Research article Received: 20 January 2014
Revised: 17 April 2014
Accepted: 19 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3418
Simultaneous determination of ten underivatized biogenic amines in meat by liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)† Veronica Sirocchi, Giovanni Caprioli, Massimo Ricciutelli, Sauro Vittori and Gianni Sagratini* Biogenic amines (BAs) are considered to be important indicators of freshness and quality in food. In this work, an analytical method for analyzing ten underivatized BAs in meat by performance liquid chromatography-tandem mass spectrometry has been developed. Comparison between ion trap and triple quadrupole as mass analyzers indicated that the latter provides greater sensitivity and selectivity. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.987–0.999, and the limits of detection and limits of quantification were in the range of 0.002–0.1 mg l 1 and 0.008–0.5 mg l 1, respectively. Once validated, the method was used to analyze the concentrations of BAs in 16 commercial meat samples, for evaluating the freshness of food through the study of BA indices, i.e. biogenic amine index (BAI) and the ratio spermidine/spermine (SPD/SPE). The results indicated that all the samples were fresh, with a BAI lower than 1.49 mg kg 1 and a SPD/SPE ratio lower than 0.41 in each case. This methodology for testing the freshness of meat has potential for quality control applications along the entire production chain of meat products. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: biogenic amines; meat; liquid chromatography; tandem mass spectrometry; triple quadrupole
Introduction
J. Mass Spectrom. 2014, 49, 819–825
* Correspondence to: Gianni Sagratini, University of Camerino, School of Pharmacy, via S. Agostino 1, 62032 Camerino, Italy. E-mail: [email protected] †
This article is part of the Journal of Mass Spectrometry special issue entitled “3rd MS Food Day” edited by Gianluca Giorgi. School of Pharmacy, University of Camerino, via S. Agostino 1, 62032, Camerino, Italy
Copyright © 2014 John Wiley & Sons, Ltd.
819
Biogenic amines (BAs) are basic nitrogenous compounds present in food and produced by different mechanisms, such as decarboxylation of amino acids (catalyzed by the decarboxylase enzymes contained in various microorganisms) or by the normal cellular metabolism of tissues.[1] In low concentrations, BAs are essential for many physiological functions, but in high concentrations they can generate toxicological problems such as migraines, headaches, gastric and intestinal problems, and pseudo-allergic responses[2] and thus are considered important indicators of food freshness and quality. Two key indicators, the biogenic amine index, or BAI, which is the sum (mg kg 1) of putrescine, cadaverine, histamine and tyramine, and the spermidine/spermine ratio (SPD/SPE), are used to assess food quality.[3] Such an important entity as the European Union, with its Rapid Alert System for Food and Feed, has affirmed the value of BA assessment in food quality control. In 2013, it published its analysis of 10 years worth of notifications recorded in its database and reported that the average histamine concentrations in fish products, tracked from 2002 to 2012, was between 1000 and 2000 mg kg 1 in 12% of cases and above 2000 mg kg 1 in 11% of cases.[4] Recently, a study published by Erim F.B.[5] reported that, after fish products, meat is the food matrix most studied for the presence of BAs, considered markers of freshness and hygiene during storage of this product. Another study emphasized that high levels of BAs and the nitrates and nitrites added as preservatives in meat products are associated with the potential for forming carcinogenic nitrosamines.[6]
Over the years, various methods have been proposed for analyzing BAs in food with the use of high performance liquid chromatography (HPLC) coupled to various detectors. The most frequently used methods are based on derivatization reactions and separation processes followed by ultraviolet (UV) or fluorescence detection.[2] In these cases, the derivatization step is fundamental because most BAs lack chromophore groups. The most used derivatizating agents are o-phtalaldeyde (OPA),[7] benzoyl chloride,[8] dansyl chloride[9] and 6-aminoquinolylN-hydrosuccinimidyl carbamate.[10] HPLC can be used in combination with mass spectrometry (MS) or a MS tandem system (MS/MS) for analyzing derivatized BAs in food matrices such as cheese, fermented meat,[11] beer[12] and wines.[13] But often the derivatization step is time consuming and, as an accessory chemical reaction, yields poor recoveries and low sensitivity. For these reasons, it is preferable to analyze underivatized BAs in food. To our knowledge, very few methods using an HPLC coupled to an MS system for analyzing underivatized BAs in food have been published, and none of them have focused on BAs in meat. Gosetti et al.[14] analyzed six BAs in typical Piedmont cheeses by HPLC-MS/MS (ion trap (IT)) and compared the results
V. Sirocchi et al. with the HPLC-UV method, while Calbiani et al.[15] proposed an analytical method for analyzing histamine, β-phenylethylamine and tyramine in cheese by matrix solid-phase dispersion followed by HPLC-MS/MS (triple quadrupole (TQ)). Millàn et al.[16] proposed a method for determining eight BAs in wine by HPLC-MS/MS (IT) and Sagratini et al.[9] reported an HPLC-MS/MS (TQ) method for determining BAs in fish from Valencia (Spain). The aim of this work was to develop an analytical method that simultaneously determines ten underivatized BAs, namely, spermidine, cadaverine, putrescine, histamine, agmatine, spermine, tyramine, serotonin, phenylethylamine, tryptamine, in fresh meat by using solid phase extraction (SPE) and an HPLC-MS/MS system. Moreover, the performances of two mass analyzers, the IT and the TQ, were studied and compared. Finally, the method was applied to analyze the BA levels in 16 commercial meat samples, and the results were used to formulate the BA quality indices of the samples, as indicators of their freshness.
