Hydrophilic interaction liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry for determination of nuclear and cytoplasmatic contents of nucleotides, nucleosides and their nucleobases in food yeasts

Abstract

Hydrophilic interaction liquid chromatography using a zwitterionic phase, coupled with high-resolution mass spectrometry based on quadrupole and time-of-flight mass analyser (HILIC-Q-TOF-MS) is applied for the determination of four nucleobases (adenine, thymine, guanine and uracil), five nucleosides (adenosine, 5´-methyluridine, guanosine, cytidine and uridine) and five nucleotides (cytidine 5´-monophosphate, uridine 5´-monophosphate, adenosine 5´-monophosphate, inosine 5´-monophosphate and guanosine 5´-monophosphate) in food yeasts. Sample treatment based on the lysis of yeast using an ultra-turrax homogenizer allows the pellet and supernatant to be differentiated with low-speed centrifugation (1000 g). The pellet contains the nucleus (nuclear fraction), while the supernatant contains, among others, the organelles present in the cytoplasm (cytoplasmatic fraction). Both fractions were treated with perchloric acid in order to extract nucleobases, nucleosides and nucleotides and analysed by HILIC-Q-TOF-MS. The method was validated following international guidelines and detection limits were in the 2.5–22 ng mL−1 range (62–550 ng g−1, for 200 mg yeast). Five different Saccharomyces cerevisiae food yeasts were analysed and nucleotide concentrations of 0.6–570 µg g−1 and 5.6–924 µg g−1 were found in the nuclear and cytoplasmatic fractions of all samples. Nucleosides and nucleobases were not found in any sample.

Keywords

• Hydrophilic interaction liquid chromatography
• Quadrupole-time-of-flight mass spectrometry
• Nucleobases
• Nucleosides
• Nucleotides
• Food yeasts