Performance evaluation of a fluorescamine-HPLC method for determination of histamine in fish and fish products

A method for the quantification of histamine in fish and fish products using tandem solid-phase extraction and fluorescence derivatization with fluorescamine was previously developed. In this study, we improved this analytical method to develop an official test method for quantification of histamine in fish and fish products, and performed a single laboratory study to validate it. Recovery tests of histamine from fillet (Thunnus obesus), and two fish products (fish sauce and salted and dried whole big-eye sardine) that were spiked at the level of 25 and 50 μg/g for T. obesus, and 50 and 100 μg/g for the two fish products, were carried out. The recoveries of histamine from the three samples tested were 88.8-99.6% with good repeatability (1.3-2.1%) and reproducibility (2.1-4.7%). Therefore, this method is acceptable for the quantification of histamine in fish and fish products. Moreover, surveillance of histamine content in food on the market was conducted using this method, and high levels of histamine were detected in some fish products.     

Determination of histamine in fish products by capillary electrophoresis and ion‐pair liquid chromatography with diode‐array detection

A method for histamine determination in fish and fish products by capillary electrophoresis and ion‐pair liquid chromatography has been developed. Histamine was extracted from different fish samples with 10% trichloroacetic acid, and the filtrate was directly applied to capillary electrophoresis apparatus. A preliminary purification through a solid phases extraction cartridge (Sep‐pak C₁₈) was necessary for HPLC analysis. Histamine was detected ‘on‐line’ at 210 nm by both approaches. HPLC procedure allowed accurate (±2.2%) and repeatable (±1.6%) determination of histamine in fish products at appropriate levels (20–100 mg kg⁻¹) to meet current regulations. © 1999 Society of Chemical Industry     

Histamine levels in commercially important fresh and processed fish of Oman with reference to international standards

Histamine was investigated in fresh (378), frozen (441), canned (290) and dried (24) fish samples of scombroid and non-scombroid species of Oman using a high performance liquid chromatography with a fluorescence detector. Of the 1133 fish samples tested, histamine was detected in 551 samples with a detection rate of about 41.8% among fresh fish, 61.0% of frozen fish, 78.9% of canned fish and 91.6% of dried fish samples and the mean histamine levels were 2.6, 5.8, 3.1 and 104 mg kg⁻¹, respectively. A total of 3.7% and 0.79% of the total samples exceeded the FDA and EU regulatory limits for histamine. Imported dried anchovies contained high histamine levels. The study confirms that post catching and commercialisation practices of seafood are adequate, warranting good quality fish and may not cause histamine risk to consumer in terms of human diet. While necessary monitoring may be done for imported dried fish products.     

Occurrence of histamine in canned tuna fish from Iran

Histamine fish poisoning is a significant public health and safety concern and also a trade issue. Histamine was investigated in canned tuna fish in Qazvin province from Iran by ELISA method. Histamine was detected in 29.37 % canned tuna fish samples. The overall mean level of histamine was 8.59 ± 14.24 ppm and the concentration was between 2.51 and 74.56 ppm. Except four samples (2.50 %) of 47 positive canned tuna fish samples, histamine content of other positive samples was in compliance with Food and Drug Administration (FDA) legislation (lower than 50 ppm). Most canned tuna fish contain histamine with high mean value was produced in summer (52.50 %) and spring (47.50 %) seasons, the mean levels of histamine were 6.30 and 12.62 ppm in these positive samples, respectively. The results of this study indicate that the most canned tuna fish produced in Qazvin province of Iran has histamine levels lower than the allowable limit suggested by FDA. Further studies should be carried out to investigate the presence of this toxin and other biogenic amines in different fish and sea food products.     

高效液相色谱-紫外检测法测定水产品中组胺含量

目的对高效液相色谱法测定水产品(鲭鱼)中组胺含量的试验方法进行改进,建立一种提取方法简单、衍生物稳定、操作方便的检测方法。方法用0.4 mol/L高氯酸溶液提取水产品中的组胺,采用10 mg/m L丹磺酰氯溶液作为衍生剂进行衍生,用甲醇、水的流动相梯度洗脱,高效液相色谱-紫外检测法测定水产品中的组胺含量。结果该方法提取完全、操作简单、峰型理想、组胺出峰干扰少,回收率大于90%,方法检出限为50 mg/kg。结论该方法能够满足水产品中组胺含量的测定要求,是一种理想的组胺检测方法。     

Simultaneous determination of eight underivatised biogenic amines in fish by solid phase extraction and liquid chromatography-tandem mass spectrometry