Experimental Materials and standards BAs, namely spermine tetrahydrochloride (SPE, C10H26N4·4HCl, >98%, CAS No. 306-67-2), spermidine trihydrochloride (SPD, C7H17N3 3HCl, >98%, CAS No. 334-50-9), cadaverine dihydrochloride (CAD, C5H14N2 2HCl, >98%, CAS No. 1476-39-7), putrescine dihydrochloride (PUT, C4H12N2 2HCl, >98%, CAS No. 333-93-7), histamine dihydrochloride (HIS, C5H9N3·2HCl, >99%, CAS No. 56-92-8), tyramine hydrochloride (TYR, C8H11NO·HCl, >98%, CAS No. 60-19-5), 2-phenylethylamine hydrochloride (PHE, C8H11N·HCl, >98%, CAS No. 156-28-5), tryptamine hydrochloride (TRY, C10H12N2·HCl, >99%, CAS No. 343-94-2), serotonin hydrochloride (SER, C10H12N2O·HCl, >98%, CAS No. 153-98-0), and agmatine sulfate salt (AGM, C5H14N4·H2SO4, >97%, CAS No. 2482-00-0) were supplied by Sigma-Aldrich (Milano, Italy). Trichloroacetic acid (TCA) for analysis was from Sigma-Aldrich (Milano, Italy). Individual stock solutions of BAs were prepared by dissolving 10 mg of each compound in 10 ml of HCl 0.1 M (Merck Darmstadt, Germany) and were then stored in glassstoppered bottles at 4 °C. Standard working solutions, at various concentrations, were prepared daily by appropriate dilution of different aliquots of the stock solutions with water. HPLC-grade methanol was supplied by Sigma-Aldrich (Milano, Italy). Deionized water (
Revised: 17 April 2014
Accepted: 19 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3418
Simultaneous determination of ten underivatized biogenic amines in meat by liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)† Veronica Sirocchi, Giovanni Caprioli, Massimo Ricciutelli, Sauro Vittori and Gianni Sagratini* Biogenic amines (BAs) are considered to be important indicators of freshness and quality in food. In this work, an analytical method for analyzing ten underivatized BAs in meat by performance liquid chromatography-tandem mass spectrometry has been developed. Comparison between ion trap and triple quadrupole as mass analyzers indicated that the latter provides greater sensitivity and selectivity. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.987–0.999, and the limits of detection and limits of quantification were in the range of 0.002–0.1 mg l 1 and 0.008–0.5 mg l 1, respectively. Once validated, the method was used to analyze the concentrations of BAs in 16 commercial meat samples, for evaluating the freshness of food through the study of BA indices, i.e. biogenic amine index (BAI) and the ratio spermidine/spermine (SPD/SPE). The results indicated that all the samples were fresh, with a BAI lower than 1.49 mg kg 1 and a SPD/SPE ratio lower than 0.41 in each case. This methodology for testing the freshness of meat has potential for quality control applications along the entire production chain of meat products. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: biogenic amines; meat; liquid chromatography; tandem mass spectrometry; triple quadrupole
Introduction
J. Mass Spectrom. 2014, 49, 819–825
* Correspondence to: Gianni Sagratini, University of Camerino, School of Pharmacy, via S. Agostino 1, 62032 Camerino, Italy. E-mail: [email protected] †
This article is part of the Journal of Mass Spectrometry special issue entitled “3rd MS Food Day” edited by Gianluca Giorgi. School of Pharmacy, University of Camerino, via S. Agostino 1, 62032, Camerino, Italy
Copyright © 2014 John Wiley & Sons, Ltd.
