Optimization of extraction solvents, solid phase extraction and decoupling for quantitation of free isoprenoid diphosphates in Haematococcus pluvialis by liquid chromatography with tandem mass spectrometry.

Isoprenoid diphosphates are important precursors actively participating in many downstream metabolisms; they are often in modified forms, e.g., protein-coupled or esterified form. Therefore, in vivo level of free isoprenoid diphosphates is quite low, ˜0.07 nmol/g fresh weight in plants. In order to directly measure the isoprenoid diphosphate pool during stress-induced accumulation of astaxanthin in Haematococcus pluvialis, the present study optimized several pretreatment procedures to enrich free isoprenoid diphosphates for high-pressure liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) detection. Specifically, different extraction solvents, e.g., water, methanol, chloroform, and mixture of water, methanol, and chloroform (1:1:1, V/V/V), and solid phase extraction (SPE) columns (OASIS@ WAX and HLB Cartridges) were compared; and gentle decoupling by NaOH or trifluoroacetic acid (TFA) was introduced to release free isoprenoid diphosphates. Results found that solvent mixture of water, methanol and chloroform (1:1:1, V/V/V) showed the highest extraction efficiency (RE) for five isoprenoid diphosphates, ranging from 76.83% to 92.43%; HLB column showed the balanced recoveries ranging from 75.29% to 87.54%; and incubation with low NaOH (˜4.7 mmol/L) at 4 °C significantly increased detectable isoprenoid diphosphates in algal cells, some of which were undetectable or in trace level before NaOH decoupling. The method was applied to H. pluvialis cells under various stresses. Low levels of isoprenoid diphosphates were determined in most of the stresses used, e.g., 0.19 ± 0.09 to 0.98 ± 0.06 mg/g fresh weight (FW) for IPP/DMAPP, 0.35 ± 0.07 mg/g FW for GGPP and undetectable for FPP and GPP; while isoprenoid diphosphates were significantly accumulated in the dark to 3.27 ± 0.05, 0.17 ± 0.09, 1.81 ± 0.16 and 0.58 ± 0.07 mg/g FW for IPP/DMAPP, GPP, FPP and GGPP, respectively. These results implied that isoprenoid diphosphates were exhausted by downstream carotenogenesis under stress. Our work emphasizes NaOH decoupling for exact quantitation of in vivo isoprenoid diphosphates.