Attila Frank Torma (Presenter)
LGC LTD
Bio: I work in the Chemical Measurement and Calibration Team at LGC as a Researcher. LGC is the designated National Measurement Institute for chemical measurements in the UK. Currently I am doing a PhD in the SI-traceable quantification of clinically relevant proteins from plasma using Liquid Chromatography Mass Spectrometry. The work is aimed at the development of reference measurement procedures for cardiac hormones and the production of certified reference materials to expedite standardization in clinical chemistry. I hold an MSc in Chemical Engineering from the Budapest University of Technology and Economics. I joined the Chemical Measurement and Calibration Team in 2008. In my spare time, I read chemistry. I am the face of SI-traceable quantification.
Authorship: Attila F. Torma
LGC, Queens Road, Teddington TW11 0LY
| Short Abstract Brain Natriuretic Peptide (BNP) is a cardiac marker that is routinely measured by immunoassays to diagnose heart failure. Large variability between immunoassay results highlighted the need for standardization in clinical diagnostics based on traceability to the International System of Units (SI). I present the development and validation of an LC-MS/MS method for its quantification in plasma by using isotope dilution mass spectrometry (IDMS), multi stage clean-up and signal enhancement by post column infusion of dimethyl sulfoxide (DMSO). The method was applied to analyze National External Quality Assessment Scheme samples and enabled the direct comparison of immunoassay and mass spectrometry results. |
Long Abstract
Development of a Reference Method for the Quantification of the Cardiac Marker 1-32 B-Type Natriuretic Peptide
Attila F. Torma
LGC, Queens Road, Teddington TW11 0LY
INTRODUCTION:
B-type Natriuretic Peptide (BNP) is a 32 amino acid long cardiac hormone routinely measured by immunoassays for the diagnosis of heart failure (HF). Immunoassay results can vary up to 45%[1] due to cross reactivity and lack of calibrator characterization. Potential route to achieve comparability in clinical diagnostics is standardization of BNP measurements through the development of reference methods and certified reference materials (CRM). Isotope dilution mass spectrometry (IDMS) is an established primary method that uses isotopically labelled internal standards to assign concentration with low uncertainties. Issues associated with the quantification of BNP by mass spectrometry include the very low concentrations of BNP in plasma (lower decision limit 100pg/g), the difficulties with measuring large intact peptides by electrospray ionisation (ESI) MS and poor stability of BNP in plasma (half-life: 22.6 min). Here we present the development and validation of an LC-MS/MS method for its quantification in plasma by using IDMS, multi stage clean-up and signal enhancement by post column infusion of dimethyl sulfoxide (DMSO). The method was applied for the analysis of samples from the UK National External Quality Assessment Scheme (NEQAS) to provide direct comparison between results obtained by LC-MS/MS and three different immunoassay platforms.
METHODS:
Quantification is performed on a Triple Quadrupole (QqQ) system using BNP primary calibrator, isotopically labelled internal standard (BNP*, +30Da) and selected reaction monitoring (SRM). To increase sensitivity, 0.25% dimethyl sulfoxide (DMSO) was infused post column. The extraction method is based on protein precipitation (PPT) followed by solid phase extraction (SPE). The BNP stock solution was characterized for peptidic impurities and degradation products on a Quadrupole Time of Flight (Q-TOF) instrument. Traceability to the International System of Units (SI) is maintained by quantification of BNP primary standard solution by amino acid analysis using amino acid CRMs and GC-MS[2].
RESULTS
An iterative optimization of the LC, MS and sample clean-up conditions was performed to achieve the limit of detection and quantification required for analyzing clinical samples. With the final LC-MS/MS method 6fmol/g (20.8pg/g) BNP in aqueous solutions was detectable using an injection volume of 10µL. Extensive oxidation of methionine residues was observed during sample clean-up. Q-TOF full scan experiments were performed to identify methionine oxidation and SRM transitions were set up on the QqQ to monitor for the presence of oxidized BNP. The addition of methionine as a scavenger to the extraction solvents reduced oxidation below 1%. PPT solvent composition was optimized to reproducibly precipitate the majority of high abundance proteins. Enzymatic degradation of BNP in plasma was prevented by the addition of 0.5% formic acid to the precipitation solution.
The method was validated in the range of 52 and 530 pg/g (15-155 fmol/g). The lowest calibration point was set at 52 pg/g (15fmol/g) corresponding to half of the BNP concentration at the lowest clinical decision limit. The method was applied to 9 samples from the UK NEQAS Cardiac Marker scheme. Immunoassay results are always higher than the results of the LC-MS/MS method. The SI-traceable quantified BNP primary calibrator was also sent to UK NEQAS to be used for the preparation of plasma spikes and distributed through the Cardiac Marker Scheme. Whereas a slightly lower uncertainty was observed when the BNP primary calibrator was used the agreement between the diverse platforms did not improve.
Discrepancy between immunoassays and immunoassays vs LC-MS/MS results can be due to both the difference in the amount of BNP in the calibrants provided by the manufacturers and the different specificity of the assays for degradation products in plasma.
CONCLUSIONS
Here we developed a reference method for the quantification of BNP in plasma at the clinically relevant concentrations. The expanded uncertainty of the method was 15%. This uncertainty can be improved through additional characterization of the starting material, further optimization of the sample clean-up procedure and by applying exact matching IDMS. The method was successfully applied for the quantification of BNP in the UK NEQAS samples and the results obtained by applying the LC-MS/MS method were consistently lower than the immunoassay results confirming the aspecificity of the assays. Further experiments are currently on-going on UK NEQAS and patient samples to identify the potential utility and format of a certified reference material for standardization of BNP measurements.
References & Acknowledgements:
REFERENCES
1. Clerico, A. et al. State of the art of BNP and NT-proBNP immunoassays: The CardioOrmoCheck study. Clin. Chim. Acta 414, 112–119 (2012).
2. Pritchard, C., Torma, F. A., Hopley, C., Quaglia, M. & O’Connor, G. Investigating microwave hydrolysis for the traceable quantification of peptide standards using gas chromatography–mass spectrometry. Anal. Biochem. 412, 40–46 (2011).
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