Determination of estrogens in pregnant women
10μL or 20μL sample of the serum is added to acetonitrile (100μL) containing [2,4,6,6,9-2H5]-E1 (D5-E1) (100pg), vortex mixed for 30 sec and centrifuged at 1500×g (4°C, 5 min). The supernatant is diluted with water (400μL) and purified using a Strata®-X cartridge. After successive washing with water (2mL) and 30% methanol (2mL), estrogens are eluted with ethyl acetate (1mL) and evaporated. To a solution of estrogen in acetone (40μL), 1 (10μg) in acetone (10μL) and 1M NaHCO3 (10μL) are added, and the mixture is then incubated at 60°C for 1 h. The reaction mixture is diluted with 50% methanol (500μL) and passed through a Strata®-X cartridge for desalting. After washing with water (2mL), the derivatized estrogen is eluted with ethyl acetate (1mL) and evaporated. To the PPZ-derivative, methyl iodide (100μL) is added. The mixture is incubated at 60°C for 30 min, and then excess reagent is evaporated off. The methylated PPZ-derivative is dissolved in methanol-10mM ammonium formate (1:1, v/v), an aliquot of which is subjected to LC-MS(/MS).
Labeling and detection of amino acids and peptides
An ethanol solution of diisopropylamine and acetonitrile solution of 2 are added sequentially to an aqueous solution of amino acids (or peptides) and left for 30 min. An appropriate amount of this sample solution is put to a MALDI plate. After drying naturally, mass spectra are acquired in negative mode.
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