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Sigma-Aldrich offers kits, standards and procedures for measuring TVB, TMAO and biogenic amines in fish tissue
By Rainer Walz, rwalz@europe.sial.com
One of the most important applications of analytical chemistry is for the assessment of food quality, safety and nutritional value. Within the food industry, there are thousands of methods to determine levels of specific compounds that are indicators of some aspect of the food in question. Many of these tests are dictated by regulatory agencies. The focus of this article is the measurement of compounds that are indicators of the freshness of seafood products.
A challenge to the fisheries industry is how to maintain freshness of the catch over the distance between the catch site and the cannery or processing plant, and until it reaches the consumer. Seafood has a notoriously short freshness span between catch and degradation; freshly caught fish that isn't quickly processed or iced rapidly develops an unpleasant “fishy” odor. (Interestingly, fresh fish has little or no odor.) This unpleasant odor arises from a variety of compounds, but mostly from amines that are formed from the breakdown of amino acids in the fish tissue. Obviously, spoiled seafood has very little commercial value, so having reliable methods for the determination of freshness and spoilage is of great importance to both producers and consumers. There are three groups of compounds that are of particular interest from an analytical standpoint as freshness indicators: total volatile bases (TVB), trimethylamine N-oxide (TMAO) and the biogenic amines index (BAI).
Total Volatile Bases (TVB)
Regulations in the EU (95/149/EC) specify a maximum value for TVB in different fish species. The analytical method described in 95/149/EC is applicable to TVB concentrations from 5 mg/100 g up to 100 mg/100 g. The volatile nitrogenous bases are extracted from the fish sample using 0.6 M perchloric acid. After neutralization with alkali the extract undergoes steam distillation. An acid receiver absorbs the volatile basic compounds in the extracted sample. The TVB concentration is determined by titration of the absorbed bases.
Sigma-Aldrich offers a convenient, ready-to-use kit that makes TVB determination faster and easier than ever. The kit contains all the reagents needed for 10 determinations of TVB according to the EU method. The suitability of each pre-made reagent was thoroughly tested and verified. Concentrations and specifications of the perchloric acid, sodium hydroxide and boric acid solutions were specially designed for this method. Using these pre-made solutions saves a great amount of time (Table 1) allowing the analyst to increase the number of samples analyzed per day. A fully detailed protocol is provided with the Kit.
Table 1: Components of TVB in Fish Kit
|
Catalog No.
|
Brand
|
Pack Size
|
Product Name
|
Concentration
|
|
Fluka |
1 Kit |
TVB Fish Kit (enough for 10 analyses)* |
|
| 08612 |
Fluka |
4 x 250 mL |
Perchloric acid solution |
6 g/100 mL |
| 55973 |
Fluka |
1 x 100 mL |
Sodium hydroxide solution |
20 g/100 mL |
| 55293 |
Fluka |
5 x 250 mL |
Boric acid solution |
3 g/100 mL |
| 85390 |
Fluka |
1 x 10 mL |
Silicone anti-foaming agent |
30g in 100 mL water emulsion |
| 34607 |
Riedel-de Haën |
1 x 30 mL |
Phenolphthaleine solution |
1 g/100 mL 95 % ethanol |
| 36083 |
Riedel-de Haën |
1 x 30 mL |
Tashiro Indicator |
3 g in 1 L 95 % ethanol |
| 09700 |
Fluka |
1 x1g |
Ammonium chloride |
> 99.5% |
|
*Standard solutions are available in kit only and cannot be ordered individually.
Trimethylamine N-oxide (TMAO)
The second freshness indicator is trimethylamine N-oxide (TMAO). Seafood naturally contains TMAO, an osmolyte that is believed to counteract the deleterious effects of the high intracellular concentrations of urea in fish, sharks and rays. Ammonia and trimethylamine (TMA) are products of microbiological decomposition of TMA. Some fish, while being stored on ice, also develop formaldehyde and dimethylamine (DMA) as decomposition products of TMAO. The decomposition indicators, i.e. MNA, DMA and TMA can be measured using the TVB kit. Yet, TMAO must be evaluated using a different procedure.
