Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC-MS/MS

A liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of 5,10-methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5-methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC-MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut-off membrane, 10kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC-MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right- and left-sided tumors follows different pathways. Copyright (c) 2012 John Wiley & Sons, Ltd.