SPME-GC METHOD FOR THE DETERMINATION OF VOLATILE AMINES AS INDICES OF FRESHNESS IN FISH SAMPLES

Abstract

Volatile amines including, methylamine, dimethylamine and trimethylamine have been widely used for measuring freshness of seafood. In the present work an SPME-GC-FID method to evaluate the volatile amine contents in fish samples stored under controlled conditions was developed. The optimum conditions for the extraction of amines were as follows: 50/30 µm DVB/Carboxen/PDMS fibre, extraction time and temperature of 15 min and 21°C. In addition, total volatile basic nitrogen (TVB-N) was determined by a titration method. Results showed good agreement between two determinations. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Volatile amines including, methylamine, dimethylamine and trimethylamine have been widely used for measuring freshness of seafood. In the present work an SPME-GC-FID method to evaluate the volatile amine contents in fish samples stored under controlled conditions was developed. The optimum conditions for the extraction of amines were as follows: 50/30 µm DVB/Carboxen/PDMS fibre, extraction time and temperature of 15 min and 21°C. In addition, total volatile basic nitrogen (TVB-N) was determined by a titration method. Results showed good agreement between two determinations.

Key words: Freshness, fish quality parameters, SPME-GC, TVB-N, volatile amines.

INTRODUCTION

Fish quality has been commonly evaluated by sensory and chemical tests. Among chemical indices, volatile amines including, methylamine (MA), dimethylamine (DMA) and trimethylamlne (TMA) have been widely used as indices of freshness and spoilage of seafood.

Different techniques including colorimetrie assays, gas chromatography and capillary electrophoresis among others have been reported in the literature for their determination. In recent years solid-phase microextraction coupled with gas chromatography (SPME-GC) appears as a promising methodology for the analysis of volatile and semi-volatile compounds. This technique combines simultaneously the extraction and pré-concentration steps. Besides, it is a fast technique and no solvents are required. Simple and rapid methods for the estimation of freshness in seafood for quality control laboratories are required (Pena-Pereira et al., 2010; Chan et al, 2006).

The aim of this work is to develop an SPME-GC-FID method to determine volatile amines in fish samples. Additionally, total volatile basic nitrogen (TVB-N) was determined according Commission Regulation (EC) No 2074/2005.

MATERIALS AND METHODS

Chemicals and analytical standards

Hydrochloric acid and sodium hydroxide were supplied by Merck and Panreac, respectively. The analytical standards: Trimethylamlne hydrochloride (CAS 593-817), dirnethylamlne hydrochloride (CAS 506-59-2) and propylamine hydrochloride (CAS 556-53-6) used as internal standard were obtained from Aldrich and methylamine hydrochloride (CAS 593-5 1- 1) was from Fluka, Analytical. Water used for all solutions was obtained from Milli-Q water purification system (Millipore) (Bedford, MA USA). Stock standard solutions of amines at a concentration of 1000 mg/ L were prepared in 0.5 N HCl and stored at 4 0C: Working standard solutions were prepared by dilution.

Samples

Fish samples of roundnose grenadier [Coryphaenoides rupestris) were stored in ice and inside a refrigerator at 5 °C.

SPME procedure

Amines were extracted from fish samples as follows: 20 mL of 0.5 N HCl were added to 30 g of sample, and mixed in a vortex (Minishaker, IKA) for five min. Then, the mixture was centrifuged (Hettich Zentrifugen) at 5000 rpm for 10 min at 5 °C. The supernatant was removed and made up to 10 mL in a volumetric flask containing the internal standard. 500 µLof the resulting solution and 1 mL of 15 N NaOH was transferred into 20 mL headspace vial and a small magnetic stirring bar was also added to the vial before was capped. Then, the SPME fibre was exposed to the headspace of the sample for 15 min. The fibre was conditioned into the GC injector at 270 0C during 1 h following the supplier's instructions prior the first use.

A SPME fibre coated with a 50/30 µ?? layer of divinylbenzene-carboxen-polydimethylsiloxane (DVD-CAR-PDMS) (Supelco, Bellefonte, PA, USA) was used for extraction of amines.

