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Optimization of cheese sample preparation methodology for free amino acid analysis by capillary isotachophoresis
Journal of Food Composition and Analysis 40 (2015) 136–142
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Original Research Article
Optimization of cheese sample preparation methodology for free amino acid analysis by capillary isotachophoresis Aneta Jastrze˛bska *, Anna Marlena Piasta, Edward Szłyk Faculty of Chemistry, Nicolaus Copernicus University in Torun´, 7 Gagarin Street, 87-100 Torun´, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 April 2014 Received in revised form 11 December 2014 Accepted 6 January 2015 Available online 16 February 2015
The objective of this work was the evaluation of different conditions for the selected free amino acids extraction from cheese samples, followed by capillary isotachophoresis analysis. Parameters of the extraction: concentration and type of extraction reagents (solvents: methanol, ethanol and mineral acids: perchloric acid, trichloroacetic acid, sulphuric acid and hydrochloric acid), time and temperature, and isotachophoretic separation conditions (voltage, time) were studied. Repeatability, reproducibility and stability of this method at different conditions are discussed. The best yield of free amino acids was obtained after triple extraction (30 min each process) with 0.1 M hydrochloric acid at 50 8C. Histidine, lysine, arginine, ornithine, tyrosine, and phenylalanine were determined using calibration curves and the standard additions method in different types of cheeses. The highest level of amino acids was determined in semi-hard cheese (2626.3 mg 100 g 1 – calibration curve, and 2679.2 mg 100 g 1 – standard additions method), whereas the lowest were found in sheep cheese (426.3 mg 100 g 1 and 455.7 mg 100 g 1, respectively), and Gouda type cheese samples (441.5 mg 100 g 1 and 472.8 mg 100 g 1, respectively). ß 2015 Elsevier Inc. All rights reserved.
Keywords: Cheese amino acids Sample preparation Histidine Lysine Arginine Ornithine Tyrosine Phenylalanine Optimization of extraction Capillary isotachophoresis Food analysis Food composition
1. Introduction During cheese ripening, several compounds including free amino acids (FAAs) and biogenic amines are produced from the catabolism of proteins (McSweeney and Sousa, 2000; Pinho et al., 2001). The amount of FAAs in cheese depends on many factors, such as the proteins quantity in a raw material used for production, activity of the proteolytic enzymes in dairy technologies, and microorganisms involved in these processes (McSweeney, 2004; Kabelova´ et al., 2009). Due to the role of free amino acids as precursors of biogenic amines and other compounds found in the fermented food, several methods of FAAs analysis were described (Callejo´n et al., 2010; Fiechter et al., 2013; Gorostiza et al., 2004; Izco et al., 2002; Jia et al., 2011; Kabelova´ et al., 2009; Molna´r-Perl, 2000, 2003; Pinho et al., 2001; Subramanian et al., 2011). The most popular method is a high-performance liquid chromatography (HPLC) after pre-column samples derivatization by dansyl
* Corresponding author. Tel.: +48 566114786; fax: +48 566542477. E-mail address: [email protected] (A. Jastrze˛bska). http://dx.doi.org/10.1016/j.jfca.2015.01.004 0889-1575/ß 2015 Elsevier Inc. All rights reserved.
chloride, dabsyl chloride, o-phtalaldehyde, 9-fluorenylmethyl carbonyl chloride, phenylthiohyantoin, 6-aminoquinolyl N-hydroxysuccinimidyl carbamate and others (Callejo´n et al., 2010; Fiechter et al., 2013; Jia et al., 2011; Ko˝ ro¨s et al., 2008; Mayer and Fiechter, 2013; Molna´r-Perl, 2003; Rubio-Barroso et al., 2006). However, amino acids derivatization imposes complicated sample preparation method (Molna´r-Perl, 2003). In order to reduce this inconvenience capillary isotachophoresis (ITP) method was applied for analyses of free amino acids in foods. This method has high sensitivity and precision and enables analysis of samples which are problematically separated by HPLC. Capillary isotachophoresis is particularly useful for the analysis of non-UV absorbing low molecular weight compounds, such as amino acids, due to the conductivity detection. ITP offers possibility of the analyte preconcentration and preseparation from interfering compounds. Furthermore, amino acids can be analyzed as cations or anions depending on the electrolyte type used (Gebauer and Bocˇek, 2000; Gebauer et al., 2011; Mala´ et al., 2013). Application of the capillary isotachophoresis for analysis of amino acids (AAs) in food samples was described by Kvasnicˇka and Kra´tka´ (2006), Kvasnicˇka (1999), Kvasnicˇka and Voldrich (2000) and Jastrze˛bska et al. (2013).
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The precision and accuracy of amino acids analyses in food samples depend on the extracting methodology. According to the literature data, various solvents have been used for extraction of FAAs from food, including cheese samples (Fiechter et al., 2013; Gorostiza et al., 2004; Jastrze˛bska et al., 2013; Kabelova´ et al., 2009; Pinho et al., 2001; Pintado et al., 2008). However, it is hard to decide which extraction process is optimal for the ITP method. Consequently, the present work was focused on the optimization of cheese samples preparation methodology for isotachophoretic analysis of FAAs. The following free amino acids were chosen: histidine (His), lysine (Lys), arginine (Arg), ornithine (Orn), tyrosine (Tyr) and phenylalanine (Phe), which are known as precursors of principal biogenic amines found in cheeses (histamine, cadaverine, agmatine, putrescine, tyramine and phenylethylamine). The impact of the type and concentration of extraction reagent, time and temperature of extraction step on the efficiency of the amino acids extraction and on the isotachophoretic separation conditions (voltage, time) was studied. We applied two methods for quantitative analysis (calibration curves and standard additions method) of FAAs by ITP and obtained results were compared by F-Snedecor and paired t-tests. To the best of our knowledge, despite many applications of the ITP method in food analysis, the separation methodology of free amino acids from cheese samples has not been described.
