by Isotachophoretic Method with Simple Sample Preparation
Aneta Jastrzbska & Marzanna Kurzawa & Anna Piasta & Edward Szyk Received: 23 August 2011 / Accepted: 6 December 2011 / Published online: 24 December 2011 #Springer Science+Business Media, LLC 2011 Abstract A one-dimensional capillary isotachophoretic method in cationic system of the separation has been applied for histamine determination in food samples. The proposed electrolyte system consisted of 0.01 M potassium hydroxide with L-valine to pH09.9 as the leading electrolyte and 0.02 M 2-amino-2-hydroxy- methyl-propane-1,3-diol adjusted to pH08.3 with 0.1 M hydrochloric acid as terminating electrolyte. Proposed method was characterized by linearity range 5 50 mg L 1 and R 2 00.9982, accuracy (recoveries ranged from 95% to 102%), detection (2.10 mg L 1 ), and quan- tification (7.01 mg L 1 ) limits. The sample preparation for proposed electrophoretic method included only simple extraction with trichloroacetic acid with filtration and derivatisation stage are avoided. The histamine concen- tration was determined in meat (turkey, chicken, beef and pork) and meat products (ripened sausage and dry-cured ham), fish (smoked salmon and mackerel), and different kind of mildew and mold ripened cheeses samples. The histamine content ranged from not detected level for fresh meat to 29.63 mg 100 g 1 for cheese samples. The reversed phase HPLC was applied as reference method and the F-Snedecor test and the t test were employed to compare the precision and accuracy of the both methods. Positive correlations were found between the two analytical methods for histamine determination in food products. The obtained results indicate that the proposed electrophoretic method is simple, precise, accu- rate, and convenient. Keywords Histamine determination . Isotachophoretic method . Food samples . HPLC Introduction Biogenic amines (BA) are present in living organisms at low levels where they are responsible for many essential biolog- ical functions (Bodmer et al. 1999; Saccani et al. 2005; Hernndez-Jover et al. 1997). Their presence in food, espe- cially in fish, cheese, and meat products, varies by a great extent depending on technological processes and microbial factors. In fact their formation mainly depends on the activity of microbial decarboxylases and in a minor role on the endogenous amino acid decarboxylase activities (Galgano et al. 2009; Talon and Leroy 2011). For this reason, the content of biogenic amines can be relatively high in fer- mented food, especially in raw materials (meat and dairy products) presenting a high content of proteins combined with high proteolytic activity (Talon and Leroy 2011). BA can be generated during storage or processing of food rich in proteins by thermal or bacterial enzymatic decarbox- ylation of amino acids. Hence, the composition and number of biogenic amines gives information about the freshness of food. Consequently, they are important indicators of food quality and hygiene and high level of BA is usually consid- ered as an index of poor manufacturing practices (Galgano et al. 2009; Mazzoli et al. 2009; Sml et al. 2003). Histamine (HIS), beta-phenylethylamine, tyramine, trypt- amine, putrescine, cadaverine, spermine, and spermidine are the most important biogenic amines identified in food and beverages. The high concentration of these compounds in food causes different effects in susceptible individuals (Mazzoli et al. 2009; Kaniou et al. 2001; Vinci and Antonelli 2002; Gosetti et al. 2007). Among BA, histamine A. Jastrzbska (*) : M. Kurzawa : A. Piasta : E. Szyk Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., 87-100 Toru, Poland e-mail: aj@chem.uni.torun.pl Food Anal. Methods (2012) 5:10791087 DOI 10.1007/s12161-011-9345-7 is the most important substance responsible for intoxications and allergies (Gosetti et al. 2007; Bomke et al. 2009; Hungerford 2010). Moreover, HIS is neither volatile nor destroyed by cooking; hence, it is difficult to remove it from food, what diminishes food quality. This amine