GC-FID analysis

Analyses were carried out on a GC 8000 TOP CE Instruments gas Chromatograph equipped with a flame ionization detector (FID). Amines were separated on an Rtx®-VolatileAmine (30 m ? 0.32 mm I.D.) column.

The GC conditions were as follows: the injector temperature was 200 0C, and the split ratio 1:25. Helium was used as carrier gas and the flow rate was 1.3 mL/ min. Detector temperature was kept at 250 0C. The ramp temperature was initially set at 40 0C (5 min) then increased at the rate of 10 °C/mln until 120 0C and held for 2 min. The software Chrom-Card for Windows was used for data processing.

Total volatile basic nitrogen (TVB-N)

TVB-N was determined according Commission Regulation (EC) No 2074/2005. The method involves distilling an extract deproteinised by perchloric acid. Results were expressed as mg / 1 00 g.

RESULTS AND CONCLUSIONS

Solld-Phase-MlcroExtraction (SPME) has been successfully applied to extract the volatile amines from fish samples. It is a simple, fast and solvent-free technique that combines the extraction and pre-concentration into one step.

A triple-coated fibre 50/30 µ?? DVB/Car/PDMS has demonstrated to be suitable for the analytes studied. A complete extraction was achieved after 15 min of exposure.

Fig. 1 shows a chromatogram of volatile amines of a fish sample. The compounds were identified by comparison of their retention times with those of pure standards.

For quantitative analysis a series of volatile amines standards of known concentrations were used. The calibration curves were constructed based on mg/L concentration levels ranging between 0.5-10 mg/L for MA, 0.25-10 mg/L for DMA and 0. 1-10 mg/L for TMA. A constant amount of the internal standard was added. Each point of the calibration curve is the average of three peak-area measurements.

The equations and determination coefficients obtained were the follow: y=0.0305x+0.0106 (R2 =0.9991) for MA; y=0.1387x+0.0183 (R2=0.9961) for DMA andy= 0.1796x-0.0163 (R2=0.9847) for TMA.

Detection limits, calculated in accordance with ACS Guidelines (defined as a Signal three times the height of the noise level) were 0.25 mg/L for MA, 0. 1 mg/L for DMA and 0.05 mg/L for TMA.

Once the analytical conditions were established the method was applied to determine volatile amines in fish samples stored in ice and inside a refrigerator at 5 °C in three consecutive days. The TVB-N was also determined. The samples were analyzed in triplicate and the values were expressed as mg N/100g sample.

The results obtained were the following: Day 1: (MA) 0.5691 ±0.3242; (DMA) 0.3636±0.0028; (TMA) 0.0310±0.0219; (TVB-N) not detected; Day 2: (MA) not detected; (DMA) 2.3436±0.0600; (TMA) 0.0060±0.0009; (TVB-N) 1.33±0.05 and Day 3: (MA) no detected; (DMA) 1.6055±0.0045; (TMA) 0.0060±0.0014; (TVB-N) 1.49±0.73. The MA content decreases along the time whereas the DMA and the TVB-N levels increase progressively during storage. Results showed good agreement between two determinations.

ACKNOWLEDGEMENTS

The study was financially supported by Xunta de Galicia (Project n° 1 OTALO 1 OCT) . R. Sendón is grateful to the "Parga Pondal" Program financed by "Conselleria de Innovación e Industria, Xunta de Galicia" for her postdoctoral contract. The authors are also grateful to C. Casal, P. Blanco and G. Hermelo for their excellent technical assistance.

References

REFERENCES

Chan, ST., Yao, M.W.Y., Wong, Y.C., Wong, T., Mok, CS., Sin, D.W.M. 2006. Evaluation of chemical indicators for monitoring freshness of food and determination of volatile amines In fish by headspace solid-phase microextraction and gas chromatography-mass spectrometry. Eur. Food Res. Technol. 224: 67.

Commission Regulation (EC) No 2074/2005, Off. J. Eur. Commun. L338 (2005) 27.

Pena-Pereira, F., Lavilla, I., Bendicho, C. 2010. Colorimetrie assay for determination of trimethylamlnenitrogen (TMA-N) in fish by combining headspace-single-drop microextraction and microvolume UV-Vis spectrophotometry. Food Chem. 119: 402.