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Accurate quantification of plasma glucagon levels in humans is necessary for understanding the physiological and pathological importance of glucagon. Although several immunoassays for glucagon are available, they provide inconsistent glucagon values owing to cross-reactivity of the antibodies with peptides other than glucagon. To overcome this limitation, we developed a novel method to measure glucagon levels by a liquid chromatography (LC)-high-resolution mass spectrometry (HRMS) assay via parallel reaction monitoring (PRM) without immunoaffinity enrichment. Using stable isotope-labeled glucagon as an internal standard and 200 μL of plasma, the lower limit of quantification was 0.5 pM. This method was applied to measure plasma glucagon levels during the oral glucose tolerance test (OGTT) and meal tolerance test (MTT) in healthy volunteers, and its results were compared with those of sandwich enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). During the OGTT, this method showed significant suppression of plasma glucagon levels, and similar patterns were observed with sandwich ELISA and RIA. In contrast, during the MTT, plasma glucagon levels were slightly elevated according to the LC-MS/MS and sandwich ELISA results and were reduced according to RIA results. Our newly developed LC-MS/MS method overcomes a lack of specificity among currently available immunoassays for glucagon and may contribute to a better understanding of the importance of glucagon. Graphical abstract Flowchart for the extraction and quantification of glucagon in human plasma, and plasma glucagon responses in healthy volunteers quantified by the present LC-MS/MS, sandwich ELISA, and RIA during OGTT and MTT.

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In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [(13) C2 ,(15) N2 ]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [(13) C2 ,(15) N2 ]xanthine as a substrate, [(13) C2 ,(15) N2 ]UA as an analytical standard, and [(13) C3 ,(15) N3 ]UA as an internal standard. The [(13) C2 ,(15) N2 ]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R(2)  = 0.998, weighting of 1/x(2) ) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 μL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma.

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