一种利用反相高效液相色谱法测定乳酸菌发酵液中苯乳酸的方法及其评价

为建立一种反相高效液相色谱法(Reversed phase high performance liquid chromatography,RP-HPLC)检测乳酸菌发酵液中苯乳酸含量的方法,系统考察了3种固定相、2种流动相有机相、5种水相及其比例对苯乳酸RP-HPLC检测的影响并对方法进行了评价。确定了以双封端C₁₈色谱柱(4.6 mm×250 mm,5 μm)为固定相,0.05%三氟乙酸(Trifluoroacetic acid,TFA)-乙腈,体积比75∶25,等度洗脱,流速1 mL/min,检测波长210 nm,柱温30 ℃。在0.2~5.0 mg/L内线性良好,r=0.9998,回收率99.88%~101.08%,检出限0.1400 mg/L,定量限0.4600 mg/L,1 d精密度和重复性相对标准偏差(Relative standard deviation,RSD)为0.30%和0.64%,30 d精密度和重复性RSD为0.59%和0.79%,30 d内苯乳酸标准储备液稳定性RSD为2.45%,乳酸菌发酵液中苯乳酸合成量为0.1400 mg/L时,其扩展不确定度为0.0046,95%的置信区间,包含因子k=2。选用短乳杆菌P3(Lactobacillus brevis)和植物乳杆菌(Lactobacillus plantarum)ATCC8014发酵液为检测对象,培养120 h苯乳酸合成量分别为77.58和62.60 mg/L。结果表明,在该方法下苯乳酸能够稳定出峰,检测效率高,检测成本低,操作简单,结果准确可靠,适合检测大批量样品,可为苯乳酸的相关研究提供理论依据。

A Method for the Determination of Phenyllactic Acid in Lactic Acid Bacteria Fermentation Broth by Reversed Phase-High Phase Liquid Chromatography and Its Evaluation

A reversed phase high performance liquid chromatography(RP-HPLC)method for the determination of phenyllactic acid(PLA)in fermentation broth of lactic acid bacteria(LAB)was established and optimized.The effects of three stationary phases,two organic phases,five water phases and their proportions on the determination of PLA by HPLC were investigated. The double capped C₁₈ column(4.6 mm×250 mm,5 μm)was selected as the stationary phase,isoelution with 0.05% TFA acetonitrile(75∶25,v/v).The flow rate was 1 mL/min,the detection wavelength was 210 nm,and the column temperature was 30 ℃. The results showed that the linear range of the calibration curve for PLA was 0.2~5.0 mg/L with a correlation coefficient of 0.9998. The recovery rate was 99.88%~101.08%,LOD was 0.1400 mg/L,LQD was 0.4600 mg/L,relative standard deviation(RSD)of precision and repeatability were 0.30% and 0.64% in 1 d,precision and repeatability RSD were 0.59% and 0.79% in 30 d,stability of PLA standard stock solution RSD was 2.45%. Under the 95% confidence interval(coverage factor k=2),when the amount of PLA in the fermentation broth was 0.1400,the expanded uncertainty was 0.0046 mg/L. The yield of PLA synthesizd by Lactobacillus brevis P3 and Lactobacillus plantarum ATCC8014 were 77.58 and 62.60 mg/L after 120 h of culture,respectively. The results demonstrate that PLA can be detected stably,with high efficiency,low cost,simple operation,accurate and reliable results under the proposed method.