does not affect organoleptic characteristics, but clearly induces food intolerance in an increasing number of human populations and caused the potential risk to health (Bodmer et al. 1999; Akbari-adergani et al. 2010). Therefore, a regulation level on histamine in food has been set by many countries or organizations (Hwang et al. 2003; Peng et al. 2008). The Polish Standards (in agreement with The European Union (EU) 2007/642/WE) (Polish Monitor 2009) regulate the permissible content of histamine in fish and fish products from the families Scombridae, Clupeidae, Eugraulidae, and Coryphaenidae. These prod- ucts must fulfill the following requirements: (1) the mean value must not exceed 10 mg 100 g 1 ; (2) two samples may have a value of more than 10 mg 100 g 1 but less than 20 mg 100 g 1 ; (3) no sample may have a value exceeding 20 mg 100 g 1 . However, fish belonging to these families which have undergone enzyme ripening treatment in brine may exhibit higher histamine levels but not more than twice the above values (2040 mg 100 g 1 ). According to Talon and Leroy (Talon and Leroy 2011), this regulation is already obligatory in the fish industry in EU and may be extended to fermented food in the future. Because of its potential risk to health, the research on the simultaneous and rapid analysis of BAs in a variety of biological matrices is of the widest interest. The number and variety of methods developed for histamine and other amines determination in food samples is impressive and reviewed by nal (nal 2007) and Karoviov and Kohajdov (Karoviov and Kohajdov 2005). According to Cinquina et al. (Cinquina et al. 2004), methods proposed for amines determination may be divided into two classes: simple method with short time analysis and low sensitivity and complicated method with pre- or post-column derivatization that is accompanied by good sensitivity and specificity, long analysis times, nonreproducibility, and problems of derivati- zation products stability. The most popular methods for biogenic amines determi- nation are various chromatographic techniques, such as: gas chromatography (GC) (Hwang et al. 2003), liquid chroma- tography (high-performance liquid chromatography (HPLC) and UHPLC) coupled with different detection (Galgano et al. 2009; Sml et al. 2003; Kaniou et al. 2001; Gosetti et al. 2007; Bomke et al. 2009; Hungerford 2010; Peng et al. 2008; Dadkov et al. 2009; Aygn et al. 1999; Latorre- Moratalla et al. 2009; Sun et al. 2011), ion chromatography (Saccani et al. 2005; Favaro et al. 2007). Most of the separation methods for histamine determination applied de- tection schemes based on pre-column derivatization to produce fluorescent products or strong chromophores, but direct UV detection of imidazole ring has also been applied (Hungerford 2010; Cinquina et al. 2004). According to literature (Cinquina et al. 2004; Dadkov et al. 2009; Steiner et al. 2009; Gallardo et al. 1997; Oguri et al. 1997), capillary electrophoresis is an excellent alternative to HPLC methods for the analysis of BA in complex matrices. The fluorimetric methods based on the formation of a fluores- cent derivative with ortho-phthalaldehyde were widely used for the analysis of histamine because of their great sensitivity and selectivity (Adamou et al. 2005; Larionova et al. 2009; Leszczyska et al. 2004). As the chromatographic and fluorescence analysis of biogenic amines is complicated, further analysis meth- ods have been elaborated. The immunochemical method (enzyme-linked immunosorbent assay (ELISA)) (Aygn et al. 1999; Leszczyska et al. 2004), colorimetric (Patange et al. 2005), electrochemical method (Akbari-adergani et al. 2010), or biosensors (Keow et al. 2007; Ito et al. 2009) for rapid assessment of histamine level were studied for this purpose. Although the histamine content of food has been the sub- ject of considerable analysis in the past years, the problem of searching new simple methods for the HIS determination is still fundamental and interesting. It is related to the fact, that most of methods