一种新的柱前衍生RP-HPLC法分析酱腌菜中的生物胺

Analysis of Biogenic Amines in Pickled Vegetable by a New Pre-column Derivatization of RP-HPLC

摘要:

以4-fluoro-3-nitrobenzo-trifluoride(FNBT)为衍生剂，N,N-diisopropylethyl-amine(DIPEA)为催化剂，建立了一种新的柱前衍生-反高效液相色谱检测法（RP-HPLC）同时测定酱腌菜中的8种生物胺(组胺、色胺、β-苯乙胺、腐胺、酪胺、尸胺、亚精胺及精胺)。采用硼酸盐缓冲溶液为衍生介质，在60 ℃下衍生反应30 min，使用C18色谱柱分离，以乙腈和超纯水为流动相梯度洗脱，流速1.00 mL/min，紫外检测波长242 nm。在0.5~100 mg/L浓度范围内，8种生物胺呈现良好的线性相关（r2≥0.999），方法的检出限(LODs)为0.013~0.053 mg/L，定量限(LOQs)为0.044~0.176 mg/L，加标平均回收率为93.47%~99.87%，相对标准偏差(RSD)为0.47%~3.67%。采用该方法对26种市售酱腌菜中的生物胺进行了分析，发酵型酱腌菜中的生物胺总量范围为40.57~692.82 mg/kg，非发酵型酱腌菜中的生物胺总量范围为9.86~270.93 mg/kg。发酵型酱腌菜中8种生物胺单体的平均含量均高于非发酵型酱腌菜。该方法简单、准确和可靠，可适用于酱腌菜中生物胺的快速分析。

Abstract:

A new pre-column derivatization of reversed-phase high performance liquid chromatographic (RP-HPLC) method for the determination of eight biogenic amines (histamine, tryptamine, β-phenylethylamine, tyramine, putrescine, cadaverine, spermidine and spermine) in pickled vegetable was established using 4-fluoro-3-nitrobenzo-trifluoride (FNBT) as the derivatizing agent and N,N-diisopropylethyl-amine (DIPEA) as the catalyst. The derivatization of biogenic amines was conducted by FNBT and DIPEA with borate buffer solution at 60 ℃ for 30 min, and then the resultant mixture was separated on a C18 column with mobile phase (acetonitrile and ultrapure water) at a flow rate of 1.00 mL/min. The UV detection wavelength was 242 nm. Eight biogenic amines showed a good linear correlation (r2≥0.999) in the concentration range of 0.5~50 mg/L with the limits of detection (LODs) of 0.013~0.053 mg/L and the limits of quantification (LOQs) of 0.044~0.176 mg/L. The mean recovery was in the range of 93.47%~99.87%, and the relative standard deviations (RSDs) were 0.47%~3.67%. This method was used to analyze the biogenic amines in the 26 commercial pickled vegetable, and the results showed that the total content of biogenic amine in fermented and non-fermented pickled vegetable samples were 40.57~692.82 mg/kg and 9.86~270.93 mg/kg, respectively; the average content of eight monomer biogenic amines in fermented pickled vegetable samples were higher than those of non fermented pickled vegetable samples. This developed method was simple, sensitive and reliable, and it was suitable for the rapid analysis of biogenic amines in pickled vegetable.