2. Materials and methods 2.1. Reagents and apparatus L-Histidine (His), L-valine (Val), L-ornithine monohydrochloride (Orn), L-lysine monohydrochloride (Lys), L-arginine monohydrochloride (Arg), L-tyrosine (Tyr), L-phenylalanine, (Phe) a,a,atris-(hydroxymethyl)-aminomethane (TRIS), hydroxyethylcellulose (HEC, average mol wt 90.000), b-alanine (BALA), ethanol and diethyl ether were purchased from Sigma Aldrich (Poznan´, Poland), whereas hydrochloric acid, sulphuric acid, potassium acetate, acetic acid (HAc), methanol, ethanol, trichloroacetic acid, perchloric acid and barium hydroxide were from Alchem (Torun, Poland). All reagents were of analytical grade. Deionised water (<0.30 mS, HLP Smart 2000, Hydrolab, Wis´lina, Poland) was used for all solutions. Isotachophoretic separations were performed using a Villa Labeco EA 100/101 isotachophoretic analyzer equipped with a sample valve with fixed internal loop (30 mL) and conductometric detector (Villa Labeco, Spisˇska´ Nova´ Ves, Slovakia). The PTFE pre-separation capillary (160 mm length; 0.8 mm I.D.) was connected with PTFE analytical capillary (90 mm; 0.3 mm I.D.). The isotachopherograms were evaluated with the ITPPro 32 software supplied with analyzer (KasComp Ltd., Slovakia). Food samples were extracted using an orbital shaker (Chemland, Starogard Szczecin´ski, Poland) and next centrifuged by an MPW-351 laboratory centrifuge (RPM/RCF 18,000/24,088 g, angle 308, falcon tubes 50 mL) (MPW Med. Instruments, Warsaw, Poland).
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The standard solutions of amino acids were prepared in a 100 mL volumetric flask, brought to the mark with 0.1 M hydrochloric acid and analyzed as the cationic form. The amino acids in the standard solution and in cheese samples were identified by the relative step height (RSH) parameter (Jastrze˛bska et al., 2013). Subsequently, in order to verify the stability of the RSH measurements, the five working mixtures (M1–M5), containing all tested amino acids in different concentrations, were analyzed. Concentrations of His, Lys, Arg, Orn, Phe and Tyr in the mixtures were as follows: 20, 30, 40, 50, 60, 70 mg L 1 (M1); 70, 60, 50, 40, 30, 20 mg L 1 (M2); 50, 40, 20, 60, 70, 30 mg L 1 (M3); 20, 20, 20, 70, 70, 70 mg L 1 (M4) and 70, 70, 70, 20, 20, 20 mg L 1 (M5), respectively. The obtained RSH values are expressed as an average value of all measurements confidence interval (p = 95%). Precision of RSH 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 tested mixtures of AAs three times per day. The intralaboratory reproducibility was determined by triple analysis of the amino acids mixtures solutions during 5 consecutive days. The calibration curves were constructed using eight standard solutions of tested amino acids in the concentration range: 10– 100 mg L 1, which were prepared in 10 mL volumetric flasks by appropriate dilution of the stock solutions of Orn, Lys, His, Arg, Phe and Tyr (100 mg L 1) in 0.1 M hydrochloric acid. Each concentration level was determined in triplicate. Accuracy was determined using recovery test based on the analysis of mixtures (M1–M5) containing all amino acids. Each mixture of AAs standard solutions was determined in triplicate and the obtained recovery data are presented as the mean value from all measurements. To reduce matrix effects standard additions as a second calibration method was applied. The analytical calibration procedure was performed on each cheese blank sample spiked at six equispaced concentration levels of all tested amino acids. Then, blank and spiked cheese samples were extracted, analyzed by the ITP method and the analytical results were calculated using the measured signal and plotted versus concentration of the added AAs standards (Miller and Miller, 2000). 2.3. Cheese samples Eight brands of cheeses produced in Poland, and distributed by main suppliers on the Polish market, were purchased from different local stores and tested. Samples included hard cheese: Emmental (27%, w/w fat, 29%, w/w protein – declaration of manufacturer, DM), Parmesan (32%, w/w fat, 41%, w/w protein – DM), Gouda (26%, w/w fat and 25%, w/w protein – DM); semi-hard cheese: Limburg (21%, w/w fat, 20%, w/w protein – DM); soft cheese: surface mould-ripened: Brie (31%, w/w fat, 17%, w/w protein – DM), and blue cheese: Bleu Gourmet (42%, w/w fat, 13%, w/w protein – DM); sheep milk blue cheese: Rokfort (31%, w/w fat, 18%, w/w protein – DM), and smoked sheep’s cheese: Oscypek (name under the EU protected Designation of Origin geographical indication). All samples (0.5–1 kg) were kept frozen until analysis.
2.2. Conditions of ITP analysis 2.4. Optimization of the sample preparation methodology The isotachophoretic analysis of FAAs was performed with two electrolyte systems: (I) Leading Electrolyte (LE): 0.01 M potassium acetate + 2% hydroxyethylcellulose (HEC) with 0.1 M acetic acid to pH = 6 and Terminating Electrolyte (TE): 0.01 M b-alanine (BALA) and (II) LE: 0.02 M a,a,a-tris-(hydroxymethyl)-aminomethane (TRIS) + 1% HEC with 0.01 M hydrochloric acid to pH = 8 and TE: 0.01 M valine with 0.1 M barium hydroxide to pH = 10, driving current 200 mA of the preseparation capillary and 100 mA for the analytical capillary.