required complicated and expensive instru- mentation, toxic reagents, time consuming operations, cum- bersome sample preparation and the derivatization reaction products have a short life time. Moreover, in the complex matrix samples, due to the high contents of protein and fat, the presence of potentially interfering compounds, and the occurrence of several BA simultaneously imposed problems in the analysis. In the literature, we have found only few reports on biogenic amines in different food samples determination by capillary isotachophoresis (Karoviov et al. 2003; Rubach et al. 1981). This electrophoretic method has many advantages (simplicity and short time of analysis) for deter- minations of many ions in complex matrices and is known as an analytical method with high reproducibility and can be competitive to other separation techniques (Kvasnika 2000; Jastrzbska 2011). Moreover, conductometric detec- tion permitted on biogenic amines determination based on their electrophoretic properties. For this reason, we applied capillary isotachophoretic (ITP) method with conductometric detection for determina- tion of histamine in food samples after simple sample prep- aration. The proposed method was validated in terms of linearity, sensitivity, precision, and recovery. To prove the versatility of the method, the analysis was carried out on three groups of food widely consumed all over the world (meat and meat products, fishes, and cheeses). Furthermore, these products belong to the usually studied ones in terms of 1080 Food Anal. Methods (2012) 5:10791087 the biogenic amines content. Our results from isotachopho- resis were compared with those obtained by reversed-phase (RP)-HPLC method. Because, amines are strong organic bases it is very useful to take advantage of this feature for their sepa- ration from sample matrix. The extraction of amines represents the critical step of the process and it influ- ences negatively the analytical recoveries. The aim of the extraction is to remove interfering compounds from the matrix, but during this step losses of BA must be as little as possible. Many different solvents have been used for the extraction of amines from the matrix, such as hydrochloric acid, trichloroacetic acid, perchloric ac- id, methanol, and other organic solvents (Vinci and Antonelli 2002). For this reason, the optimization of extraction procedures was described. Experimental Reagents All reagents were of analytical or HPLC grade. Histamine dihydrochloride (HIS), L-valine, methanol, 2-amino-2- hydroxymethyl-propane-1,3-diol (TRIS), and acetonitrile were purchased from Sigma-Aldrich (Pozna, Poland). Am- monium acetate, potassium hydroxide, hydrochloric acid, and trichloroacetic acid (TCA) were obtained from Alchem (Toru, Poland). Redistilled water was used in all solutions preparation (specific conductivity, <10 S). Apparatus Isotachophoretic separations were performed using a Villa Labeco EA 100/101 isotachophoretic analyzer equipped with a conductometric detector. The PTFE pre-separation capillary (900.8 mm, I.D.) was connected with PTFE analytical capillary (1600.3 mm, I.D.). Samples of 30-l fixed volume were injected via a sample valve by internal sample loop. The isotachopherograms were evaluated with the software supplied with analyzer (KasComp Ltd., Slovakia). The HPLC system, equipped with an autosampler SIL- 20AC HT and a photodiode multi-wavelength detector (SPD-M20A Prominence Diode Array Detector), SHI- MADZU (Kyoto, Japan) was applied. Analyses were carried out on Discovery C18 Supelco column (5-m particle size, 1504.8 mm), maintained at 25 C. Food products samples were centrifuged by a laboratory centrifuge (max speed, 9,000 rpm; RFC, 8693g; angle, 30; and falcon tubes, 50 mL; MPW, Warsaw, Poland). Food samples All food samples were purchased from different local mar- kets and collected in Table 1. Prior to analysis, purchased samples were cut up, homog- enized in household food grinder with a plate of 3-mm diameter holes. Sample Preparation for ITP and HPLC Methods