Determination Of γ-aminobutyric Acid In Human Plasma And Its Application In A Pharmacokinetic Study

Background y-aminobutyric acid(GABA)is a non protein amino acid which is widely distributed in animals.It is the major inhibitory neurotransmitter in the central nervous system,accounting for 30-40%of all the synaptic neurotransmitters,which is mainly converted from glutamic acid by glutamic acid decarboxylase.There is evidence that abnormal GABA levels can easily cause neurological diseases such as Alzheimer’s disease,Parkinson’s disease,autism and acute post-traumatic stress disorder.People are prone to be anxious or other unpeaceful emotions if lack of GABA.Therefore,GABA is added as a supplement to many health care products,functional beverages and wine to help people supply GABA.These indicate that GABA may have the potential to be developed as a new drug,but there is no report about human pharmacokinetic study after oral administration of GABA.Objective To develop and validate a rapid,sensitive and convenient liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the determination of GABA in human plasma.The method should be applied to a pharmacokinetic study of GABA tablets in healthy Chinese volunteers.Method After extraction from human plasma by protein precipitation with acetonitrile,GABA and d2-GABA(IS)were separated on a Luna HILIC column(100 mm X 3.0 mm,3 μm)using an isocratic mobile phase of water:acetonitrile:formic acid(20:80:0.12,v/v/v)with a flow rate of 0.5 mL·min-1.The inject volume was 5.0 μL and a chromatographic total run time of 3.0 min was achieved.Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode(MRM)in positive electrospray ionization using the transitions of m/z 10→69 for GABA and m/z 106→71 for d2-GABA.The first period,plasma samples were obtained from subjects after oral administration of placebo tablets.The second period,samples were obtained from subjects after an oral administration at dose of 2000 mg GABA.The last period,each subject received multi dose administration(2000 mg each time,three times a day,last for 7 days),on the 8th day,samples were collected from subjects after an oral administration of 2000 mg GABA.Results The method was linear in the concentration range of 5.00 to 1000 ng·mL-1 and the lower limit of quantification was 5.00 ng·mL-1.The intra-and inter-day precision in terms of the relative standard deviation were less than 9.9%.The accuracy was in the range of(0.9%to 4.3%,within the acceptable limits across all concentrations.The method was successfully applied to a pharmacokinetic study of GABA tablets in 11 healthy Chinese volunteers.After single and multiple oral administation of GABA,mean peak plasma levels(Cmax)of 1050 ±1016 ng·mL-1 and 1423± 1808 ng·mL-1 and Tmax of 1.43 ±0.58 h and 1.14 ± 0.42 h were observed.The mean t1/2z value of 7.33 ± 4.34 h and 8.46 ± 3.42 h were obtained,AUC0-t were calculated to be 1166 ± 797 ng·h·mL-1 and 1481 ± 1465 ng·h.mL-1。Conclusion The LC-MS/MS method is sensitive,rapid,accuracy and suitable for the determination of GABA in human plasma and the method is suitable for the pharmacokinetic study of GABA tablets.