Organic solvents (methanol and ethanol) and mineral acids of different concentrations: perchloric acid (1–7%), trichloroacetic acid (1–7%), sulphuric acid (5–15%), hydrochloric acid (0.05, 0.1, 0.5 and 1.0 M) (Gorostiza et al., 2004; Kabelova´ et al., 2009; Kvasnicˇka and Kra´tka´, 2006; Pinho et al., 2001; Subramanian et al., 2011) were used for the amino acids extraction. The obtained results were compared regarding the extracting efficiency by the recovery study. The influence of the extraction parameters on the
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ITP separation quality was studied. Extraction with organic solvents was carried out at 20–40 8C for 60 min, and with mineral acids at 20–70 8C for 60 min, except hydrochloric acid (20–70 8C, 20–60 min). The experiments were performed with the cheese samples: Emmental, Limburg and Rokfort representing hard, soft and blue cheeses, respectively. These samples were spiked with selected amino acids: aliphatic (Lys), heterocyclic (His) and aromatic (Phe). The amino acids (50 mg of Lys, His and Phe per 100 g sample) were added before extraction. Every time, five laboratory samples of each cheese (1 – blank sample, 2–4 – cheese samples spiked with single amino acid, and 5 – cheese samples spiked with all AAs) were prepared and analyzed. Prior to analysis, samples were homogenized twice at room temperature in food grinder (plate holes 3 mm diameter). Homogenized samples (15 0.0001 g) were extracted with the extraction reagent (three times – 15 mL), centrifuged and filtrated. Clear supernatants were pooled in a 50 mL flask and made up to the mark with redistilled water. Five samples of each cheese were prepared and analyzed in triplicate. 3. Results and discussion 3.1. Amino acids standard solutions analyses The typical isotachopherograms of amino acids standard solutions mixtures are presented in Fig. 1, and illustrate satisfactory separation of Lys, Orn, Arg and His for system I and Tyr, Phe and His for system II, confirmed by average values of the relative step height parameters presented in Table 1. Reasonable repeatability of RSH measurements for the electrolyte system I (CV from 0.53% to 0.91%) and for the electrolyte system II (from 1.54% to 2.54%) was obtained. The intralaboratory reproducibility of RSH values revealed CV values: 1.29–2.17% for system I and 2.17–3.28% for system II. The regression parameters of calibration curves for each AA determined by both electrolyte systems are listed in Table 2, indicated that for all analyzed AAs the linearity of L(C) dependence was satisfactory (R2 0.9990). The calculated detection (0.75–3.13 mg L 1) and quantification (2.51–10.43 mg L 1) limits confirm the range of linearity concentration for AAs analysis, whereas the mean recovery values for standard AAs solutions (97–100%) suggest satisfactory accuracy. For comparison, the presented R2 values were similar to those obtained by isotachophoretic (Jastrze˛bska et al., 2013; Kvasnicˇka, 1999; Kvasnicˇka and Voldrich, 2000; Kvasnicˇka and Kra´tka´, 2006), electrophoretic (Izco et al., 2002), and chromatographic methods (Jia et al., 2011; RubioBarroso et al., 2006) for amino acids analysis. It should be noted, that His was analyzed using both tested systems. However, for the electrolyte system II the obtained data (R2, accuracy, DL and QL) were more satisfactory. For this reason, this electrolyte composition was chosen for His quantification in cheese samples. 3.2. Optimization of the cheese samples preparation methodology Previously, we reported the histidine extraction methodology from meat and fish products using methanol (Jastrze˛bska et al., 2013). However, in the case of cheese samples, the extraction with methanol resulted in recoveries from 80 to 90% (mean recovery = 87%, CV = 7.21%). Moreover, the voltage during the isotachophoretic separation was too high and the current reduction caused elongation of the sample separation. When ethanol was applied the obtained supernatants were clear and recoveries ranged 89–96% (mean recovery = 94% and CV = 4.37%), however, separation time was longer (more than 40 min). Furthermore, in the case of extraction with solvents, the RSH values of amino acids were not stable, what excluded these reagents from further research. More
Fig. 1. The isotachopherogram of amino acids mixture, (a) system I (Leading Electrolyte, (LE): 0.01 M potassium acetate + 2% hydroxyethylcellulose + 0.1 M acetic acid; Terminating Electrolyte, (TE): 0.01 M b-alanine) and (b) system II (LE: 0.02 M a,aa-tris-(hydroxymethyl)-aminomethane + 1% hydroxyethylcellulose + 0.01 M hydrochloric acid; TE: 0.01 M Valine + 0.01 M barium hydroxide to pH = 10).
results of recovery obtained for cheese samples after solvent extraction are presented in Supplementary material (Table S.1) In the case of perchloric and trichloroacetic acids, the extracts after centrifugation were turbid and not suitable for analysis. Extraction
Table 1 The relative step height (RSH) values for standard solution of tested amino acids. Amino acid
Relative step height XRSH
Within-day CVRSH (%)
Between-days CVRSH (%)
Electrolyte system I LE: 0.01 M CH3COOK + 2% HEC + 0.1 M CH3COOH to pH = 6; TE: 0.01 M BALA Lys 0.220 0.004 0.79 1.85 Orn 0.291 0.004 0.53 2.09 Arg 0.403 0.007 0.91 2.17 His 0.510 0.006 0.60 1.29 Electrolyte system II LE: 0.02 M TRIS + 1% HEC + 0.01 M HCl; TE: 0.01 M Valine + 0.1 M Ba(OH)2 to pH = 10 Lys + Arg + Orn 0.390 0.024 2.54 3.12 Tyr 0.451 0.002 1.67 2.17 Phe 0.520 0.003 1.54 2.87 His 0.670 0.005 1.87 3.25 Note: HEC – hydroxyethylcellulose; BALA – b-alanine; TRIS – a,aa-tris(hydroxymethyl)-aminomethane; CV – coefficient of variation (%), X – average value with confidence limit (tn 1 s/n(1/2); p = 95%). Each sample was analyzed in triplicate.
