Two‐dimensional gas chromatography with heart‐cutting for isotope ratio mass spectrometry analysis of steroids in doping control

Abstract

The accuracy and precision of gas chromatography combustion isotope ratio mass spectrometry (GC‐C‐IRMS) measurements are highly dependent on analyte purity. Reliable analysis of urinary steroids for doping control therefore requires extensive and time‐consuming sample preparation (i.e. liquid chromatography fraction collection) prior to GC‐C‐IRMS analysis. The use of two‐dimensional GC (GC‐GC) with heart‐cutting (Deans Switch) as a possible approach to reduce the sample purification required for IRMS analysis is described herein. The system uses a low thermal mass oven (LTM) incorporated into an existing GC‐C‐IRMS system. GC‐GC allowed the use of a cyanopropyl/phenyl column in the first dimension to optimize the separation of underivatized steroids, while a phenyl‐methylpolysiloxane column in the second dimension focuses the selectively cut analytes into narrower peaks for more sensitive and reliable MS analysis. In addition, to confirm analyte identity, eluent from the second GC was split, with 20 % entering a scanning MS, and 80 % flowing to the IRMS. As a proof concept, the developed method was then used to analyze a single spot urine (5 ml) from an individual receiving T therapy (2 × 50 mg sachets of Testogel®). The T delta value (−27.8 ‰, [T] = 38 ng/ml) was clearly distinct from 11‐ketoetiocholanolone (−22.5 ‰) (used as an endogenous reference compound (ERC)), indicating T as being of exogenous origin. The simultaneous analysis by the scanning MS yielded a full scan mass spectrum of the same chromatographic peak, thus confirming the peak to be T. Copyright © 2012 John Wiley & Sons, Ltd.

IRMS delta values (13C) of nandrolone and testosterone products available in the UK: Implications for anti‐doping

“…This is not statistically significant (one sample t-test, p > 0.05) compared to the laboratoryassigned value (−29.6‰). 20 This step is also important to monitor co-elution of interferents with the target steroids which, if present, may alter the measured carbon isotope value. 10,19 All δ 13 C values for steroids reported in this manuscript are for the free steroid, with correction for the influence of the acetate moiety done using a mass balance approach.…”

“…10 Splitting of GC eluents into the MSD and IRMS alleviates this problem by allowing simultaneous acquisition of full scan mass spectra of chromatographic peaks along with isotope ratio analysis. 20 This step is also important to monitor co-elution of interferents with the target steroids which, if present, may alter the measured carbon isotope value. All chromatograms were found to be free of interfering compounds, with an example of the GC-C-IRMS chromatogram presented in Figure 1 and the simultaneously-acquired GC-MS data presented in Figure 2.…”

Origin Identification of Naturally Occurring Hormones in Dope Analysis

“…Two-dimensional gas chromatography with heart-cutting for an isotope ratio mass spectrometry analyzer for doping control 25) . Abuses of performance-enhancing agents and the manipulation or cheating of test systems are prohibited in order to protect fairness in sports and athletes' health according to ethical considerations.…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…[69] In agreement with the aforementioned assays, urine samples were enzymatically hydrolyzed and purified by SPE; however, due to the use of GC-GC employing a fistdimension column with cyanopropyl/phenyl coating and a second-dimension column with phenyl-methylpolysiloxane stationary phase material, HPLC fractionation could be entirely omitted. [69] In agreement with the aforementioned assays, urine samples were enzymatically hydrolyzed and purified by SPE; however, due to the use of GC-GC employing a fistdimension column with cyanopropyl/phenyl coating and a second-dimension column with phenyl-methylpolysiloxane stationary phase material, HPLC fractionation could be entirely omitted.…”

“…An alternative approach to minimize workload and sample preparation effort was demonstrated by Brailsford et al using two-dimensional gas chromatography with heart-cutting for IRMS analysis. [69] In agreement with the aforementioned assays, urine samples were enzymatically hydrolyzed and purified by SPE; however, due to the use of GC-GC employing a fistdimension column with cyanopropyl/phenyl coating and a second-dimension column with phenyl-methylpolysiloxane stationary phase material, HPLC fractionation could be entirely omitted. Sufficient sensitivity was guaranteed by PTV-supported large-volume sample injection and peak focusing on the second dimension GC column, while the required peak purity was accomplished by precise heart-cutting of the target analyte(s).…”