819
Biogenic amines (BAs) are basic nitrogenous compounds present in food and produced by different mechanisms, such as decarboxylation of amino acids (catalyzed by the decarboxylase enzymes contained in various microorganisms) or by the normal cellular metabolism of tissues.[1] In low concentrations, BAs are essential for many physiological functions, but in high concentrations they can generate toxicological problems such as migraines, headaches, gastric and intestinal problems, and pseudo-allergic responses[2] and thus are considered important indicators of food freshness and quality. Two key indicators, the biogenic amine index, or BAI, which is the sum (mg kg 1) of putrescine, cadaverine, histamine and tyramine, and the spermidine/spermine ratio (SPD/SPE), are used to assess food quality.[3] Such an important entity as the European Union, with its Rapid Alert System for Food and Feed, has affirmed the value of BA assessment in food quality control. In 2013, it published its analysis of 10 years worth of notifications recorded in its database and reported that the average histamine concentrations in fish products, tracked from 2002 to 2012, was between 1000 and 2000 mg kg 1 in 12% of cases and above 2000 mg kg 1 in 11% of cases.[4] Recently, a study published by Erim F.B.[5] reported that, after fish products, meat is the food matrix most studied for the presence of BAs, considered markers of freshness and hygiene during storage of this product. Another study emphasized that high levels of BAs and the nitrates and nitrites added as preservatives in meat products are associated with the potential for forming carcinogenic nitrosamines.[6]
Over the years, various methods have been proposed for analyzing BAs in food with the use of high performance liquid chromatography (HPLC) coupled to various detectors. The most frequently used methods are based on derivatization reactions and separation processes followed by ultraviolet (UV) or fluorescence detection.[2] In these cases, the derivatization step is fundamental because most BAs lack chromophore groups. The most used derivatizating agents are o-phtalaldeyde (OPA),[7] benzoyl chloride,[8] dansyl chloride[9] and 6-aminoquinolylN-hydrosuccinimidyl carbamate.[10] HPLC can be used in combination with mass spectrometry (MS) or a MS tandem system (MS/MS) for analyzing derivatized BAs in food matrices such as cheese, fermented meat,[11] beer[12] and wines.[13] But often the derivatization step is time consuming and, as an accessory chemical reaction, yields poor recoveries and low sensitivity. For these reasons, it is preferable to analyze underivatized BAs in food. To our knowledge, very few methods using an HPLC coupled to an MS system for analyzing underivatized BAs in food have been published, and none of them have focused on BAs in meat. Gosetti et al.[14] analyzed six BAs in typical Piedmont cheeses by HPLC-MS/MS (ion trap (IT)) and compared the results
V. Sirocchi et al. with the HPLC-UV method, while Calbiani et al.[15] proposed an analytical method for analyzing histamine, β-phenylethylamine and tyramine in cheese by matrix solid-phase dispersion followed by HPLC-MS/MS (triple quadrupole (TQ)). Millàn et al.[16] proposed a method for determining eight BAs in wine by HPLC-MS/MS (IT) and Sagratini et al.[9] reported an HPLC-MS/MS (TQ) method for determining BAs in fish from Valencia (Spain). The aim of this work was to develop an analytical method that simultaneously determines ten underivatized BAs, namely, spermidine, cadaverine, putrescine, histamine, agmatine, spermine, tyramine, serotonin, phenylethylamine, tryptamine, in fresh meat by using solid phase extraction (SPE) and an HPLC-MS/MS system. Moreover, the performances of two mass analyzers, the IT and the TQ, were studied and compared. Finally, the method was applied to analyze the BA levels in 16 commercial meat samples, and the results were used to formulate the BA quality indices of the samples, as indicators of their freshness.
Experimental Materials and standards BAs, namely spermine tetrahydrochloride (SPE, C10H26N4·4HCl, >98%, CAS No. 306-67-2), spermidine trihydrochloride (SPD, C7H17N3 3HCl, >98%, CAS No. 334-50-9), cadaverine dihydrochloride (CAD, C5H14N2 2HCl, >98%, CAS No. 1476-39-7), putrescine dihydrochloride (PUT, C4H12N2 2HCl, >98%, CAS No. 333-93-7), histamine dihydrochloride (HIS, C5H9N3·2HCl, >99%, CAS No. 56-92-8), tyramine hydrochloride (TYR, C8H11NO·HCl, >98%, CAS No. 60-19-5), 2-phenylethylamine hydrochloride (PHE, C8H11N·HCl, >98%, CAS No. 156-28-5), tryptamine hydrochloride (TRY, C10H12N2·HCl, >99%, CAS No. 343-94-2), serotonin hydrochloride (SER, C10H12N2O·HCl, >98%, CAS No. 153-98-0), and agmatine sulfate salt (AGM, C5H14N4·H2SO4, >97%, CAS No. 2482-00-0) were supplied by Sigma-Aldrich (Milano, Italy). Trichloroacetic acid (TCA) for analysis was from Sigma-Aldrich (Milano, Italy). Individual stock solutions of BAs were prepared by dissolving 10 mg of each compound in 10 ml of HCl 0.1 M (Merck Darmstadt, Germany) and were then stored in glassstoppered bottles at 4 °C. Standard working solutions, at various concentrations, were prepared daily by appropriate dilution of different aliquots of the stock solutions with water. HPLC-grade methanol was supplied by Sigma-Aldrich (Milano, Italy). Deionized water (