A. Jastrze˛bska et al. / Journal of Food Composition and Analysis 40 (2015) 136–142 Table 2 Linear regression calibration parameters of selected amino acids determination by capillary isotachophoretic method (linearity range: 10–100 mg L 1). Amino acids
a
Lys Orn Arg His (system I) His (system II) Tyr Phe
0.330 0.405 0.248 0.273 0.248 0.217 0.089
R2
b 0.246 3.536 1.259 0.1152 3.136 1.07 0.194
0.9999 0.9990 0.9990 0.9986 0.9991 0.9996 0.9999
DL (mg L
QL (%)
Recoverya
2.51 7.31 7.84 10.43 8.30 6.69 6.80
99 99 100 97 100 100 98
1
)
0.75 2.19 2.34 3.13 2.49 2.01 2.04
Note: a – slope of regression line; b – intercept of regression line, R2 – coefficient of determination, DL – Detection Limit calculated as; (y + 3 sy/x)/b, where y – the intercept of the calibration line, sy/x – standard deviation in the y-direction of the calibration line; QL – Quantification Limit calculated as (10 sy/x/b). Each concentration level was analyzed in triplicate. Recovery was calculated as mean value of (obtained concentration/theoretical concentration) 100%. a Analysis of standard solutions mixtures (M1–M5) containing all amino acids.
with sulphuric acid resulted in clear solutions and the RSH values were stable but isotachophoretic separation revealed unsatisfactory repeatability (CV = 9.17%), and accuracy (mean recovery = 93%, min recovery = 81% and max recovery = 96%). The exemplary results of recovery for cheese samples extracted with 5% sulphuric acid are presented in Supplementary material (Table S.1). The best results of extraction and optimal isotachophoretic separation conditions (voltage, time, stability of RSH value, and zone length measurement) were obtained when hydrochloric acid solution (0.05–1.0 M) was used as the extraction reagents; the results about the recovery values using this reagent in different conditions for the extractions are listed in Table 3. Full recovery
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results obtained for individual amino acids are presented in Supplementary material (Table S.2). In a first step, the influence of the concentration of the used hydrochloric acid was evaluated by the recovery values (mean, min, max) of the extraction at 20 8C for 60 min (Table 3), and all results are presented in Supplementary material (Table S.2). The differences between the mean recoveries and CV values for 0.1 M and 0.5 M solution were very small. However, maximum recovery (109%) obtained for 0.5 M hydrochloric acid suggests that combination of these parameters (concentration, temperature and time of extraction) was not appropriate for more complicated samples (Supplementary material Table S.2). Similar results were observed for 1 M hydrochloric acid where recovery varied from 95% (for samples spiked with Phe) to 125% for cheese samples spiked with all AAs (Table 3 and Table S.2). It may suggest the release of amino acids from protein. When 0.05 M hydrochloric acid was used the recovery decreased, hence a 0.1 M solution was chosen for the AAs extraction. In the next stage, the impact of the temperature on the recovery was tested in experiments of extraction using 0.1 M hydrochloric acid at 60 min. The mean recovery values for AAs extracted at 50 8C and 60 8C were almost identical. However, the CV was above 5% for 60 8C, while mean value of CV was below 3.2% at 50 8C. At 70 8C, the hydrolysis process onset was observed, resulting in high recovery (120%) and CV values (8.24%). On the other hand, lower recoveries than 95% were obtained using temperatures of extraction below 50 8C, so these temperatures were not suitable for cheese sample extraction. The last step of cheese samples preparation optimization was the choice of extraction time, this was evaluated in experiments using 0.1 M hydrochloric acid and 50 8C in the extraction. We proposed triple extraction, where time of a single
Table 3 Recovery values obtained for optimization of the cheese sample preparation methodology using hydrochloric acid as the extraction reagent. CV (%)b
Min recoveryc (%)
Max recoveryd (%)
2.35 2.89 3.01 2.79
87 94 93 95
96 99 109 125
0.1 M HCl, time 60 min 20 8C 93 30 8C 94 40 8C 95 50 8C 99 60 8C 99 70 8C 120 0.1 M HCl, temp. 50 8C
3.04 2.96 3.24 3.19 5.75 8.24
89 91 93 93 94 98
94 97 99 99 105 135
20 min 30 min 40 min 50 min 60 min 90 min
4.15 2.86 3.14 2.90 4.01 5.17
87 94 94 95 94 95
96 102 102 103 103 115
Mean recoverya (%) Temp. 20 8C, 0.05 M 0.1 M 0.5 M 1.0 M
time 60 min 92 98 99 110
93 99 99 99 99 105
a Mean recovery value calculated as average value for analysis of cheese samples (Emmental, Limburg and Rokfort) spiked with Lys, Him, Phe and mixture of Lys, Him and Phe (number of blank samples = 9; number of spiked cheese samples = 36). b CV values were calculated for all cheese samples used in recovery studies (number of blank samples = 3 for all kind of cheese; number of spiked cheese samples = 36). c Minimal average recovery obtained for analysis of single amino acid or mixture (number of spiked cheese samples = 9). d Maximal average recovery obtained for analysis of single amino acid or mixture (number of spiked cheese samples = 9); CV – coefficient of variation (%), Recovery = [((X2 X0)/X1) 100%], where: X0 – concentration found in the blank sample; X1 – added amount of AA; X2 – concentration of AA found in the spiked cheese sample.
Fig. 2. The isotachopherogram of cheese sample (Sheep cheese Oscypek), where: (a) system I, 1 – unknown; 2 – Lys; 3 – Orn; 4 – Arg; 5 – His; 6 and 7 – unknown and (b) system II, 1 – (Lys + Orn + Arg); 2 – Tyr; 3– Phe; 4 – His; 5 and 6 – unknown.
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process was varied from 20 to 90 min. The recovery values as a function of the extraction time (30–60 min.) were very small, hence 30 min was selected for the AAs analysis. Summarizing the discussion, the best sample preparation conditions were triple 30 min extractions with 0.1 M hydrochloric acid at 50 8C. It should be emphasized that 0.1 M hydrochloric acid is often used for food samples extraction before amino acids analysis. Flo´rez et al. (2006) discussed evaluation of FAAs and biogenic amines concentration in cheese inoculated with Penicillium roqueforti spores, and applied this reagent for the cheese sample preparation. Amino acids and biogenic amines were analyzed in beer, cheese and sausage samples by the liquid chromatography method coupled with quadrupole time-of-flight mass spectrometry after extraction with 0.1 M hydrochloric acid and derivatization with dansyl chloride (Jia et al., 2011).