Depending on the complexity of food matrix and the natural amount of free amino acids, extraction and pu- rification steps can be necessary prior to analytical determination. Because proposed ITP method with con- ductometric detection required ionic form of histamine, the sample extraction was performed in acidic medium as reported (Hernndez-Jover et al. 1997; Vinci and Antonelli 2002; Innocente et al. 2007) for food matrices with relatively low free amino acids content such as meat, fish and vegetables. According to Moret and Conte (Moret and Conte 1996), the choice of acid has to be related to the characteristics of the analyzed ma- trix. On the basis of their experience, 0.1 M HCI appears to be a good choice for the analysis of cheese, however, it is not a suitable for fish or meat products. For these samples, TCA represented a better choice because of its capacity to precipitate proteins. For this reason, in our study we tested TCA as all-purpose extraction solution in concentration from 1% to 5%. The best precision and recovery for ITP and HPLC methods were obtained for 2% of TCA. Possible differences between extracted analytes and in- terfering species were checked for the range 525 g of Table 1 Food samples analyzed by ITP and HPLC methods Sample no Type of sample Sample 1 Minced turkey breast Sample 2 Minced chicken breast Sample 3 Minced pork shoulder Sample 4 Minced beef shoulder Sample 5 Minced pork ham Sample 6 Raw beef Sample 7 Ripened sausage Sample 8 Dry-cured ham Sample 9 Smoked salmon Sample 10 Smoked mackerel Sample 11, 11a; 11b; 11c; 11d Different kind of mildew and mold ripened cheeses Sample 12 Fresh poultry meat Sample 13 Fresh pork Sample 14 Fresh beef Food Anal. Methods (2012) 5:10791087 1081 processed food samples. For ITP and HPLC methods the highest analytes/interferents ratio was noted for 15 g and consequently this amount was used in all successive experi- ments. The sample preparation procedure with all details is presented below. Procedure of Sample Preparation The food products samples (150.0001 g purchased products) were extracted with 25 mL of TCA (2%) using an orbital shaker for 30 min. The extracts were separated using centrifuge at 9000 rpm for 30 min, followed by double filtration. All extracts were transferred into 50 mL volumetric flasks, made up to the mark and analyzed with ITP and HPLC methods. In the case of HPLC, the obtained extracts were filtered through a 0.4-mm membrane prior the analyses. Additionally, for several samples, dilution with redistilled water was applied. It should be noted, that applied sample preparation proce- dure avoids a derivatization stage in the case of both method. The sample preparation procedure is simple, short and uncomplicated in comparison to the methods requiring clean solution or derivatization procedure. Determination of Histamine by ITP and HPLC Methods The ITP analysis of histamine was performed with LE (leading electrolyte): 10 mM KOH+L-valine (pH09.9) and terminating electrolyte: 0.02 M TRIS+0.1 M HCl (pH08.3). A driving current of the preseparation capil- lary was 150 A, while of analytical capillary 100 A for all samples in five repetitions. The histamine in standard solution and food samples was identified by the relative step height parameter (RSH histamine 00.15). The time of analysis was short and located between 510 min for standard solutions and 1525 min for food extracts determination, respectively. Precision of analysis was evaluated as the within-day and between-days coefficient of variation (CV) (Miller and Miller 2000). Within-day analyses were determined by injection of the histamine standard solution (20 mg L 1 ) six times per day. The intralaboratory re- producibility was determined by analysis of the standard solution during five consecutive days. The CV values for within-day and between-days determination varied from 0.98% to 4.12% and 2.544.89%, respectively, that con- firmed stability of the proposed electrolyte system. HPLC measurements were carried out using the mixture of 0.1 M ammonium