Biogenic amines on fish tissue are formed as a result of bacterial contamination and spoilage during storage. A new method based on liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using a triple quadrupole (QqQ) analyser was developed for the analysis of eight biogenic amines (cadaverine, histamine, phenylethylamine, putrescine, spermine, spermidine, tyramine and tryptamine) in fish tissues. Sample preparation was performed by extraction with trichloroacetic acid 5% and solid phase extraction clean up with STRATA X cartridge. The MS/MS method was validated and compared with a method based on the analysis of dansyl derivatives by LC and fluorescence detector (FD). MS/MS achieved higher sensitivity (from 0.02 mg kg⁻¹ for spermidine and phenylethylamine to 0.2 mg kg⁻¹ for spermine) when compared to FD (from 1 mg kg⁻¹ for putrescine and tyramine to 4 mg kg⁻¹ for histamine); MS/MS method showed higher precision too, with intraday relative standard deviations (RSDs) from 1% to 4% with respect to those obtained with FD method (from 3% to 17%).Recovery study was conducted at two different fortification levels and the average ranged from 71% to 93% for all of the studied compounds with RSDs lower than 18%. Matrix-matched standards were used to counteract matrix effect observed in MS/MS determination. The applicability of the method was demonstrated by the analysis of biogenic amines in fish obtained from commercials of Valencia.     

A comparison of the TLC-densitometry and HPLC method for the determination of biogenic amines in fish and fishery products

A comparative study on the suitability of chromatographic techniques such as thin layer chromatography (TLC)-densitometry and high performance liquid chromatography (HPLC) for the analysis of biogenic amines in fish and fishery products was carried out. The HPLC method was found to offer a good linearity (5–100 ng), sensitivity and repeatability (<3%), but required sophisticated instrumentation, technical skill and high operational cost and time. On the other hand, with a little loss in linearity (20–300 ng) and repeatability (<8%), TLC-densitometry was found to be rapid and less expensive. In addition, this method is suitable for rapid and simultaneous screening of several samples at a time. Therefore, the TLC-densitometric method can be effectively used in the fish industry to detect biogenic amines, especially the toxic histamine, and putrescine and cadaverine, which can potentiate histamine toxicity in fish and fishery products.     

Determination of histamine by capillary zone electrophoresis using a low-pH phosphate buffer: application in the analysis of fish and marine products

 Histamine levels in samples of fish and marine products were determined by capillary zone electrophoresis using phosphate buffer at pH 2.44 and UV detection at 214 nm. A plot of the area of the peaks versus histamine concentration was linear over the range of 1–100 ppm with a correlation coefficient of 0.9996. Calibration using the height of the peak also gave good correlation, with a correlation coefficient of 0.9969, at concentrations between 1 ppm and 20 ppm. The recovery of added histamine in different samples of fish and fish products was, on average, 99.65%. The method described in this work was used for the quantitation of histamine in fish and fish products. Received: 19 March 1996/Revised version: 22 July 1996     

Commercial test kits and the determination of histamine in traditional (ethnic) fish products-evaluation against an EU accepted HPLC method

Although commercial test kits are generally used and/or evaluated for determining histamine in fresh and canned fish, and fish meal, there is little information on their performance and the application for traditional fish products (TFPs), which generally differ in product properties. In this study, three quantitative (Food EIA, Veratox, Histaquant) and four qualitative (Histasure, Histameter, Transia qualitative and semi-quantitative) commercial histamine test kits were evaluated against HPLC method for detecting histamine in several traditional fish products. Among the quantitative kits, Histamine Food EIA showed the best correlation with HPLC method for TFPs (R² = 0.9132) as well as good recoveries ranging between 89 (±4.11)% and 117 (±1.50)%. Although good recoveries were also observed with Veratox kit, poor correlation was found with HPLC. Poor correlation and low recoveries were also observed with Histaquant. Histasure and Transia tube histamine kits showed good agreement with HPLC results. However, the detection limit for Transia qualitative kit is 100 ppm, and for Histasure and Transia semi-quantitative kits can be set to 50 ppm. Therefore, Histasure and Transia semi-quantitative kits are found more suitable for either HACCP monitoring histamine in seafood processing plants or regulatory purposes according to Food Drug and Administration (FDA) legislation for TFPs. However, Transia semi-quantitative kit should be used in caution for histamine cut off values <50 ppm. This study shows that each test kit can represent different performance for determining histamine in TFPs according to product type, and therefore new commercial test kits should be evaluated against an approved analytical method before applications in future for these types of products.     

