation by measuring boron ¹¹B/¹⁰B isotope amount ratios has been used to correct for the effects of instrumental mass bias. Absolute ¹³C/¹²C ratios have been measured and corrected for instrumental mass bias and full uncertainty budgets have been calculated using the Kragten approach. Alternatively raw ratios have been measured for four isotopic NIST RMs and used for external calibration. This work proves the suitability of the method for the measurement of carbon isotope ratios within the natural range of variation of organic carbon compounds. The potential of the method for the production of isotopic reference standards to be used in metabolic studies, sports science and forensic testing is shown. Measured/corrected value for carbon isotope ratios traceable to the SI for the compound of interest and its combined uncertainty budget (including instrumental error and each parameter contributing to Russell expression for mass bias correction) were found to be 0.010853 ± 0.000011. The main advantages of the method versus conventional Isotope Ratio Mass Spectrometry (IRMS) measurements are that the carbon isotope ratios are measured as C⁺ instead of CO₂⁺ and therefore an oxygen correction is not required. Moreover, absolute carbon isotope ratios can be measured and expressed as ¹³C/¹²C instead of δ¹³C (relative differences in ‰ between a reference standard and the sample).