In our study, the obtained mean recovery (99%) and CV values (<3%) for the proposed cheese samples extraction and isotachophoretic analysis indicate that this methodology exhibits satisfactory accuracy and precision. For comparison, Ko˝ ro¨s et al. (2008) discussed acidic extraction of AAs and biogenic amines from cheese samples applying hydrochloric, trichloroacetic, sulfosalicylic and perchloric acids. The authors concluded that maximum extraction yield, regarding their efficiency in terms of the amino acids and amines analysis, was obtained with 1 M perchloric acid after 10 min and double protein residue centrifugation. Moreover, methodology revealed average recovery of AAs = 101.5% and relative standard deviation (RSD) = 3.2%. Analysis of free amino acids and biogenic amines in cheese samples by RP-HPLC was described by Pinho et al. (2001). Perchloric acid (0.2 M) was used for sample preparation and proposed methodology included
Table 4 Results of tested free amino acids determination in cheese samples by capillary isotachophoretic method. Sample
Amino-acids
Calibration curve X (mg 100 g
1
)
Standard addition method CV (%)
X (mg 100 g
1
)
CV (%)
F-Test
t-Test
Fcrit = 6.39
tcrit = 2.77
Sheep cheese Oscypeka
His Phe Lys Arg Orn Tyr
42.3 2.2 157.2 2.9 163.0 4.0 29.1 2.3 12.4 1.0 22.3 0.9
4.27 1.48 1.96 6.40 6.68 3.49
50.1 1.1 162.0 2.4 171.7 3.0 31.4 1.3 15.2 0.7 25.3 1.3
1.83 1.20 1.40 3.42 3.68 4.15
3.82 1.42 1.77 2.95 2.06 1.82
0.94 0.21 0.09 1.15 0.48 2.31
Parmesana
His Phe Lys Arg Orn Tyr
78.1 1.3 267.1 5.2 129.2 5.0 30.1 1.7 below QL below QL
1.33 1.56 3.10 4.64 – –
81.1 0.9 269.0 1.0 134.9 4.8 33.4 0.9 6.0 0.3 8.4 0.6
0.87 0.31 2.89 2.21 4.99 5.82
2.12 24.7 1.05 3.60 – –
0.64 1.59 2.01 0.34 – –
Emmentala
His Phe Lys Arg Orn Tyr
66.1 3.0 232.0 7.5 51.3 0.9 246.1 8.0 nd 12.5 0.7
3.66 2.60 1.75 2.64 – 4.57
72.1 1.2 249.0 1.5 61.4 1.3 253.7 4.5 – 20.3 1.1
2.60 0.48 1.70 1.43 – 4.51
6.22 25.5 1.97 3.17 – 2.61
1.88 0.77 2.41 2.07 – 2.15
Goudaa
His Phe Lys Arg Orn Tyr
75.5 3.0 224.4 10.3 125.1 1.3 16.5 0.5 nd below QL
3.18 3.70 1.02 3.19 – –
85.6 3.0 234.0 8.1 127.5 3.1 16.6 0.5 – 9.1 0.4
2.78 2.79 1.99 2.23 – 4.00
1.01 1.61 3.13 1.22 – –
0.57 1.99 1.85 2.53 – –
Limburga
His Phe Lys Arg Orn Tyr
952.0 13.9 1555.1 15.4 73.5 2.4 32.8 1.8 nd 12.9 0.9
1.17 0.80 2.67 4.47 – 5.50
965.0 15.2 1575.1 7.1 81.5 1.3 41.4 1.5 – 16.2 0.5
1.27 0.36 1.25 3.05 – 2.33
1.19 4.75 3.69 1.36 – 3.64
2.29 2.88 3.69 1.54 – 0.30
Bleu Gourmeta
His Phe Lys Arg Orn Tyr
22.4 1.0 1910.0 35.3 200.4 4.4 24.8 1.7 nd below QL
3.78 1.49 1.77 5.34 – –
24.9 0.6 1917.0 6.0 208.0 3.4 30.5 1.0 – 11.6 0.4
1.79 0.25 1.33 2.85 – 5.80
3.44 34.8 1.62 2.54 – –
2.47 0.55 0.75 0.49 – –
Briea
His Phe Lys Arg Orn Tyr
353.7 12.9 452.4 12.3 109.3 6.2 29.5 1.6 nd 27.5 1.2
2.94 2.20 4.56 4.55 – 3.91
379.2 10.7 457.1 11.8 119.1 3.1 36.4 1.1 – 30.4 1.0
2.27 2.08 2.09 2.51 – 2.67
1.46 1.09 3.98 2.20 – 1.79
1.75 1.12 3.97 0.51 – 1.74
Rokforta
His Phe Lys Arg Orn Tyr
502.6 24.1 169.0 9.7 59.3 3.6 47.1 2.1 nd 19.2 0.8
3.86 4.60 4.83 3.58 – 3.65
512.2 11.8 171.0 4.9 65.5 3.5 52.4 1.9 – 21.2 0.7
1.86 2.32 4.32 3.02 – 2.85
4.13 3.85 1.04 1.15 – 1.29
0.88 0.69 2.59 0.86 – 1.09
Note: X – average value [mg 100 g 1] with confidence limit (tn 1 s/n1/2); p = 95%); CV – coefficient of variation (%); nd – not detected. a For each type of cheese five samples were extracted and each samples were analyzed in triplicate.
