Quantification of 13C pyruvate and 13C lactate in dog blood by reversed-phase liquid chromatography-electrospray ionization mass spectrometry after derivatization with 3-nitrophenylhydrazine

Injection of hyperpolarized C-13-labelled pyruvate (C-13 pyruvate) is under evaluation as an agent for medical metabolic imaging by measuring formation of C-13 lactate using magnetic resonance spectroscopy of the C-13 nuclei. A quantitative method for analysis of these C-13-labelled substances in dog blood was needed as part of the development of this agent and we here describe a liquid chromatography-mass spectrometry method for that purpose. Immediately after blood collection, the blood proteins were precipitated using methanol added internal standard ([U-C-13]pyruvate and [U-C-13] lactate). Prior to analysis, the compounds were derivatized using 3-nitrophenylhydrazine. Following separation on a Supelco Discovery HS C18 column, 13C pyruvate and C-13 lactate were detected using negative electrospray ionization mass spectrometry. Calibration standards (4.5-4500 mu M 13C pyruvate and 9-9000 mu M C-13 lactate) and added internal standard were used to make the calibration curves, which were fitted to a non-linear equation y = a + bx + cx(2) and weighted with a weighting factor of 1/y(2). The analytical lower limit of quantification of C-13 pyruvate and C-13 lactate was 4.5 and 9 mu M, respectively. The total precision of the method was below 9.2% for 13C pyruvate and below 5.8% for C-13 lactate. The accuracy of the method showed a relative error less than 2.4% for C-13 pyruvate and less than 6.3% for C-13 lactate. The recoveries were in the range 93-115% for C-13 pyruvate and 70-111% for 13C lactate. Both substances were stable in protein-free supernatant when stored for up to 3 weeks in a -20 degrees C freezer, during three freeze/thaw cycles, and when stored in an autosampler for at least 30 h. (c) 2007 Published by Elsevier B.V.