甲苯萃取-原子荧光光谱法检测鱼肉中甲基汞

目的建立甲苯萃取-原子荧光光谱检测鱼肉中甲基汞的方法。方法样品经48%氢溴酸水解、甲苯萃取、0.05 mol/L硫代硫酸钠反萃取后,应用AFS-9230原子荧光光谱法进行测定,同时经此前处理后的样品采用DMA-80直接测汞仪进行测定,与原子荧光光谱法的结果进行对比。结果原子荧光光谱法以及直接测汞仪直接测定的结果与标准物质定值吻合,确证该方法的可靠性,适用于鱼、贝等水产品中甲基汞的测定。采用该方法对鱼肉中总汞与甲基汞成分分析标准物质GBW10029以及英国食品分析水平评估计划(food analysis performance assessment scheme,FAPAS)的罐装鱼肉样品(样品编号07189)进行了检测,检测结果与参考物定值吻合,回收率分别为96.1%,97.5%。结论该方法操作简单,灵敏度高,重现性好,适用于鱼肉中甲基汞含量的定量检测。     

Histamine contents of fermented fish products in Taiwan and isolation of histamine-forming bacteria

Twenty-seven imported fermented fish products from Southeast Asian countries and sold in the supermarkets in Taiwan, including fish sauce, fish paste and shrimp paste, were tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, total volatile basic nitrogen, trimethylamine, and aerobic plate count in all samples ranged from 4.8% to 6.5%, 16.2% to 45.3%, 51 to 275 mg/100 g, 5.4 to 53.9 mg/100 g and 1.0 to 4.2 log CFU/g, respectively. The average content for each of eight different biogenic amines in all samples was less than 90 ppm, except for histamine which has an average content of 394 ppm in fish sauce, 263 ppm in fish paste, and 382 ppm in shrimp paste. Most of the tested fermented fish products (92.6%) had histamine levels greater than the FDA guideline of 50 ppm, while seven of them (25.9%) contained >500 ppm of histamine. Although Bacillus coagulans and Bacillus megaterium were identified as the two histamine-producing bacteria capable of producing 13.7 and 8.1 ppm of histamine, respectively, in trypticase soy broth broth supplemented with 1.0% l-histidine, they were not determined to be the main contributors to histamine accumulation in these fermented fish products.     

Histamine fish poisoning revisited

Histamine (or scombroid) fish poisoning (HFP) is reviewed in a risk-assessment framework in an attempt to arrive at an informed characterisation of risk. Histamine is the main toxin involved in HFP, but the disease is not uncomplicated histamine poisoning. Although it is generally associated with high levels of histamine (> or =50 mg/100 g) in bacterially contaminated fish of particular species, the pathogenesis of HFP has not been clearly elucidated. Various hypotheses have been put forward to explain why histamine consumed in spoiled fish is more toxic than pure histamine taken orally, but none has proved totally satisfactory. Urocanic acid, like histamine, an imidazole compound derived from histidine in spoiling fish, may be the "missing factor" in HFP. cis-Urocanic acid has recently been recognised as a mast cell degranulator, and endogenous histamine from mast cell degranulation may augment the exogenous histamine consumed in spoiled fish. HFP is a mild disease, but is important in relation to food safety and international trade. Consumers are becoming more demanding, and litigation following food poisoning incidents is becoming more common. Producers, distributors and restaurants are increasingly held liable for the quality of the products they handle and sell. Many countries have set guidelines for maximum permitted levels of histamine in fish. However, histamine concentrations within a spoiled fish are extremely variable, as is the threshold toxic dose. Until the identity, levels and potency of possible potentiators and/or mast-cell-degranulating factors are elucidated, it is difficult to establish regulatory limits for histamine in foods on the basis of potential health hazard. Histidine decarboxylating bacteria produce histamine from free histidine in spoiling fish. Although some are present in the normal microbial flora of live fish, most seem to be derived from post-catching contamination on board fishing vessels, at the processing plant or in the distribution system, or in restaurants or homes. The key to keeping bacterial numbers and histamine levels low is the rapid cooling of fish after catching and the maintenance of adequate refrigeration during handling and storage. Despite the huge expansion in trade in recent years, great progress has been made in ensuring the quality and safety of fish products. This is largely the result of the introduction of international standards of food hygiene and the application of risk analysis and hazard analysis and critical control point (HACCP) principles.     

