Notes

1. The precolumn derivatization and the column apparatus were at room temperature.

2. OPA/MCE reagent was not used within 24 h of preparation.

3. In OPA method, to achieve a better separation between amines and between amines and acidic and neutral amino acids, the time of analysis was sacrificed (50 min). The acidic and neutral amino acids are those present at higher levels than amines eluted first and into the chromatogram. However, the simple preparation of the sample and the rapid derivatization of the amines considerably reduce time and effort.

4. The formation of o-phthaldialdehyde derivatives was performed automatically with reac-tant solution 100-150-folds higher in concentration than the amino nitrogen. The determination of amino nitrogen was performed using the method described previously (5).

5. The entire gradient cycle lasted for 62 min, including the time necessary for stabilization of the column after each injection. The analysis time was only 50 min in the OPA method.

6. Quantification by the external standard method, used in the OPA method, is based on the linearity of the detector response: a double concentration of product led to a peak of double area. The fluorescence exhibited a linear correlation with the concentration in the range 1-10 mg/L. A good linear regression between peak area and concentration for each biogenic amine was obtained, with correlation coefficients ranging from 0.985 to 0.998

Opa Microbiology

Fig. 1. HPLC profile of amines in standard solution (OPA method). 1, agmatine; 2, histamine; 3, tyramine; 4, putrescine; and 5, cadaverine. The elution time was between 11.23 and 42.27 min. Agmatine was the first amine eluted, followed by histamine, tyramine, putrescine, and cadaverine. A relative standard deviation (RSD) for retention time was less than 4.00%. An RSD of less than 6.00% for the response factor was obtained. Agmatine and putrescine had an RSD for the response factor greater than average (2.99%) (see Notes 11-13).

Fig. 1. HPLC profile of amines in standard solution (OPA method). 1, agmatine; 2, histamine; 3, tyramine; 4, putrescine; and 5, cadaverine. The elution time was between 11.23 and 42.27 min. Agmatine was the first amine eluted, followed by histamine, tyramine, putrescine, and cadaverine. A relative standard deviation (RSD) for retention time was less than 4.00%. An RSD of less than 6.00% for the response factor was obtained. Agmatine and putrescine had an RSD for the response factor greater than average (2.99%) (see Notes 11-13).

for the OPA method. This confirms the accuracy of this method for determining biogenic amine content.

7. Some modifications of this procedure were carried out: inclusion of agmatine, omission of centrifugation by filtration using a filter of 0.22 ^m to avoid microbial spoilage, increase of column length (higher plate number), and use of a UV detector.

8. In the Dansyl method the linearity was tested by dansylating the standard solutions at different concentrations. Dansylated amines showed linear responses in the concentration range 1-10 mg/mL, with correlation coefficients ranging from 0.980 to 0.998. The greatest variation in results was observed in the case of cadaverine.

9. The total run time was 35 min including the washing time. Washing was essential to maintain column performance in the Dansyl method.

Times Column Metod

Fig. 2. HPLC profile of amines and amino acids (OPA method). 1, aspartic acid; 2, glutamic acid; 3, asparagines; 4, serine; 5, glutamine; 6, histidine; 7, glycine; 8, threonine; 9, arginine; 10, alanine; 11, tyrosine; 12, agmatine; 13, methionine; 14, valine; 15, tryptophan; 16, histamine; 17, phenylalanine; 18, isoleucine; 19, leucine; 20, ornithine; 21, tyramine; 22, lysine; 23, putrescine; 24, cadaverine. Using the OPA method and beginning the gradient with 6 min (0% B) and 11 min (15% B), it is possible to separate and quantify amines and amino acids from a sample in a single chromatogram (see Note 14).

