Internal Standards for Mass Spectrometry: When Gravimetric Analysis is Not Enough Michael Gelb (Presenter) Univ. of Washington Presenter Bio: Mike Gelb is a Professor of Chemistry and Biochemistry at the Univ. of Washington. In the area of mass spectrometry he developed the first Isotope-Coded Affinity Tags (ICAT) used in quantitative proteomics. More recently his laboratory has developed the main technology used for worldwidenewborn screening and diagnosis of lysosomal storage diseases (done by tandem mass spectrometry).

Authors: Michael H. Gelb
University of Washington

Gravimetric measurement is often used to prepare stock solutions of analytes and internal standards of known absolute concentrations. This is valid only if the standards are known to be pure by weight, and this is often not the case. In a recent multi-reference laboratory comparison of the lipid biomarker psychosine in dried blood spots, large differences in analyte concentrations were reported, and the problem was due to the use of gravimetric measurement and commercial reagents that were not pure by weight. The problem was resolved by quantification of stock solutions by quantitative nuclear magnetic resonance, a technique that allows the absolute moles of analyte in a solution to be measured. The problem encountered may be widespread in the field of mass spectrometry and the clinical laboratory, and a careful discussion of these issues is warranted.

Problem

Gravimetric measurement of reagents to prepare stock solutions for mass spectrometry is often used with reagents that are not pure by weight. Vendors often provide purity information that may be miss-interpreted to mean that the compound is pure by weight.

Method Information

It is important to consider the criteria by which standards from commercial and other sources are reported to be pure, because many purity analyses do not ensure that the compounds are pure by weight. Quantitative nuclear magnetic resonance is one of the few appropriate methods that allows measurement of the true absolute concentration of analytes including internal standards in stock solutions used in clinical assays. These considerations and methods were applied to a recent multi-reference laboratory measurement of the lipid biomarker psychosine (an important biomarker for diagnosis of Krabbe disease).

Troubleshooting Steps

Poor agreement between psychosine concentrations in dried blood spots measured in 4 different reference laboratories was obtained when gravimetric measurement was used to prepare stock solutions. The problem was resolved when quantitative nuclear magnetic resonance was used to determine the absolute concentration of reagents in stock solutions (1). The study has widespread significance in the use of mass spectrometry in the clinical laboratory, and it is important that these data are made widely available to the community.

Outcome

Great care is needed in the interpretation of specification data sheets for standards provided from all sources including vendors. Very often, there is no guarantee that the compound is pure by weight. Without assurance of weight purity, a method such as quantitative nuclear magnetic resonance is needed for high confidence knowledge of the true absolute concentration of analytes in stock solutions. This is especially important in the use of mass spectrometry in the clinical laboratory in cases where the goal is to determine the absolute concentration of analytes in biological samples.

References:

1. Gelb, M. H. (2018) Absolute Amounts of Analytes: When Gravimetric Methods Are Insufficient.

Clin Chem. 64:1430-1432.

Acknowledgement:

This work was supported by a grant from the National Institutes of Health (R01 DK067859).