acetate + acetonitrile + methanol (90:5:5) as a mobile phase (isocratic elution) and detection at 0224 nm. The chromatographic data were recorded and processed by the LC solution ver- sion 1.23 SP. Table 2 Linear regression calibration parameters of histamine deter- mination by ITP and HPLC methods (n05) Parameter ITP HPLC Linearity range (mg L -1 ) 550 10100 Slope (b) 0.5863 16082 Standard deviation of slope (S b ) 0.0874 149 Intercept (a) ()0.7422 134040 Standard deviation of intercept (S a ) 0.2810 9220 Standard deviation of regression (S r ) 0.7011 0.6818 Detection limit (DL; mg L 1 ) 2.10 2.05 Quantification limit (QL; mg L 1 ) 7.01 6.82 Coefficient of determination R 2 0.9982 0.9997 Where: DL0(y+3s y/x )b 1 , QL010s y/x b 1 A histamine B histamine C histamine D histamine Fig. 1 The isotachopherograms of food samples: a dry-cured ham sample, b minced pork ham sample, c cheese sample, and d smoked salmon sample 1082 Food Anal. Methods (2012) 5:10791087 Results and Discussion Calibration Curves for Histamine Determination by ITP and HPLC Calibration curves were constructed using nine calibration solutions of histamine and results were calculated as an aver- age of five replicates. Calibration points were established by measuring the zone length (L) or peak areas (A) versus stan- dard concentration (c). The regression parameters of calibra- tion curves are listed in Table 2. The linearity for histamine determination by ITP and HPLC methods was satisfactory and comparable with values reported by other authors (Akbari-adergani et al. 2010; Latorre-Moratalla et al. 2009; Steiner et al. 2009; Oguri et al. 1997; Karoviov et al. 2003). The Table 3 Results of histamine determination (mg 100 g 1 purchased products (pp)) in food samples by ITP (n05) Where: X is the average value (mg 100g 1 pp), confidence limit (0(t n1 s/n 1/2 ), p095%); CV coefficient of variation (%), nd not detected, n number of samples Samples X (mg 100 g 1 ) CV (%) Fresh poultry meat nd Fresh pork nd Fresh beef nd Minced turkey breast 1.110.035 1.38 Minced chicken breast nd Minced pork shoulder 1.180.052 3.57 Minced beef shoulder 1.650.11 2.89 Minced pork ham 6.200.29 1.22 Raw beef nd Ripened sausage 19.200.83 1.88 Dry-cured ham 6.410.38 2.55 Smoked salmon 19.830.75 1.64 Smoked mackerel 18.250.72 2.46 Different kind of mildew and mold ripened cheeses 25.600.55 0.94 15.240.55 2.95 9.180.27 2.39 29.631.04 2.83 3.900.18 3.59 Table 4 Results of histamine determination (mg 100 g 1 pur- chased products (pp)) in food samples by HPLC (n05) For abbreviations, see Table 3 Samples X (mg 100g 1 ) CV (%) Fresh poultry meat nd Fresh pork nd Fresh beef nd Minced turkey breast 2.510.14 2.47 Minced chicken breast nd Minced pork shoulder 2.570.20 6.42 Minced beef shoulder 3.320.28 7.11 Minced pork ham 14.200.72 2.21 Raw beef nd Ripened sausage 21.821.39 2.75 Dry-cured ham 13.140.22 0.73 Smoked salmon 20.830.95 3.67 Smoked mackerel 20.201.24 2.68 Different kind of mildew and mold ripened cheeses 27.341.51 2.40 17.410.72 3.31 11.510.62 4.33 31.801.56 3.97 5.030.31 5.03 Food Anal. Methods (2012) 5:10791087 1083 detection limits of the studied and HPLC methods were sufficiently low for the determination of histamine in food samples. In the case of quantification limit, values were slightly higher than the lowest concentration of the calibration curve. The last value indicates that the low- est concentration of histamine solution cannot be deter- mined with required precision. The Determination of Histamine in Food Samples by Isotachophoretic Method The isotachopherograms of food samples are presented on Fig. 1 and results are listed in Table 3. Meat and Meat Products As reported in Table 3 fresh meat (pork, poultry, and beef), raw beef, and minced chicken breast samples revealed level of histamine below the detection limit. In the case of remain- ing meat samples, the amount of histamine ranged from 1.11 mg 100 g 1 for minced turkey breast (sample 1) to 6.20 mg 100 g 1 for minced pork ham (sample 5). In our study, the maturing meat products (dry-cured