Robert Wardle (Presenter)
Bio: I am currently employed at Waters Corporation, Wilmslow, UK as a Senior Clinical Applications Scientist within the Scientific Operations group. I perform research and development activities for Waters applications and products in the Health Sciences field, with my primary area of research being vitamins, in particular vitamin D metabolites and more recently estrogens.
Authorship: Robert M Wardle, Nayan Mistry and Lisa J Calton
Waters Corporation, Wilmslow, United Kingdom
Ion suppression in LC-MS/MS methods can be considered the dark side of the force, possibly leading to analytical sensitivity, selectivity and accuracy goals not being met. Here we discuss the routine measurement of 17β-Estradiol (E2) and Estrone (E1) for clinical research as an example. Testing was performed to investigate the source of a disturbance in the force (labware, biological matrix or solvents), chromatographically resolve it from the analytes and identify the interferent using MS scanning techniques and accurate mass analysis. For Research Use Only, Not for use in diagnostic procedures.
Ion suppression in LC-MS/MS methods can be considered the dark side of the force, possibly leading to analytical sensitivity, selectivity and accuracy goals not being met. Here we discuss an example of ion suppression when quantifying 17β-Estradiol (E2) and Estrone (E1) in serum for clinical research applications.
Samples were prepared by performing a liquid/liquid extraction using a mixture of hexane and ethyl acetate. Following centrifugation, the organic layer was removed and evaporated to dryness. Samples were then reconstituted in methanol and water prior to injection.
Chromatographic separation was performed using a Cortecs Phenyl, 2.1 x 50mm column and a mobile phase gradient consisting of water, methanol, acetonitrile and ammonium fluoride.
Detection was performed on a Xevo TQ-XS system in electrospray negative mode, with precursor and product ions of m/z271.2 > 145 (qualifier ion of m/z183) for E2 and m/z269.2 > 145 (qualifier ion of m/z183) for E1.
Qualitative matrix effect testing was performed by conducting a post column infusion of E2 and E1 when analyzing extracted samples from six individuals and a water (blank) extract. Ion suppression or enhancement effects were monitored at the retention time of the analytes. Whilst little to no ion suppression was observed for E2, ion suppression was observed at the retention time of E1 in all six matrix samples and in the extracted water sample. However, a solvent injection showed no suppression effect for E1 whilst performing post column infusion. This would suggest that the interference was introduced during the sample preparation procedure and not from the biological matrix, mobile phase or reagents.
To determine the possible cause of the ion suppression, the microfuge tubes used to perform the sample extraction were investigated. Water was extracted in glass vials and compared to samples extracted in microfuge tubes. Suppression was not observed in samples prepared in glass vials but was present in those prepared in microfuge tubes, confirming that these tubes are the source of the interferent causing the ion suppression.
To try and identify the potential agent causing the issue, extracted samples were analyzed using a Xevo G2-XS (Q-Tof) instrument in MSe acquisition mode to obtain accurate mass spectral data for precursor and product ions. The spectral data obtained was compared to online libraries and a possible identity for the interferent was found, FCM substance number 784, which is a clearing agent used in the manufacture of plastics.
As the interferent may be present in other plastic labware and sample collection devices, chromatographic separation of the interference from the analytes was evaluated. The current method employed a gradient separation using 50:50 methanol:acetonitrile (MeOH:MeCN) as mobile phase B. When changing this to 100% MeOH, the interferent co-eluted with E2. Through a series of experiments, a mobile phase comprising of 80:20 MeOH:MeCN was selected to ensure the interferent did not co-elute with the analytes of interest.
The source of the interferent causing a tremor in the force for E1 was found to be the microfuge tubes used in the extraction process. Further testing revealed a possible identity of the compound, FCM substance number 784, which is used as a clarifying agent in polypropylene. This contaminant was chromatographically resolved from both E2 and E1 to prevent interference, which could potentially be present in other polypropylene labware and sample collection devices.
For Research Use Only, Not for use in diagnostic procedures.
References & Acknowledgements:
|Salary||yes||Micromass UK Ltd.|
IP Royalty: no
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