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 (13)C-labelled pyruvate ((13)C pyruvate) is under evaluation as an agent for medical metabolic imaging by measuring formation of (13)C lactate using magnetic resonance spectroscopy of the (13)C nuclei. A quantitative method for analysis of these (13)C-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-(13)C]pyruvate and [U-(13)C]lactate). Prior to analysis, the compounds were derivatized using 3-nitrophenylhydrazine. Following separation on a Supelco Discovery HS C18 column, (13)C pyruvate and (13)C lactate were detected using negative electrospray ionization mass spectrometry. Calibration standards (4.5-4500 microM (13)C pyruvate and 9-9000 microM (13)C 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 (13)C pyruvate and (13)C lactate was 4.5 and 9 microM, respectively. The total precision of the method was below 9.2% for (13)C pyruvate and below 5.8% for (13)C lactate. The accuracy of the method showed a relative error less than 2.4% for (13)C pyruvate and less than 6.3% for (13)C lactate. The recoveries were in the range 93-115% for (13)C pyruvate and 70-111% for (13)C 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.
Uran
Steinar
S
Research and Development, GE Healthcare, Nycoveien 2, N-0401 Oslo, Norway.
Landmark
Kristin Eitrem
KE
Hjellum
Gro
G
Skotland
Tore
T
D000818:Animals; D002247:Carbon Isotopes; D002853:Chromatography, Liquid; D004285:Dogs; D007202:Indicators and Reagents; D019344:Lactic Acid / Q000097:blood*; D008970:Molecular Weight; D010659:Phenylhydrazines / Q000737:chemistry*; D019289:Pyruvic Acid / Q000097:blood*; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization
C523491:3-nitrophenylhydrazine; D002247:Carbon Isotopes; D007202:Indicators and Reagents; D010659:Phenylhydrazines; D019344:Lactic Acid; D019289:Pyruvic Acid