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以LC/MS/MS分析γ-胺基丁酸（GABA）之方法確效及發芽稻米之含量分析
Development and Validation of a Quantitative Method for γ-aminobutyric Acid (GABA) in Germinated Rice Using High-Performance Liquid Chromatography Tandem Mass Spectrometer (LC/MS/MS)
摘要
γ-胺基丁酸（γ-aminobutyric acid, GABA）是一種非蛋白質胺基酸，常用之檢驗方法是以HPLC/螢光偵測器執行，其缺點為樣品需進行衍生，基質干擾易影響其分析專一性，因此，本實驗擬建立高效液相層析串聯質譜儀（LC/MS/MS）分析GABA的方法，並進行方法確效。實驗以米糠粉為基質，於超音波震盪萃取後，以LC/MS/MS進行分析。發現GABA的滯留時間為2.01分鐘，與α-aminobutyric acid滯留時間重疊，經確立MS/MS之偵測條件，以m/z 104＞m/z 69為其定性離子對，m/z 104＞m/z 87為定量離子對，可以明確有效地加以分離和定量。標準曲線的線性方程式為y=1910.8x + 6005.9，R^2=0.9993，線性範圍在25-200 ppb。準確度評估以添加試驗進行，於米糠粉中添加97 ppb及388 ppb之GABA標準品，其回收率分別為80.74%及93.81%。重複性之變異係數（CV）分別在2.69%和4.15%。97 ppb添加試驗中，三天的中間精密度之變異係數分別為7.48%、8.93%和2.69%；388 ppb添加試驗則為0.45%、3.41%和4.15%。偵測極限（LOD）為2.0 ppb，定量極限（LOQ）為6.6 ppb，顯示所建立之LC/MS/MS分析方法可以穩定有效的分析糙米樣品中的GABA成分。以建立之方法分析七種糙米樣品的GABA含量，其值約在4.78-15.67 mg/100g dry rice之間，其中以台梗2號、9號和4號為較高，而台中秈10號則最低。經發芽0.3 cm - 0.5 cm後的糙米所含GABA含量有顯著的增加，台梗2號和台中秈10號的GABA含量分別增加至31.44 mg/100g dry rice和30.52 mg/100g dry rice，增加約2-6倍。
並列摘要
γ-aminobutyric acid (GABA) is a non-protein amino acid ubiquitous in food matrices. Determination of GABA by HPLC with fluorescence detector has been mainly used. However, the sample needs to be derivated with orthophthalaldehyde (OPA) and the specificity is affected by the matrix interference. Therefore, the purpose of this study was to establish a high-performance liquid chromatography tandem mass spectrometer (LC/MS/MS) method for analyzing GABA. The method validation will be also evaluated. The results indicated that the retention time of GABA was at 2.01 minutes, which overlapped with the α-aminobutyric acid in the chromatogram. Thereafter, the effective separation and quantitation detection conditions by MS/MS, with m/z 104＞ m/z 69 as the qualitative ion pair and m/z 104＞ m /z 87 as quantitative ion pair, were established. The linear equation of the standard curve is y = 1910.8 x + 6005.9, R^2 = 0.9993, and the linear range is 25-200 ppb. Accuracy evaluation was conducted by standard addition method. When 97 ppb and 388 ppb of GABA standard were added to rice bran powder, the recoveries were 80.74% and 93.81%., respectively. For the repeatability, the coefficient of variation (CV) was at range of 2.69% to 4.15%. The coefficient of variation of 3 days intermediate precision were 7.48%, 8.93%, and 2.69% in the 97 ppb added group, while there were 0.45%, 3.41%, and 4.15% in the 388 ppb added group. The limit of detection (LOD) is 2.0 ppb and the limit of quantification (LOQ) is 6.6 ppb. Collectively, the established LC/MS/MS analysis method can determine the GABA content in brown rice effectively. Then, the GABA content of seven brown rice was determined. The contents were at range of 4.78-15.67 mg/100g dry rice, while Taigen brown rice No. 2, 9, and 4 had higher content, and Taichung Indica brown rice No. 10 had the lowest content. The GABA content of brown rice increased significantly after germinated in 0.3 cm-0.5 cm. The GABA contents of germinated Taigen brown rice No. 2 and Taichung Indica brown rice No. 10 increased to 31.44 mg/100g dry rice and 30.52 mg/100g dry rice, respectively, which was about 2-6 times of ungerminated brown rice.