Development and application of LC/HRPS for quantification of adenine nucleotides, creatine phosphate, and creatine in sturgeon spermatozoa

Abstract

The objective of this study was to investigate the applicability of liquid chromatography coupled with high resolution product scan (LC/HRPS) mass spectrometry for simultaneous quantification of adenine nucleotides, creatine phosphate, and creatine levels after fixation of sturgeon sperm by perchloric acid. This approach has been used for the determination of adenine nucleotides, creatine, and creatine phosphate in biological tissues, but no applications to sperm cells have been reported to date. The results of method validation showed that specific correction factors for the calculation of macroergic phosphate content in sperm cells extracted by perchloric acid should be used to get accurate concentration values. The proposed LC/HRPS method is beneficial for the analysis of adenine nucleotides because it allows simultaneous quantification of all target analytes at low concentrations in a single run. This is an advantage compared to conventional methods based on colorimetric or chemiluminescent assays and even compared to sophisticated methods based on high resolution nuclear magnetic resonance.

Keywords

• macroergic phosphates
• high resolution mass spectrometry
• extraction
• method validation
• sperm
• chondrostean fishes