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 13C-labelled pyruvate (13C pyruvate) is under evaluation as an agent for medical metabolic imaging by measuring formation of 13C lactate using magnetic resonance spectroscopy of the 13C nuclei. A quantitative method for analysis of these 13C-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-13C]pyruvate and [U-13C]lactate). Prior to analysis, the compounds were derivatized using 3-nitrophenylhydrazine. Following separation on a Supelco Discovery HS C18 column, 13C pyruvate and 13C lactate were detected using negative electrospray ionization mass spectrometry. Calibration standards (4.5–4500 μM 13C pyruvate and 9–9000 μM 13C lactate) and added internal standard were used to make the calibration curves, which were fitted to a non-linear equation y = a + bx + cx2 and weighted with a weighting factor of 1/y2. The analytical lower limit of quantification of 13C pyruvate and 13C lactate was 4.5 and 9 μM, respectively. The total precision of the method was below 9.2% for 13C pyruvate and below 5.8% for 13C lactate. The accuracy of the method showed a relative error less than 2.4% for 13C pyruvate and less than 6.3% for 13C lactate. The recoveries were in the range 93–115% for 13C 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 °C freezer, during three freeze/thaw cycles, and when stored in an autosampler for at least 30 h.