Fig. 2. HPLC profile of amines and amino acids (OPA method). 1, aspartic acid; 2, glutamic acid; 3, asparagines; 4, serine; 5, glutamine; 6, histidine; 7, glycine; 8, threonine; 9, arginine; 10, alanine; 11, tyrosine; 12, agmatine; 13, methionine; 14, valine; 15, tryptophan; 16, histamine; 17, phenylalanine; 18, isoleucine; 19, leucine; 20, ornithine; 21, tyramine; 22, lysine; 23, putrescine; 24, cadaverine. Using the OPA method and beginning the gradient with 6 min (0% B) and 11 min (15% B), it is possible to separate and quantify amines and amino acids from a sample in a single chromatogram (see Note 14).

Fig. 3. HPLC profile of amines in standard solution (Dansyl method). 1, putrescine; 2, cadav-erine; 3, agmatine; 4, histamine; 5, 1,7 diaminoheptane; and 6, tyramine. The elution time lay within the range of 12.90 to 17.24 min. The elution order was putrescine, cadaverine, agmatine, histamine, and tyramine. The internal standard 1,7 diaminoheptane eluted between histamine and tyramine. Values for the relative standard deviation (RSD) ranged from 2.08 to 5.30%. An RSD ranging from 1.30 to 3.50% for the response factor was obtained. Putrescine and cadaverine had an RSD greater than the average (2.30%; see Notes 11-13).

Fig. 3. HPLC profile of amines in standard solution (Dansyl method). 1, putrescine; 2, cadav-erine; 3, agmatine; 4, histamine; 5, 1,7 diaminoheptane; and 6, tyramine. The elution time lay within the range of 12.90 to 17.24 min. The elution order was putrescine, cadaverine, agmatine, histamine, and tyramine. The internal standard 1,7 diaminoheptane eluted between histamine and tyramine. Values for the relative standard deviation (RSD) ranged from 2.08 to 5.30%. An RSD ranging from 1.30 to 3.50% for the response factor was obtained. Putrescine and cadaverine had an RSD greater than the average (2.30%; see Notes 11-13).

10. In the Dansyl method, an internal standard was used. It involves a compound not present in the sample as an internal standard. Internal standardization compensates for variations in conditions during sampling and derivatization, as well as for variations in injection volumes and retention times during the chromatographic run.

11. The peaks of the biogenic amines were satisfactorily resolved, and there were no interfering peaks, so simple observation of the chromatograms suggests the presence of these compounds.

12. Without derivatization the amines would elute as broad peaks shown by fluorescence detection. UV absorbance detection is only possible for the heterocyclic and aromatic amines; therefore derivatization is necessary for the detection of aliphatic amines and for increased sensitivity.

13. Programmed elution with a polarity gradient was necessary to obtain optimum separation and quantification of the corresponding amines owing to the wide range of polarities of these molecules.

14. Our results using the OPA method point out the possibility of obtaining good resolution for concentrated samples of biogenic amines and amino acids, without the interference in the analysis from the other less concentrated biogenic amines, which are next to these compounds in the chromatogram.

References

1. Halasz, A., Barath A., Simon-Sarkadi, L., and Holzapfel, W. (1994) Biogenic amines and their production by microorganisms in food. Trends Food Sci. Technol. 5, 42-49.

2. Marce, R. M., Callull, M., Guasch, J., and Borrull, F. (1989) Determination of free amino acids in wine by HPLC using precolumn derivatization with phenylisothiocyanate. Am. J. Enol. Vitic. 40, 194-198.

3. Gonzalez de Llano, D., Polo, M. C., and Ramos, M. (1991) Production, isolation and identification of low molecular mass peptides from blue cheese by high performance liquid chromatography. J. Dairy Res. 58, 363-372

4 Eerola, S., Hinkkanen, R., Lindfors, E., and Hurvi, T. (1993) Liquid chromatographic determination of biogenic amines in dry sausages. J. AOAC Int. 76, 575-577.

5. Doi, E., Shibata, D., and Matoba, T. (1981) Modified colorimetric ninhydrin methods for peptidase assay. Anal. Biochem. 118, 173-184.

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