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A rapid UHPLC-HILIC method for algal guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and the potential separation
Hui Jin1, Yong Min Lao1, Jin Zhou1
1Shenzhen Public Platform of Screening & Application of Marine Microbial Resources, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China; Shenzhen Key Laboratory for Coastal Ocean Dynamic and Environment, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China.
Abstract:
A fast and facile hydrophilic interaction liquid chromatography (HILIC) method was developed and applied to quantify physiologically important ppGpp and its analogues in a tough sample, the astaxanthin-accumulating alga Hameatococcus pluvialis. The method is able to analyze simultaneously seven nucleotides, including ppGpp at the order of pmol g-1 cells within 12 min. Mechanism on the elution order was investigated. It was found that 1) phosphate salt competed for the amide groups on the HILIC column with the phosphate groups of the nucleotides; 2) intramolecular hydrogen bonds might contribute to the elution order by offsetting and reducing the number of free hydrogen acceptor/donor of the nucleotide molecules interacting with the amide groups. This is the first HILIC method for ppGpp, which is feasible and applicable to a wide range of samples, especially tough samples, e.g., algae and plants.