Food matrices and samples. For the evaluation of HPLC analytical method, 4 different agricultural products, including apple juice, Juk (Korean traditional porridge made of rice), corn oil and peanut butter, were selected as food matrices by a research steering committee, which consists of 11 principal investigators involved in a total dietary survey in Korea, based on their water and fat contents (i.e., non-fatty liquid, non-fatty solid, fatty liquid and fatty solid), which commonly affect and interfere with the performance of HPLC analytical methods. The samples were purchased from retail markets in Sejong city in Korea.
Four different types of fermented agricultural products, including soy sauce, Gochujang, Cheonggukjang and Doenjang, were purchased from 18 supermarkets in 9 different metropolitan cities with populations of more than one million in Korea and transported to the preparation laboratory within the day of collection. The collected products were homogenized to form a composite sample of each product type within 2~3 days. The frozen aliquots of composite samples in sealed containers were delivered to analytical laboratory within a day. After arrival, the samples were immediately subjected to analyses of biogenic amines. Thus, in this study, only one composite sample prepared by Korea Heath Industry Development Institute was subject to analysis for each food item.
Preparation of standard stock solution and working solution. All standard chemicals, including histamine dihydrochloride, β-phenylethylamine hydrochloride, putrescine dihydrochloride, tyramine hydrochloride, cadaverine dihydrochloride, tryptamine, spermine tetrahydrochloride and spermidine trihydrochloride (Sigma Chemical Co., St. Louis, MO, USA), were of analytical grade. Standard stock solutions of biogenic amines were separately prepared at 10,000 mg/L concentration in distilled water. Working solutions at concentrations of 100 or 1000 mg/L were prepared by diluting 100 μL of 1000 μL of each stock solution in distilled water to bring to a final volume of 10 mL.
Extraction of samples for HPLC analysis. Analysis of biogenic amines in the samples of spiked food matrices and fermented agriculture products was carried out based on the procedure of Ben-Gigirey et al. (13) (link). Briefly, 20 mL of 0.4 M perchloric acid (Sigma) were added to 5 g of the samples, and the mixture was homogenized using a vortex mixer (Vortex-Genie, Scientific industries, Inc., Bohemia, NY, USA), reacted in a cold chamber at 4℃ for 2 hr, and centrifuged at 3000 ×g at 4℃ for 10 min. The supernatant was collected, and the residue was extracted again with an equal volume of 0.4 M perchloric acid. Both supernatants were combined, and the final volume was adjusted to 50 mL with 0.4 M perchloric acid. The extract was filtered through Whatman paper No. 1 (Whatman; GE Healthcare UK Limited, Buckinghamshire, UK).
Derivatization of extracts and standards. Derivatization of biogenic amines was carried out according to the procedures developed by Ben-Gigirey et al. (13) (link). One milliliter of extract or standard solution prepared as above was mixed with 200 μL of 2 M sodium hydroxide and 300 μL of saturated sodium bicarbonate solution. Two milliliters of a dansyl chloride (Sigma) solution (10 mg/mL) prepared in acetone were added to the mixture, which was then incubated at 40℃ for 45 min. Residual dansyl chloride was removed by adding 100 μL of 25% ammonium hydroxide. After incubation for 30 min at 25℃, the volume of mixture was adjusted with acetonitrile to 5 mL. Finally, the mixture was centrifuged at 3000 ×g for 5 min, and the supernatant was filtered through 0.2 μm-pore-size filters (Millipore Co., Bedford, MA, USA). The filtered supernatant was kept at −25℃ until assayed by HPLC.
Chromatographic separations. Chromatographic separation of biogenic amines was carried out according to the procedures developed by Ben-Gigirey et al. (13) (link) with minor modifications. An HPLC unit (YL 9100, Young Lin Instrument, Co., Anyang, Korea), equipped with a UV-Vis detector (YL 9120, Young Lin) and Autochro-3000 software (Young Lin) was employed. A Nova-Pak C18 4 μm column (150 mm × 3.9 mm, Waters, Milford, MA, USA) was used for the separation of biogenic amines, and the samples were eluted with a step gradient of ammonium acetate (0.1 M; Sigma; solvent A) and acetonitrile (SK chemicals, Ulsan, Korea; solvent B) as the mobile phases at the flow rate of 1 mL/min. The program was set for a linear gradient starting from 50% of solvent B to reach 90% of the solvent at 19 min. The sample volume injected was 10 μL and monitored at 254 nm.
Spiking recovery test. The recovery test was performed by spiking food matrices with eight biogenic amines at three fortification levels of 10, 100 and 500 mg/kg. The reference spiked samples were pretreated and analyzed as described above. Recovery rate was calculated by comparing the amount of each biogenic amine detected in the spiked sample with the amount of each standard material added to sample. The analysis was conducted in quintuplicate.
Validation parameters. The method for the quantitation of biogenic amines in agricultural products was validated in terms of the analytical parameters of selectivity, linearity, precision, accuracy, recovery, limit of detection (LOD) and limit of quantification (LOQ) following conventional protocols from international guidelines (14 ,15) . The analysis was conducted in quintuplicate.