sausages and ham) revealed higher amount of histamine than fresh or minced meat. The different concentration of BA in fresh and meat products can be explained by the properties of meat substrates and by microbial flora with different biochemical potentiality for amino acids metabo- lism (Galgano et al. 2009). On the other hand, the obtained contents of HIS in minced meat suggested decrease of quality or long time of storage before sale. There are several studies on the determination of biogenic amines in meat samples but usually level of putrescine, cadaverine, tyramine, and spermine was dis- cussed as coefficients for storage time and temperature as well as for the microbiological quality of meat (Kaniou et al. 2001; Vinci and Antonelli 2002; Rokka et al. 2004). Fresh and processed pork contains high levels of adrenalin, spermidine and spermine but low levels of noradrenalin, putrescine, histamine, cadaverine, and tyramine (Karoviov and Kohajdov 2005). According to authors (Kaniou et al. 2001; Vinci and Antonelli 2002; Rokka et al. 2004), some of amines can be formed during the storage of fresh meat. Further- more, during manufacturing of dry-cured and cooked meat products several technological factors, such as pH, temperature, and salt concentration are key factors in the onset and the rate of amino enzymatic reactions and their synergic effect (Saccani et al. 2005). Favaro et al. (Favaro et al. 2007) found HIS in dry fermented sausages 5. 7 mg 100 g 1 and dry-cured bel l y 11.4 mg 100 g 1 with CV values 10% and 6%, respec- tively. The histamine in different food samples deter- mined by Karoviov et al. (Karoviov et al. 2003) was detected in four samples, while the highest level was in frankfurters (14.67 mg 100 g 1 ). Hernndez- Jover et al. (Hernndez-Jover et al. 1997) applied HPLC method for determination of BA in fresh pork and beef meat and only spermidine and spermine were found in both types of meat. Authors suggested that these amines are naturally occurring in fresh pork and beef meat, and 0 50 100 150 200 250 0 1 2 3 4 5 6 7 8 9 min A 1 2 0 40 80 120 160 0 1 2 3 4 5 6 7 8 9 min min B C 2 1 1 2 m A U m A U m A U Fig. 2 The chromatograms of food samples: a cheese sample, b smoked salmon sample, and c minced pork ham sample; 1 histamine and 2 histidine Table 5 Results of standard addition method (n015) parameter ITP HPLC Precision (CV %) 1.103.47 1.989.17 Accuracy (recovery %) 95102 101110 For abbreviations, see Table 3 1084 Food Anal. Methods (2012) 5:10791087 their formation is not due to food spoilage or fermen- tation processes. The range of HIS level in dry-cured ham and ripened products was from below detection limit to 15 mg 100 g 1 and to 35.7 mg 100 g 1 , re- spectively. The levels of histamine in meat products determined by Saccani et al. (Saccani et al. 2005) were as follows: 060 mg 100 g 1 (fresh pork meat), 10 40 mg 100 g 1 (dry-cured sausage), 070 mg 100 g 1 (dry-cured ham), and 0110 mg 100 g 1 (cooked ham). Samples of Cheeses In contrast to the comparable contents of HIS in meat samples, the observed level of this amine in cheese samples demonstrated wide fluctuations from 3.90 mg 100 g 1 to 29.63 mg 100 g 1 . It should be noted that long ripened cheeses are major sources of dietary biogenic amines. The formation and presence of amines depend on a variety of factors including the presence of substrate and microbial enzymes, temperature, pH, salt and water content, presence of enhancing substances and catabolism of amines. Accord- ing to Bodmer et al. (Bodmer et al. 1999), in cheese pro- duction the increase of histamine content was observed with maximum level at 2,500 ppm in aged cheese. It is known that dairy products are good examples to demonstrate the undesired increase of histamine content during non-proper food processing (Bodmer et al. 1999). A competitive direct enzyme-linked immunosorbent assay for HIS determination in cheese was proposed by Aygn et al. (Aygn et al. 1999) and obtained mean values were as follows: 32.2 mg 100 g 1 for hard cheese, 3.3 mg 100 