Studies On The High-performance Liquid Chromatography Techniques For Biogenic Amine Analysis In Food

Biogenic amines are microbial decarboxylation products of free amino acids and are widely distributed among various types of foods, especially in fermented or bacterially contaminated foods. Biogenic amines in food are considered as a public health concern because of their potential toxicity; also, they have been proposed as microbiological quality indicators of certain foods. For these reasons, biogenic amine analysis has now become an important and routine part of food contaminant analysis and of food quality analysis. The main objective of this study was to develop improved or noval high-performance liquid chromatography (HPLC) methods/techniques for biogenic amine analysis in food. The main results and conclusions are listed as follows.1. For the first time, a good number of samples (n= 39) of semi-dry and semi-sweet Chinese rice wine produced in the Shaoxing region were analyzed for 9 biogenic amines, using a reversed-phase HPLC-UV method based on pre-column benzoylation. The main biogenic amines found, in descending order of mean concentration, were serotonin, putrescine, tyramine, cadaverine, and histamine. The total biogenic amine contents were variable, ranging from 29.3 to 260 mg/L with 115 mg/L on average. Statistical analyses indicated that:(1) good Pearson's correlation relationships were found between some amines, suggesting that their formation may be governed by the same factors under the same mechanisms; (2) higher contents of serotonin were found in semi-sweet rice wine samples, possibly because serotonin is of rice origin; (3) samples from some brewers had a higher level of biogenic amines, which might be attributed to the raw materials and/or brewing practice.2. Important modifications to the benzoylation derivatization procedure have been made. (1) The employment of a continuous vortex-mixing significantly enhanced the reaction rate between sparingly soluble benzoyl chloride and biogenic amines in aqueous solutions. The proposed derivatization reaction takes only 2 min, in contrast to existing procedures which require 15-45 min, making it one of the rapidest pre-column derivatization methods. (2) The derivatization yields of some amines (including histamine and tyramine) were also greatly improved by continuously vortex-mixing, allowing more sensitive detection; under continuously vortex mixing, the level of NaOH addition was crucial and excessive addition of NaOH consumed most of benzoyl chloride molecules, decreasing the yields of some biogenic amines. (3)The addition of NaOH and acetonitrile after derivatization and a following 1-min vortex-mixing efficiently and rapidly decomposed excess benzoyl chloride and eliminated the interference from the main byproducts benzoic acid, benzoic anhydride, and methyl benzoate, thus preventing the column from damage and improving the separation and qualification and quantification. The new benzoylation method was successfully applied to the analysis of 10 biogenic amines in beer samples. The whole method consisting of the sample cleanup, benzoylation, separation, and detection was validated.3. A chaotropic chromatography method was proposed for analyzing six aromatic or heterocyclic biogenic amines of greatest toxicological importance. The main problems associated with the reversed-phase separation of these protonated amines were poor or even no retention and mass overloading peak-tailing. Both the two problems were successfully addressed by adding the chaotropic salt KPF6 into an acidic mobile phase. However, an excessive concentration of KPF6 led to a slightly decrease of retention. The dependences of the logarithmic retention factor (log k) of amines on the acetonitrile content in the mobile phase (<p) were not always reasonably linear, especially for histamine dication which exhibited a rather irregular retention behavior:in the low ?-region, the retention increased with (p. A set of optimized chromatographic conditions were obtained for analyzing three different food samples (vinegar, mackerel, and baijiu). Under the conditions the retention, peak shape, and peak efficiency of the six amines, and the resolution between the amines and interferents were very satisfactory. The method detection limits were in the range 0.06-0.15?g/mL, comparable to, or even lower than, those of derivatization based HPLC-UV methods.4. A red wine sample was used for the investigation of biogenic amine analysis by high-temperature liquid chromatography (HTLC). The main results are as follows: For moderate high-temperature (say,?80?) operation, conventional HPLC equipment requires little key adaptation except for experimentally choosing an appropriate length (e.g.,0.5-1 m) of SS tubing as mobile-phase preheater. Biogenic amines as benzoyl derivatives showed good thermostability on the thermally rugged Zorbax StableBond C8 column. With the aid of DryLab simulation promising conditions of temperature and gradient for separation can be thoroughly and rapidly examined. The optimization of the wine sample separation demonstrates the importance of temperature as a selectivity variable in method development, and the ease of exploring temperature selectivity constituents a clear advantage of HTLC. Operating the column at elevated temperatures complicates the optimization of flow rate because higher flow rates require longer preheat tubing and a readjustment of the oven temperature may be required to maintain the same retention and selectivity while increasing flow rate. The optimized HTLC method offered a high sample resolution of Rs=1.83 (eight biogenic amines and eight interferents) in 6.5-min injection-to-injection time at a high flow rate of 3 mL/min (with a system pressure drop lower than 200 bar). The HTLC methodology outperforms existing fast liquid chromatography methodologies in terms of analysis time, sample resolution or selectivity, equipment cost, and reliability.Altogether, the improved benzoylation derivatization method can be applied for the routine analysis of biogenic amines in food, while for analyzing aromatic or heterocyclic amines, the chaotropic chromatography method is simpler and more convenient and the analytical accuracy is independent on that of the derivatization method. The HTLC method is accessible to the average laboratory for fast and high-throughput analysis; besides, the temperature selectivity may be useful to tackle the challenging separation of difficult (complex) samples.