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30 min homogenization in ultrasonic bath and centrifugation. The obtained average repeatability was less than 4%, whereas recovery was above 90%. The recoveries for AAs in cheese samples after triple 30 min extraction with 0.6 M perchloric acid and UHPLC analysis reported by Fiechter et al. (2013) varied from 83% (for arginine) to 120% (for gamma-aminobutyric acid) with repeatability in the range: 1.0–4.5%. Izco et al. (2002) proposed capillary electrophoresis for analysis of organic acids, lactose and some of the amino acids in cheese samples and in other dairy products. Sample preparation included stirring for 15 min with buffer consisting of 4.5 mM sulphuric acid and boric acid. Studies of the recoveries for Asp, Tyr, Leu, Phe, and Trp, determined by capillary electrophoresis were: 98.9%, 104.0%, 99.6%, 102.4% and 97.8%, respectively. The ITP method was proposed by Kvasnicˇka and Kra´tka´ (2006) for the analysis of L-theanine in green tea and supplements and reported recovery was 99%. Kvasnicˇka (1999) achieved 95% recovery of 3-methylhistidine for meat and meat products analysis, while precision of the method, expressed as RSD value, was 1.5%. In our previous paper we proposed ITP for histidine analysis in meat and fish samples, and the average recovery was 98% with the coefficient of variation varying from 0.65% to 5.75% (Jastrze˛bska et al., 2013). Summarizing the above discussion it can be concluded, that our methodology of sample preparation reveals a good performance (linearity, precision and recovery) in order to quantify AAs in cheeses. No pre-purification and cleanup steps are required before analysis. Since no significant matrix effects were found for the cheese samples, the proposed methodology of sample preparation might be applicable for the ITP detection of amino acids in cheeses. 3.3. Application of the proposed methodology for AAs analysis in cheese samples The typical isotachopherograms for cheese samples are presented in Fig. 2. The FAAs quantification in cheese samples was performed by the calibration curves and standard additions methods (Table 4). The FSnedecor test and paired t-test were employed to compare the precision and accuracy of the used methods (Miller and Miller, 2000). As expected, tested samples varied substantially in amino acids contents, which can be related to manufacturing procedures and an intrinsically different degree of proteolysis. The highest total level of AAs was obtained for Limburg type cheese (calibration curve: 2626.3 mg 100 g 1 and standard additions method: 2679.2 mg 100 g 1). Similar levels of tested six AAs were achieved for blue cheese (Gourmet). The latter is in good agreement with results obtained by Kabelova´ et al. (2009). On the contrary, low amino acids concentrations were found in sheep cheese (426.3 mg 100 g 1 for calibration curves and 455.7 mg 100 g 1 for standard additions method) and Gouda samples (441.5 mg 100 g 1 and 472.8 mg 100 g 1, respectively). Results listed in Table 4 indicates that majority AAs were Phe, Lys, His and Arg in the analyzed samples. It is evident, that Phe and Lys are in prevailing amounts in Bleu Gourmet cheese, His in Limburg cheese and Arg in Emmental cheese. Tyr was detected in five samples at similar levels (12.4–27.5 mg 100 g 1), whereas Orn only in Oscypek and Parmesan. Kabelova´ et al. (2009) studied FAAs content in similar surface mould-ripened cheese (President) and determined: 20 mg 100 g 1 of His, 150 mg 100 g 1 of Arg, 40 mg 100 g 1 of Tyr, 350 mg 100 g 1 of Lys and 280 mg 100 g 1 of Phe. The results presented by Ko˝ ro¨s et al. (2008) suggested that the prevailing AAs in cheese samples were phenylalanine, lysine, aspartic and glutamic acid. Different results of amino acids concentration in cheese samples reported in the literature (Kabelova´ et al., 2009; Izco et al., 2002; Jia et al., 2011), and obtained in this work arise from
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the fact that FAAs composition and concentration generally depend on the milk source, manufacturing technology, the presence of natural bacteria strains and other sources of enzymes, and ripening conditions and duration. The CV values obtained for calibration curves and standard additions methods (Table 4) were below 7%, what indicates satisfactory precision for food analysis. For comparison, Prest et al. (2004) proposed the ITP method for AAs analysis and obtained repeatability and reproducibility from 1.6% to 7.6% and from 1.4% to 7.1%, respectively. The t-test indicates that there are no significant differences between the average concentrations of tested amino acids in cheese samples, assayed by studied calibration methods. The calculated F-test values were above the theoretical ones only in three cases (Table 4), indicating that there was no significant difference of precision between the methods. However, the standard additions method seems to be more appropriate for AAS analysis in cheese samples, because allows the quantification of lower concentrations as can be seen in Table 4. As example, the amount of Orn and Tyr could be quantified by the standard additions method, while the values were below the QL when the calibration curve was used for quantification. In summary, the utility of the described methodology was confirmed by the analysis of six amino acids in samples from different types of cheeses. In addition, satisfactory separation and analysis, and no matrix effect indicate that the proposed method of sample preparation and AAs quantification could be useful for analysis of many amino acids in different food samples. 4. Conclusion The optimization of a cheese sample preparation methodology for determination of selected amino acids by capillary isotachophoresis method was developed. The accuracy, precision and simplicity of separation and quantification of these compounds were satisfactory. The best results in cheese samples were obtained for triple extraction (30 min each) with 0.1 M HCl at 50 8C. Acceptable linearity, repeatability and accuracy in combination with simplicity of amino acids analysis make the proposed method a good alternative to chromatographic techniques. Moreover, the proposed methodology of sample preparation followed by ITP analysis is simpler in comparison to HPLC due to the direct injection of samples without their derivatization. The results obtained in our work are particularly important because free amino acids are commonly accepted as naturally occurring substances, and their analysis in food samples is a widely discussed current analytical problem. Acknowledgement The study was supported by the Polish Ministry of Science and Higher Education: grant No. NN 312 465640 (4656/B/P01/2011/40).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jfca.2015.01.004. References Callejo´n, R.M., Troncoso, A.M., Morales, M.L., 2010. Determination of amino acids in grape-derived products: a review. Talanta 81, 1143–1152. Fiechter, G., Sivec, G., Mayer, H.K., 2013. Application of UHPLC for the simultaneous analysis of free amino acids and biogenic amines in ripened acid-curd cheeses. Journal of Chromatography B 927, 191–200. Flo´rez, A.B., Ruas-Madiedo, P., Alonso, L., Mayo, B., 2006. Microbial, chemical and sensorial variables of the Spanish traditional blue-veined Cabrales cheese, as
