Sensitive and selective glutathione determination in probiotic bacteria by capillary electrophoresis - laser induced fluorescence

Glutathione (GSH) is a thiol playing an important role in tissue protection against oxidative stress, which has been also related to carcinogenesis in the colon; from this point of view, the development of probiotic species producing glutathione could be of great interest. In order to determine the glutathione content of some probiotic bacteria of the Bifidobacterium and Lactococcus genera, a very sensitive and selective analytical method based on capillary electrophoresis coupled to laser induced fluorescence detection was developed. The pre-treatment of cell lysate samples is very simple and feasible: protein precipitation was carried out with acetonitrile in a 1:2 volume ratio. The fluorophore 5-iodoacetamidofluorescein (5-IAF) was chosen for glutathione derivatisation; it reacts with thiols at pH 12.5, forming a fluorescent adduct which is excited by laser at 488 nm for detection. The reaction parameters were optimised in terms of temperature, time and 5-IAF/GSH molar ratio. The electrophoretic analysis was carried out using a background electrolyte composed of carbonate buffer (25 mM, pH 9.8) and applying a 30 kV voltage; a complete electrophoretic run lasts less than 7 minutes. Good linearity was found in the 2.5-500.0 ng/mL concentration range of GSH; LOD = 0.5 ng/mL. The glutathione content in probiotic cells was determined using the standard additions method in order to reduce matrix effect. The method has been fully validated and has resulted to be suitable in terms of sensitivity and selectivity for the determination of GSH in probiotic cell lysates.