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

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

Journal of pharmaceutical and biomedical analysis (2007-05-08)
Steinar Uran, Kristin Eitrem Landmark, Gro Hjellum, Tore Skotland
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 10 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 5 cm × 4.6 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 10 cm × 2.1 mm
Supelco
Discovery® HS C18 Prep Guard Cartridge, 10 μm particle size, L × I.D. 1 cm × 21.2 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 5 cm × 2.1 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 21.2 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 15 cm × 2.1 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 15 cm × 4 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 4 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 10 cm × 4.6 mm
Supelco
Discovery® HS C18 HPLC Column, 10 μm particle size, L × I.D. 25 cm × 21.2 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 15 cm × 4.6 mm
Supelco
Discovery® HS C18 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 4.6 mm
Supelco
Discovery® HS C18 HPLC Column, 3 μm particle size, L × I.D. 5 cm × 4.6 mm
Supelco
Discovery® HS C18 HPLC Column, 3 μm particle size, L × I.D. 7.5 cm × 2.1 mm
Supelco
Discovery® HS C18 HPLC Column, 3 μm particle size, L × I.D. 15 cm × 2.1 mm
Supelco
Discovery® HS C18 HPLC Column, 3 μm particle size, L × I.D. 15 cm × 4.6 mm
Supelco
Discovery® HS C18 HPLC Column, 3 μm particle size, L × I.D. 5 cm × 2.1 mm
Supelco
Discovery® HS C18 HPLC Column, 3 μm particle size, L × I.D. 7.5 cm × 4.6 mm