g 1 for semi-hard cheese, and 7.3 mg 100 g 1 for soft cheese. Samples of Fishes The histamine level has also been proposed as a chemical index of freshness of fish, poor hygienic quality of raw materi- als used and/or poor manufacturing conditions (Hwang et al. 2003). It should be noted, that fresh fish contains very low level of histamine, but the content increases with the progress of fish decomposition and processing. In this study the smoked fish samples were analyzed and observed level of HIS (Table 3) does not exceed the permitted level for fish products (Polish Monitor 2009). According to Keow et al. (Keow et al. 2007), histamine induced slightly poisoning at 840 mg 100 g 1 raw fish, moderate poisoning at >40 mg 100 g 1 , and severe at >100 mg 100 g 1 . The Determination of Histamine in Food Samples by RP-HPLC Method As a reference method RP-HPLC was applied and obtained results are listed in Table 4. The typical chromatograms of food samples are presented on Fig. 2. It is evident that obtained amounts of HIS by HPLC meth- od were higher than by ITP. The differences ranged from 1.13 mg 100 g 1 (mold ripened cheese) to 8.00 mg 100 g 1 (minced pork ham). However, the correlation between meth- ods was satisfactory. Only for two samples (minced pork ham and dry-cured ham) the difference was higher than 3.00 mg 100 g 1 . Generally, chromatographic method requires chemical derivatization of most BA due to the lack of a suitable chromophore or fluorophore group for direct detec- tion. Except that, the elution program usually consisted of the gradient system and specific columns for amines separation are often used. Also likely, the matrix interferences in complex samples cannot be eliminated efficiently applying simple sample preparation (single extraction). For this reason, the pretreatment steps (for example, solid-phase extraction clean- up) are necessary to eliminate the interferences with chro- matographic separation. In our research simple elution program without derivative and pretreatment steps was suffi- cient for good separation. Results of Statistical Analysis Precision of ITP and HPLC Methods Precision of methods for food samples was evaluated as the within-day coefficient of variations values (Miller and Miller 2000). In the case of ITP method, the CV values for HIS determination varied from 0.94% to 3.59% (Table 3), what indicated satisfactory precision of the discussed method. The relative standard deviations (RSD) at ITP determination of amines obtained by Karoviov et al. (Karoviov et al. 2003) ranged from 0.52% to 5.77% for all concentration levels. Hwang et al. (Hwang et al. 2003) proposed GC method for HIS determination in tuna and shrimp with reported precision Table 6 Comparison between ITP and HPLC methods by F and t tests (F crit 06.39 and t crit 02.31) For name of samples, see Table 1 No of samples 1 3 4 5 7 8 9 10 11 11a 11b 11c 11d F 3.20 3.23 6.05 3.28 2.13 12.2 5.00 1.19 6.50 1.26 3.28 1.97 1.96 t 1.11 0.42 0.49 7.07 1.75 0.56 1.23 1.11 1.05 1.01 0.41 Food Anal. Methods (2012) 5:10791087 1085 of 2.77.8% and 2.78.9% (for standard addition method), respectively. The RSD values obtained for cation exchange chromatographic method were as follows: 10% for dry fer- mented sausages and 6% for dry-cured belly (Favaro et al. 2007). The coefficient of variations for HPLC determination ranged from 0.73% to 7.11% (Table 4) that indicated better precision for ITP method. Comparing to the precision de- scribed by other authors (Sml et al. 2003; Kaniou et al. 2001; Aygn et al. 1999; Latorre-Moratalla et al. 2009; Steiner et al. 2009; Gallardo et al. 1997; Larionova et al. 2009; Kvasnika 2000) and obtained with reference (RP-HPLC) method one can conclude, that the proposed ITP method reveals satisfactory repeatability. Recovery Test (Standard Addition Method) In order to evaluate the accuracy of the analytical method, the food samples were spiked at two different concentrations (5.00 and 10.00 mg 100 g 1 ) of histamine to food samples, analyzed by ITP and HPLC methods and obtained