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affected by inoculation with commercial Penicillium roqueforti spores. European Food Research and Technology 222, 250–257. Gebauer, P., Bocˇek, P., 2000. Recent progress in capillary isotachophoresis. Electrophoresis 21, 3898–3904. Gebauer, P., Mala´, Z., Bocˇek, P., 2011. Recent progress in analytical capillary isotachophoresis. Electrophoresis 32, 83–89. Gorostiza, A., Cichoscki, A.J., Valduga, A.T., Valduga, E., Bernardo, A., Fresno, J.M., 2004. Changes in soluble nitrogenous compounds, caseins and free amino acids during ripening of artisanal prato cheese; a Brazilian semi-hard cows variety. Food Chemistry 85, 407–414. Izco, J.M., Tormo, M., Jime´nez-Flores, R., 2002. Rapid simultaneous determination of organic acids, free amino acids, and lactose in cheese by capillary electrophoresis. Journal of Dairy Science 85, 2122–2129. Jastrze˛bska, A., Piasta, A., Filipiak-Szok, A., Szłyk, E., 2013. Optimization of capillary isotachophoretic method for histidine determination in protein matrices. Analytical Letters 46, 1364–1378. Jia, S., Kang, Y.P., Park, J.H., Lee, J., Kwon, S.W., 2011. Simultaneous determination of 23 amino acids and 7 biogenic amines in fermented food samples by liquid chromatography/quadrupole time-of-flight mass spectrometry. Journal of Chromatography A 1218, 9174–9182. ˇ ı´zˇkova´, H., Dosta´lek, P., Melzoch, K., 2009. DeterminaKabelova´, I., Dvorˇa´kova´, M., C tion of free amino acids in cheeses from the Czech market. Czech Journal of Food Science 27, 143–150. Ko˝ ro¨s, A´., Varga, Z., Molna´r-Perl, I., 2008. Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography. Journal of Chromatography A 1203, 146–152. Kvasnicˇka, F., 1999. Isotachophoretic determination of 3-methylhistidine. Journal of Chromatography A 838, 191–195. Kvasnicˇka, F., Kra´tka´, J., 2006. Isotachophoretic determination of theanine. Central European Journal of Chemistry 4, 216–222. Kvasnicˇka, F., Voldrich, M., 2000. Isotachophoretic determination of creatinine in meat and meat products. Electrophoresis 21, 2848–2850.
Mala´, Z., Gebauer, P., Bocˇek, P., 2013. Recent progress in analytical capillary isotachophoresis. Electrophoresis 34, 19–28. Mayer, H.K., Fiechter, G., 2013. Application of UHPLC for the determination of free amino acids in different cheese varieties. Analytical and Bioanalytical Chemistry 405, 8053–8061. McSweeney, P.L., 2004. Biochemistry of cheese ripening. International Journal of Dairy Technology 57, 127–144. McSweeney, P.L., Sousa, M.J., 2000. Biochemical pathways for the production of flavor compounds in cheeses during ripening: a review. Lait 80, 293–324. Miller, J.N., Miller, J.C., 2000. Statistical and Chemometrics for Analytical Chemistry. Pearson Education Limited, England. Molna´r-Perl, I., 2000. Role of chromatography in the analysis of sugars, carboxylic acids and amino acids in food. Review. Journal of Chromatography A 891, 1–32. Molna´r-Perl, I., 2003. Quantitation of amino acids and amines in the same matrix by high-performance liquid chromatography, either simultaneously or separately. Journal of Chromatography A 987, 291–309. Pinho, O., Ferreira, I.M.P.L.O., Mendes, E., Oliveira, B.M., Ferreira, M., 2001. Effect of temperature on evolution of free amino acid and biogenic amine contents during storage of Azeita˜o cheese. Food Chemistry 75, 287–291. Pintado, A.I.E., Pinho, O., Ferreira, I.M.P.L.V.O., Pintado, M.M.E., Gomes, A.M.P., Malcata, F.X., 2008. Microbiological, biochemical and biogenic amine profiles of Terrincho cheese manufactured in several dairy farms. International Dairy Journal 18, 631–640. Prest, J.E., Baldock, S.J., Fielden, P.R., Goddard, N.J., Brown, B.J.T., 2004. Analysis of amino acids by miniaturized isotachophoresis. Journal of Chromatography A 1051, 221–226. Rubio-Barroso, S., Santos-Delgado, M.J., Martı´n-Olivar, C., Polo-Dı´ez, L.M., 2006. Indirect chiral HPLC determination and fluorimetric detection of D-amino acids in milk and oyster samples. Journal of Dairy Science 89, 82–89. Subramanian, A., Alvarez, V.B., Harper, W.J., Rodriguez-Saona, L.E., 2011. Monitoring amino acids, organic acids, and ripening changes in Cheddar cheese using Fourier-transform infrared spectroscopy. International Dairy Journal 21, 434–440.
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Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Research Article
Optimization of cheese sample preparation methodology for free amino acid analysis by capillary isotachophoresis Aneta Jastrze˛bska *, Anna Marlena Piasta, Edward Szłyk Faculty of Chemistry, Nicolaus Copernicus University in Torun´, 7 Gagarin Street, 87-100 Torun´, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 April 2014 Received in revised form 11 December 2014 Accepted 6 January 2015 Available online 16 February 2015
The objective of this work was the evaluation of different conditions for the selected free amino acids extraction from cheese samples, followed by capillary isotachophoresis analysis. Parameters of the extraction: concentration and type of extraction reagents (solvents: methanol, ethanol and mineral acids: perchloric acid, trichloroacetic acid, sulphuric acid and hydrochloric acid), time and temperature, and isotachophoretic separation conditions (voltage, time) were studied. Repeatability, reproducibility and stability of this method at different conditions are discussed. The best yield of free amino acids was obtained after triple extraction (30 min each process) with 0.1 M hydrochloric acid at 50 8C. Histidine, lysine, arginine, ornithine, tyrosine, and phenylalanine were determined using calibration curves and the standard additions method in different types of cheeses. The highest level of amino acids was determined in semi-hard cheese (2626.3 mg 100 g 1 – calibration curve, and 2679.2 mg 100 g 1 – standard additions method), whereas the lowest were found in sheep cheese (426.3 mg 100 g 1 and 455.7 mg 100 g 1, respectively), and Gouda type cheese samples (441.5 mg 100 g 1 and 472.8 mg 100 g 1, respectively). ß 2015 Elsevier Inc. All rights reserved.
