James W Howard1,2, Richard G Kay3, Ben Jones4, Jaimini Cegla4, Tricia Tan4, Steve Bloom4, Colin S Creaser2. 1. LGC Limited, Fordham, Cambridgeshire, CB7 5WW, UK. 2. Centre for Analytical Science, Department of Chemistry, Loughborough University, Leicestershire, LE11 3TU, UK. 3. Institute of Metabolic Science, University of Cambridge, Cambridgeshire, CB2 0QQ, UK. 4. Department of Investigative Medicine, Hammersmith Hospital Campus, Imperial College, London, W12 0NN, UK.
Abstract
AIM: The performance of glucagon and GLP-1 immunoassays is often poor, but few sensitive LC-MS/MS methods exist as alternatives. EXPERIMENTAL: A multiplexed LC-MS/MS method using a 2D extraction technique was developed. RESULTS: The method was established for the quantitation of endogenous glucagon (LLOQ: 15 pg/ml) and dosed GLP-1 (LLOQ: 25 pg/ml) in human plasma, and is the first such method avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier SRM. Endogenous glucagon concentrations were within the expected range, and showed good reproducibility after extended sample storage. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between methods. CONCLUSION: The LC-MS/MS method offers a viable alternative to immunoassays for quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1.
AIM: The performance of glucagon and GLP-1 immunoassays is often poor, but few sensitive LC-MS/MS methods exist as alternatives. EXPERIMENTAL: A multiplexed LC-MS/MS method using a 2D extraction technique was developed. RESULTS: The method was established for the quantitation of endogenous glucagon (LLOQ: 15 pg/ml) and dosed GLP-1 (LLOQ: 25 pg/ml) in human plasma, and is the first such method avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier SRM. Endogenous glucagon concentrations were within the expected range, and showed good reproducibility after extended sample storage. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between methods. CONCLUSION: The LC-MS/MS method offers a viable alternative to immunoassays for quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1.
Authors: Richard G Kay; Benjamin G Challis; Ruth T Casey; Geoffrey P Roberts; Claire L Meek; Frank Reimann; Fiona M Gribble Journal: Rapid Commun Mass Spectrom Date: 2018-08-30 Impact factor: 2.419
Authors: Geoffrey P Roberts; Richard G Kay; James Howard; Richard H Hardwick; Frank Reimann; Fiona M Gribble Journal: Surg Obes Relat Dis Date: 2018-02-03 Impact factor: 4.734
Authors: Kleopatra Alexiadou; Joyceline Cuenco; James Howard; Nicolai Jacob Wewer Albrechtsen; Ibiyemi Ilesanmi; Anna Kamocka; George Tharakan; Preeshila Behary; Paul R Bech; Ahmed R Ahmed; Sanjay Purkayastha; Robert Wheller; Matthieu Fleuret; Jens Juul Holst; Stephen R Bloom; Bernard Khoo; Tricia M-M Tan Journal: BMJ Open Diabetes Res Care Date: 2020-03