results are listed in Table 5. Samples were homogenized and extracted as previously described. The obtained results clearly indicate that the proposed method shows satisfactory accuracy and precision. Compar- ing both methods (ITP and RP-HPLC) it is evident that accuracy of isotachophoresis is better. Furthermore, the recoveries obtained by ITP method were in agreement with those reported in the literature (Saccani et al. 2005; Hwang et al. 2003; Cinquina et al. 2004; Latorre-Moratalla et al. 2009; Steiner et al. 2009; Gallardo et al. 1997). Recovery of histamine added to different marine products obtained by Gallardo et al. (Gallardo et al. 1997) ranged from 97.8% to 101.8%. Cinquina et al. (Cinquina et al. 2004) tested two techniques (HPLC-DAD and CE-DAD) for histamine deter- mination and recoveries obtained by spiking tuna fish samples at three different concentrations (50, 100, and 200 mg/kg) resulting in average recoveries higher than 92% and RSD< 4%for HPLC, whereas for CE the recoveries were above 85% and RSD below 3%. Similar recoveries (89115%) and RSD from 0.5% to 3.6% were reported by Steiner et al. (Steiner et al. 2009). Recovery determination of histamine from tuna by GC method ranged from 97.5% to 110.9% and for shrimp meat between 98.5% and 102.4% (Hwang et al. 2003). Re- coveries of histamine determination in fish, cheese and dry sausage samples obtained by Latorre-Moratalla et al. (Latorre- Moratalla et al. 2009) ranged from 88.80% to 95.83%. A method for simultaneous determination of underivatized bio- genic amines based on the separation by cation-exchange chromatography and suppressed conductivity coupled with mass spectrometry detection was discussed by Saccani et al. (Saccani et al. 2005), and average recoveries from meat sam- ples ranged from 85 to 97% and coefficients of variation ranged from 4.5 to 9.7%. Comparison of Results for Proposed ITP and RP-HPLC Methods by F and t Tests The F-Snedecor and the t tests were employed to compare the precision and accuracy of the used methods (Miller and Miller 2000), and the results are listed in Table 6. The calculated F test indicated that there was not a significant difference in the precision of the proposed and HPLC methods because calculated values were above the theoretical values only in two cases (samples 8 and 11). The t values calculated for the both methods were lower than the critical ones (except sample 5). The results collected in Table 6 indicated that there are no significant differences between the precision and average concentrations of HIS in the samples assayed by both analytical methods. Conclusions The proposed capillary ITP method with the conductometric detection for determination of histamine is very simple, relatively selective, highly precise and accurate. Moreover, in comparison to HPLC discussed method is relatively in- expensive, less laborious and is characterized by better statistical parameters. The time of analysis is short and located between 1525 min for food extracts determination. The sample preparation method is simple (only extraction and filtration), short and omits derivatisation stage. Further- more, fat content and non-protein nitrogenous substances (low weight peptides and free amino acids) does not inter- fere in histamine determination. The presented results reveal that proposed method can be successfully used for histamine determination in variety of samples. Therefore, it can be considered that the proposed ITP method can be usefully employed by the food industry in assessing quality of dif- ferent food products. Acknowledgment The authors acknowledge the grant from the Polish Ministry of Science and Higher Education: grant no. NN 312 465640 (4656/B/P01/2011/40) References Adamou R, Coly A, Edgar Douabal S, Saleck MLOChO, Gaye-Seye MD, Tine AE (2005) J. 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135 - Method Validation For The Quantitative Analysis of Aflatoxins (B1, B2, G1, and G2) and Ochratoxin A in Processed Cereal-Based Foods by HPLC With Fluorescence Detection.