Validation of analytical method for detection of bogenic amines in fish samples using gas chromatogarphy - flame ionization detector

• Main Author: Muhammad Abdurrahman, Munir
• Format: Thesis
• Language: English
• Published: Universiti Malaysia Sarawak (UNIMAS) 2016
• Subjects: Q Science (General); QD Chemistry
• Online Access: http://ir.unimas.my/id/eprint/27810/3/M%20Abdurrahman%20ft.pdf; http://ir.unimas.my/id/eprint/27810/
• Institution: Universiti Malaysia Sarawak

Biogenic amines are nitrogenous compounds with low molecular weights. Based on their chemical structures, they can be aliphatic (heptylamine, cadaverine and spermidine), aromatic (tyramine) or heterocyclic (histamine). Biogenic amines can be found in food and beverages which contained protein. Biogenic amines, particularly histamine, with the presence of cadaverine and putrescine can cause scombroid food poisoning. Gas chromatography coupled with flame ionization detector (GC-FID) have been validated for the determination of biogenic amines (heptylamine, histamine, tyramine, cadaverine and spermidine) in fish and fish product samples, whereas gas chromatography coupled with mass spectrometer (GC-MS) has been used to identify biogenic amines. Analysis of biogenic amines using GC is difficult owing to their lack of volatility and therefore, biogenic amines need to be derivatised before analysed using GC-FID and GC-MS. A mixture of N, O-bis (trimethylsilyl) acetamide (BSA) and rimethylchlorosilane (TMCS) were employed in this study. The chromatographic separation of GC-FID was performed on a capillary column HP-5 (30 meter, 0.25 mm and 0.25 µm stationary phase thickness), whereas GC-MS analysis was carried out on a capillary BPX-5 column (30 meter, 0.25 mm and 0.25 µm). The validation of specificity, linearity, precision, accuracy, detection limit and quantitation limit results indicate that the methods were acceptable. All review stated derivatised biogenic amines such as heptylamine, histamine, tyramine, cadaverine and spermidine appeared in GC-FID chromatogram without any impurity peak. The retention times of heptylamine, histamine, tyramine, cadaverine and spermidine after being analysed using GC-FID were 10.46, 11.13, 14.33, 14.81 and 18.88 min, respectively. The linear regression analysis data for the calibration plots showed good linear relationship of five biogenic amines ranged between r 2 = 0.9995 – 0.9999 with respect iv to peak area in the concentration range of 25 – 150 µg/mL. Good precision and accuracy were found to be below 7.87%. The detection limit of five biogenic amines obtained ranged between 1.20 – 2.90 µg/mL (3 S/N), whereas for quantification limit of five biogenic amines acquired ranged between 3.98 – 9.65 µg/mL (10 S/N). Recovery values of biogenic amines from 98.41 – 116.39 % indicate excellent accuracy of the procedure. The GC-FID method is accurate and precise and it can be successfully applied to determine the presence of biogenic amines in fish and fish product samples. Ten raw fish and salted products were sardine (Sardinella gibbosa), mackerel (Scomberomorus guttatus), gourami (Trichogester pectoralis), whiptail stingray (Himantura walga) and toli shad (Tenualosa toli), while the four canned fish samples were sardine, mackerel, salted sardine and dace (Leuciscus leuciscus). All of them were dissolved and soaked by HPLC grade water and methanol. Histamine was found in raw mackerel (S. guttatus) and sardine (S. gibbosa) with concentrations at 5.96 and 2.69 mg/kg, respectively. Salted sardine (S. gibbosa) was found to contain histamine concentration at 8.95 mg/kg. For canned fish products, histamine was only detected in dace (L. leuciscus) with concentration at 6.18 mg/kg. All concentrations of histamine were detected below 50 mg/kg (FDA regulation) which will not lead to scombroid poisoning. Cadaverine was detected in raw sardine (S. gibbosa), whiptail stingray (H. walga), salted gourami (T. pectoralis), canned mackerel (Scomberomorus sp.) and sardine (Sardinella sp.) with concentration at 4.96, 146.39, 18.80, 150.33 and 24.65 mg/kg, respectively. The content of tyramine in canned dace (L. leuciscus) should be of concern owing to the fact that the concentration was above 600 mg/kg.