PCR detection and identification of histamine-forming bacteria in filleted tuna fish samples

Total of 14 filleted yellowfin tuna fish (Thunnus albacares) sold in wholesale fish market and supermarkets in Milan, Italy, were purchased and tested to determine microbial count, histamine level, histamine-forming bacteria, and their ability to produce histamine in culture broth. Although histamine level was less than 10 ppm, many samples showed high total viable bacterial and enterobacterial counts that reached dangerous levels after temperature abuse for short periods of time. A PCR assay targeting a 709-bp fragment of the histidine decarboxylase gene (hdc) revealed that 30.5% of the 141 enteric bacteria isolated from samples were positive and potentially able to produce histamine. The hdc positive strains were mainly isolated from fish bought at wholesale fish market, where we observed several possible risk factors, such as handling in poor and non-refrigerated conditions during fillet preparation. These positive strains were identified as Citrobacter koseri/Enterobacter spp. and Morganella morganii, by 16S/23S rRNA internal transcribed spacer amplification and 16S rRNA sequence analysis. The strains showed a variable ability of histamine production, with Morganella morganii being the most active histamine-producing species. A direct DNA extraction from fish and a PCR targeting the hdc gene showed a high degree of concordance with the results obtained through microbiological and chemical analyses, and could aid in the prompt detection of potentially contaminated fish products, before histamine accumulates. PRACTICAL APPLICATION: The use of methods for the early and rapid detection of bacteria producing biogenic amines is important for preventing accumulation of these toxic substances in food products. In this study, we used a molecular approach for the detection of histamine-forming bacteria in fish. PCR-based methods require expensive equipment and a high degree of training for the user, but are fast (< 24 h) and reliable. They now represent the best predictive methods to identify a potential risk factor in fish products during processing, storage, and marketing and can be used in the investigation of risk reduction strategies.     

A risk-based sampling plan for monitoring of histamine in fish products

In 2008, the French Institute for Public Health Surveillance reported an increase in the number of histamine food poisoning outbreaks and cases in France. The aim of this study was to propose a new monitoring plan for characterizing consumers' exposure to histamine through fishery products. As fish products of concern are numerous, we proposed that the number of samples allocated for a fish category be chosen based on the risk associated with the category. Point risk estimates of histamine poisoning were assessed with the Risk Ranger tool. Fresh fish with high histidine content was found to contribute most to the number of cases. The (estimated) risks associated with the consumption of canned and deep-frozen fish appear marginal as compared with the risk associated with fresh fish with high histidine concentrations. Accordingly, we recommend excluding canned and deep-frozen fish from the monitoring plan, although these risk estimates can be biased. Within a category, samples were proportional to the relative food consumption of the different fishes. The spatial and seasonal consumption patterns were also taken into account for the design of the new monitoring plan. By testing appropriate numbers of samples from categories of fish products of concern, this plan will permit investigation of trends or comparison of product categories presenting risks of histamine poisoning.     

六类鱼制品中生物胺的HPLC法测定

针对不同类型的鱼制品建立同时检测腐胺、尸胺、精胺、亚精胺、酪胺、苯乙胺、组胺和色胺8种生物胺含量的高效液相色谱(HPLC)法。应用该方法,测定了干燥制品、油炸制品、烘烤制品、发酵制品、混揉制品以及萃取制品6类鱼干制品中的生物胺含量。结果显示,萃取制品-鱼油中未检测出生物胺,其余5类产品中生物胺含量最高的为发酵制品(73.42~70.59 mg/kg)和混揉制品(55.48~49.88 mg/kg),其次为油炸制品(17.72~14.32 mg/kg),而鱼干制品(5.28~5.17 mg/kg)和烘烤制品(4.75~4.69 mg/kg)中的生物胺含量相对较少;另外,尸胺、组胺、精胺和亚精胺四种生物胺为除萃取制品之外的5类鱼制品中普遍存在的4种生物胺成分;其中尸胺是鱼制品中变化最大、影响最为明显的单体生物胺,检测产品中尸胺含量有利于监控、评判鱼干制品的质量。     

Biogenic amines in fish, fish products and shellfish: a review

Fish, cephalopods and shellfish provide a healthy source of high-quality proteins, essential vitamins, minerals and polyunsaturated fatty acids. The beneficial effects of fish consumption on human health such as protection against coronary heart disease and certain cancer may be offset by fish decomposition and the formation of chemical contaminants such as biogenic amines. There are several toxicological effects of biogenic amines on humans, especially histamine. It is the causative agent of histamine or scombroid fish poisoning which is a significant public health problem. In individuals with diminished histamine detoxification, ingestion of even a low or moderate histamine- or tyramine-containing fish may lead to food intolerance. Biogenic amines such as putrescine, tyramine and cadaverine can potentiate histamine toxicity. Furthermore, dietary polyamine intake should be minimised in some cancer patients. Besides their potential toxicity, biogenic amines are used for the evaluation of hygienic quality of different marine and freshwater species. Spoilage pattern and biogenic amine formation are species specific. Histamine has been traditionally used as an indicator of the quality of histidine-rich fish (dark-muscle fish). On the other hand, putrescine and cadaverine are the most objective indicators of quality of histidine-poor fish (white-muscle fish), shellfish and fermented seafood products.     

