Development and validation of a method for the quantitative determination of succinate in rat blood plasma by HPLC-MS/MS

Relevance. Succinate is a central metabolite of the Krebs cycle and also a regulator of numerous processes via the specific succinate receptor SUCNR1. Succinate is an important component of several pharmacological preparations. However, its pharmacokinetics following the administration of these substances remain poorly understood. A sensitive analytical method is required to perform such studies.
Objective. To develop and validate a method for the quantitative determination of succinate in rat blood plasma.
Methods. The analysis was performed using HPLC-MS/MS — an Ultimate 3000 TSQ Fortis system and a UCT Selectra C18 column (4.6 mm × 100 mm, 3 µm, 100 Å). The separation temperature was 35 °C, and the flow rate was 0.5 mL/min. A gradient elution mode was used with a mixture of 0.1 % aqueous formic acid and 0.1 % formic acid in methanol. The injection volume was 5 µL. For sample preparation, methanol containing chloramphenicol at a concentration of 10 ng/mL as an internal standard was used. A model matrix was used during validation. For succinate detection, the following mass transitions were used in negative ionization mode: 117 m/z → 98.7 m/z, 117 m/z → 73.1 m/z.
Results. The method demonstrated selectivity, sensitivity, accuracy, precision, and linearity in the concentration range of 200–20,000 ng/mL. No carryover or matrix effect was observed. The recovery rate was over 96 %. Succinate was stable under the employed storage conditions.
Conclusion. A method for the quantitative determination of succinate in rat blood plasma has been developed and validated. This method can be used to analyze endogenous succinate concentrations and the pharmacokinetics of succinate following the administration of succinate-containing drugs.

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