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Abstract 【Introduction】
Arginine vasopressin (AVP) is a peptide hormone consisting of 9 amino acids secreted from the posterior pituitary. The main physiological functions of AVP are the antidiuretic action and vasoconstrictor action via the renal tubular V2 receptor. AVP is measured to clarify the involvement of AVP secretion in the case of water metabolism disorder and Na metabolism disorder. Conventionally, measurement of human plasma AVP concentration has been performed by radioimmunoassay. However, even now, it is very difficult to measure plasma AVP accurately. In this study, we aimed to develop LC/MS/MS based method to accurately measure endogenous AVP in human plasma.
【Methods】
A 300 µL aliquot of human plasma samples obtained from apparently healthy human subjects were mixed with 270 µL of 4％ phosphoric acid aqueous solution and 30 µL of internal standard (IS). The mixture was purified using solid phase extraction (SPE). After evaporation and reconstitution, extracts were analyzed using a AQUITY UPLC I-Class coupled to a Xevo TQ-XS mass spectrometer (Waters Corporation, Milford, MA, USA). The analytical and pre-analytical performances of the LC-MS/MS assay were evaluated by determination of precision, trueness, linearity, possible interferences, and lower limit of quantification. We also tested how the results obtained by our LC/MS/MS compare with those obtained by the conventional RIA (YAMASA CORPORATION, Japan).
【Results】
The lower limits of quantification for AVP was 0.2 pg/mL (％CV<10,%Dev<15,S/N>10). The calibration curves showed good linear response over the concentration range of 0.2-10 pg/mL for human plasma (y=0.9998x+0.0012, R2=1). Imprecision of the assay was very good: within and day to day precision (CVs) were 1.9–6.5% (0.5-9.6pg/mL, N=5) and 3.6 -7.7% (0.5-10.1pg/mL, N=5), respectively. The absolute recoveries of AVP at concentration of 0.5-10pg/mL (N=5) ranged 104-114%. Although the overall correlation between LC/MS/MS (X) and RIA (Y) was seen (Y= 0.458 X+1.0478, r= 0.94, N=32), the results obtained by LC/MS/MS did not agree well with those obtained by the RIA in the low concentration range of 4 pg/mL or less (Y=0.2473X+1.3843, r=0.37).
【Conclusions & Discussion】
We have developed a sensitive, accurate, and precise LC-MS/MS-based method for the quantitation of AVP in plasma samples obtained from healthy subjects. This method will enable the accurate determination of very low-levels of plasma AVP. We are planning to determine plasma AVP by the LC/MS/MS method in the real-world clinical samples obtained from patients with central diabetes insipidus.
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= Discovery stage.
= Translation stage.
= Clinically available.