Biogenic amines in fish,fish products and shellfish: a review

Fish, cephalopods and shellfish provide a healthy source of high-quality proteins, essential vitamins, minerals and polyunsaturated fatty acids. The beneficial effects of fish consumption on human health such as protection against coronary heart disease and certain cancer may be offset by fish decomposition and the formation of chemical contaminants such as biogenic amines. There are several toxicological effects of biogenic amines on humans, especially histamine. It is the causative agent of histamine or scombroid fish poisoning which is a significant public health problem. In individuals with diminished histamine detoxification, ingestion of even a low or moderate histamine- or tyramine-containing fish may lead to food intolerance. Biogenic amines such as putrescine, tyramine and cadaverine can potentiate histamine toxicity. Furthermore, dietary polyamine intake should be minimised in some cancer patients. Besides their potential toxicity, biogenic amines are used for the evaluation of hygienic quality of different marine and freshwater species. Spoilage pattern and biogenic amine formation are species specific. Histamine has been traditionally used as an indicator of the quality of histidine-rich fish (dark-muscle fish). On the other hand, putrescine and cadaverine are the most objective indicators of quality of histidine-poor fish (white-muscle fish), shellfish and fermented seafood products.     

Biogenic amines in cold-smoked fish fermented with lactic acid bacteria

 It has been proved in previous studies that fish can be fermented by lactic acid bacteria (LAB). Added glucose must be at concentrations of almost 1% to reduce the pH to <5.3, a level which corresponds to the safe pH values for meat products. Because some LAB and some contaminating microbes form biogenic amines, this study was conducted to examine the possible formation of biogenic amines during the fish fermentation process. Other parameters under study were sensory quality, pH value, titrated acid content, weight loss, a _w value, redox potential and microbiological counts. Three groups of fish (rainbow trout) with three different LAB inocula and a group without any inoculum were made. The fermentation of the products made with LAB succeeded. The products were sensorially accepted, the inoculated LAB grew to >8 log cfu/g, the pH reduced to 5.0–5.3 and a _w to 0.927 and the pseudomonads, the predominant flora of fish raw material, disappeared. The fish raw material and the products contained low amounts of biogenic amines with one exception: cadaverine, histamine and tyramine increased in all product groups in one experimental series (II) out of three. The highest concentrations of these amines were in the control products without any LAB inoculation. The LAB used and the contaminants isolated from the products were unable to produce cadaverine, histamine or tyramine. The appearance of these amines could therefore be caused by the non-isolated contaminants of fish raw material. Received: 18 June 1999     

鱼类罐头相关食品标准法规中组胺指标探讨

目的探讨鱼类罐头相关食品标准法规中组胺指标,为GB 7098—2015《食品安全国家标准罐头食品》中鱼类罐头组胺指标修订提供基础数据。方法整理分析国内外法规情况、我国水产品及其制品组胺中毒情况,统计分析广东省市售高组胺鱼类罐头中组胺含量、广东省成人居民鱼类罐头消费量数据及组胺参考限量值。结果我国的鱼类罐头(仅适用于鲐鱼、鲹鱼、沙丁鱼罐头)组胺限量标准为1 000 mg/kg,高于国际组织和国外的组胺限量标准;1998—2018年我国水产品及其制品组胺中毒报道案例共18例,引起组胺中毒食物检测含量最低为120 mg/kg,最高为3 820 mg/kg;广东省主要鱼类罐头生产地区采集鱼类罐头样品136份,组胺含量范围为未检出(ND)～488.8 mg/kg,组胺含量超过200 mg/kg的占3.7%(5/136),超过400 mg/kg的占1.5%(2/136);基于广东省成人居民鱼类罐头高消费人群(P97.5)消费量为120 g/次和无作用剂量(NOAEL,50 mg)得出鱼类罐头组胺参考限量值为417 mg/kg。结论 GB 7098—2015鱼类罐头组胺指标限量值高,测定鱼品种少,建议我国开展高组胺鱼类罐头组胺风险评估,修订该标准鱼类罐头组胺指标。     

Biogenic amine contents in fish and fish preserves

A method developed on the basis of highly efficient liquid chromatography of dansyl derivatives was used to study the content of biogenic amines (tyramine, histamine, cadaverine, putrescine, spermine and spermidine) in fish and canned fish. It was found that only 7 out of 149 samples contained histamine levels exceeding maximum permissible concentration (salted herring, smoked sardines, and salted Siberian salmon). No correlation was recorded between the increase of spermine content and spermidine.