Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence.
Glutathione (GSH) is a thiol with an important function in protecting tissue against the oxidative stress which has been related to carcinogenesis in the colon. For this reason the development of probiotic species producing glutathione could be of great interest. 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 has been developed. Pretreatment of cell-lysate samples is very simple--precipitation of protein with acetonitrile in 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 a laser at 488 nm for detection. The reaction conditions optimised were temperature, time, and 5-IAF/GSH molar ratio. Electrophoresis was performed with a carbonate buffer (25 mmol L(-1), pH 9.8) as background electrolyte and a voltage of 30 kV; an electrophoretic run was complete in less than 7 min. There was a good linear relationship between concentration and response in the range 2.5-500 ng mL(-1) and the LOD was 0.5 ng mL(-1). The glutathione content of probiotic cells was determined by using the standard additions method to reduce matrix effects. The method was fully validated and shown to be of suitable sensitivity and selectivity for determination of GSH in probiotic cell lysates.
Musenga
Alessandro
A
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy.
Mandrioli
Roberto
R
Bonifazi
Paolo
P
Kenndler
Ernst
E
Pompei
Anna
A
Raggi
Maria Augusta
MA
D000097:Acetonitriles / Q000737:chemistry; D001644:Bifidobacterium / Q000737:chemistry*; D002021:Buffers; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D011232:Chemical Precipitation; D019075:Electrophoresis, Capillary / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D005978:Glutathione / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D017045:Lactococcus / Q000737:chemistry*; D007834:Lasers; D019936:Probiotics / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
D000097:Acetonitriles; D002021:Buffers; D002457:Cell Extracts; D005452:Fluoresceins; C015296:5-iodoacetamidofluorescein; D005978:Glutathione; C032159:acetonitrile