Biogenic amines index (BAI)
The biogenic amines index (BAI) is the third marker of seafood freshness. BAI is based on the fact that the amount of the biogenic amines histamine, putrescine and cadaverine increases steadily after the death of the fish due to bacterial action on amino acids.
The Official Food Control Authority of the Canton of Schaffhausen (Switzerland) has recently developed an ion chromatography method for the simultaneous detection and quantitative measurement of several amines, including DMA, TMA, TMAO, histamine, putrescine and cadaverine.1,2 The method is applicable to nitrogen concentrations from 5 mg/kg fish to at least 1500 mg/kg fish. Many different species of fish have been analyzed successfully, including plaice, redfish, codfish, halibut and flounder (analyses of the examined fish samples were performed by N.Seifert, Metrohm Ltd, Switzerland).
The method is straightforward. Five grams of homogenized fish tissue and 50 mL buffer solution are combined and mixed. Each sample is prepared in duplicate. Calibration samples are prepared by spiking homogenized fish with 100, 300 or 500
µL of standard solution. An aliquot of the sample is transferred to an autosampler vial. Three different check standards are also prepared and one of the sample solutions is spiked with different concentrations of amines after the preparation to confirm the correct operation of the IC instrument. Seven-point calibration standards are prepared that bracket the expected concentrations in the samples. The solutions are stable for about one week in the refrigerator. The Kit contains all components for the analysis of TMAO and biogenic amines (Table 2).
Table 2. TMAO and Biogenic Amines Kit and Kit Components
| Catalog No.
|
Description
|
|
TMAO and Biogenic Amines Kit |
| 38270 |
Nitric acid standard solution concentrate (for 1 L of 0.1N solution) |
| 71180 |
Sodium acetate (50g) |
| 38960 |
Dimethylamine hydrochloride (2g) |
| 92277 |
Trimethylamine N-oxide dehydrate (2g) |
| 92270 |
Trimethylamine hydrochloride (2g) |
| 32810 |
Putrescine dihydrochloride (2g) |
| 33220 |
Cadaverine dihydrochloride (2g) |
| 53300 |
Histamine dihydrochloride (2g) |
| 07156 |
IC-column, C2 guard (Metrohm) |
|
The separation and measurement of the amines was performed on a 15cm Metrosep C2 IC column with 2cm guard, using nitric acid as eluent and indirect conductivity detection. The standards were injected twice. The sample solutions were injected five times each. The calculation was done by automatic integration using peak area for dimethylamine (DMA), trimethylamineoxide (TMAO), trimethylamine (TMA), putrescine, cadaverine and histamine.
A fully detailed protocol with the chromatograms of standard solution of amines, sample frozen plaice and sample fresh plaice are provided with each Kit.
Reference: R.Oechslin, et al. J. Chrom. (submitted)
Figure 1: Standard solution of amines
| No. |
Retention |
Height |
Area |
Conc. |
Name |
| |
|
|
|
|
|
| |
min |
mV |
mV*sec |
mg/kg |
|
| 1 |
4.96 |
77.89 |
810.227 |
2.046 |
DMA-N |
| 2 |
6.88 |
265.15 |
6074.601 |
13.535 |
TMAO-N |
| 3 |
8.37 |
136.93 |
4156.616 |
9.855 |
TMA-N |
| 4 |
10.82 |
38.15 |
991.843 |
2.356 |
Putrescin-N |
| 5 |
13.26 |
27.61 |
968.788 |
2.216 |
Cadaverin-N |
| 6 |
16.34 |
20.58 |
892.743 |
2.045 |
Histamin-N |
Acknowledgment: Fluka's R&D Department would like to thank to Laboratories of the Official Food Control Authority of the Canton of Schaffhausen for their support during the development of these kits.
1. Diplomasarbeit
2. G. Steil, Bestimmung von Qualitätsparameter von Dorschfilet (2002)
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