Keywords: Cheese amino acids Sample preparation Histidine Lysine Arginine Ornithine Tyrosine Phenylalanine Optimization of extraction Capillary isotachophoresis Food analysis Food composition
1. Introduction During cheese ripening, several compounds including free amino acids (FAAs) and biogenic amines are produced from the catabolism of proteins (McSweeney and Sousa, 2000; Pinho et al., 2001). The amount of FAAs in cheese depends on many factors, such as the proteins quantity in a raw material used for production, activity of the proteolytic enzymes in dairy technologies, and microorganisms involved in these processes (McSweeney, 2004; Kabelova´ et al., 2009). Due to the role of free amino acids as precursors of biogenic amines and other compounds found in the fermented food, several methods of FAAs analysis were described (Callejo´n et al., 2010; Fiechter et al., 2013; Gorostiza et al., 2004; Izco et al., 2002; Jia et al., 2011; Kabelova´ et al., 2009; Molna´r-Perl, 2000, 2003; Pinho et al., 2001; Subramanian et al., 2011). The most popular method is a high-performance liquid chromatography (HPLC) after pre-column samples derivatization by dansyl
* Corresponding author. Tel.: +48 566114786; fax: +48 566542477. E-mail address: [email protected] (A. Jastrze˛bska). http://dx.doi.org/10.1016/j.jfca.2015.01.004 0889-1575/ß 2015 Elsevier Inc. All rights reserved.
chloride, dabsyl chloride, o-phtalaldehyde, 9-fluorenylmethyl carbonyl chloride, phenylthiohyantoin, 6-aminoquinolyl N-hydroxysuccinimidyl carbamate and others (Callejo´n et al., 2010; Fiechter et al., 2013; Jia et al., 2011; Ko˝ ro¨s et al., 2008; Mayer and Fiechter, 2013; Molna´r-Perl, 2003; Rubio-Barroso et al., 2006). However, amino acids derivatization imposes complicated sample preparation method (Molna´r-Perl, 2003). In order to reduce this inconvenience capillary isotachophoresis (ITP) method was applied for analyses of free amino acids in foods. This method has high sensitivity and precision and enables analysis of samples which are problematically separated by HPLC. Capillary isotachophoresis is particularly useful for the analysis of non-UV absorbing low molecular weight compounds, such as amino acids, due to the conductivity detection. ITP offers possibility of the analyte preconcentration and preseparation from interfering compounds. Furthermore, amino acids can be analyzed as cations or anions depending on the electrolyte type used (Gebauer and Bocˇek, 2000; Gebauer et al., 2011; Mala´ et al., 2013). Application of the capillary isotachophoresis for analysis of amino acids (AAs) in food samples was described by Kvasnicˇka and Kra´tka´ (2006), Kvasnicˇka (1999), Kvasnicˇka and Voldrich (2000) and Jastrze˛bska et al. (2013).
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The precision and accuracy of amino acids analyses in food samples depend on the extracting methodology. According to the literature data, various solvents have been used for extraction of FAAs from food, including cheese samples (Fiechter et al., 2013; Gorostiza et al., 2004; Jastrze˛bska et al., 2013; Kabelova´ et al., 2009; Pinho et al., 2001; Pintado et al., 2008). However, it is hard to decide which extraction process is optimal for the ITP method. Consequently, the present work was focused on the optimization of cheese samples preparation methodology for isotachophoretic analysis of FAAs. The following free amino acids were chosen: histidine (His), lysine (Lys), arginine (Arg), ornithine (Orn), tyrosine (Tyr) and phenylalanine (Phe), which are known as precursors of principal biogenic amines found in cheeses (histamine, cadaverine, agmatine, putrescine, tyramine and phenylethylamine). The impact of the type and concentration of extraction reagent, time and temperature of extraction step on the efficiency of the amino acids extraction and on the isotachophoretic separation conditions (voltage, time) was studied. We applied two methods for quantitative analysis (calibration curves and standard additions method) of FAAs by ITP and obtained results were compared by F-Snedecor and paired t-tests. To the best of our knowledge, despite many applications of the ITP method in food analysis, the separation methodology of free amino acids from cheese samples has not been described.
2. Materials and methods 2.1. Reagents and apparatus L-Histidine (His), L-valine (Val), L-ornithine monohydrochloride (Orn), L-lysine monohydrochloride (Lys), L-arginine monohydrochloride (Arg), L-tyrosine (Tyr), L-phenylalanine, (Phe) a,a,atris-(hydroxymethyl)-aminomethane (TRIS), hydroxyethylcellulose (HEC, average mol wt 90.000), b-alanine (BALA), ethanol and diethyl ether were purchased from Sigma Aldrich (Poznan´, Poland), whereas hydrochloric acid, sulphuric acid, potassium acetate, acetic acid (HAc), methanol, ethanol, trichloroacetic acid, perchloric acid and barium hydroxide were from Alchem (Torun, Poland). All reagents were of analytical grade. Deionised water (<0.30 mS, HLP Smart 2000, Hydrolab, Wis´lina, Poland) was used for all solutions. Isotachophoretic separations were performed using a Villa Labeco EA 100/101 isotachophoretic analyzer equipped with a sample valve with fixed internal loop (30 mL) and conductometric detector (Villa Labeco, Spisˇska´ Nova´ Ves, Slovakia). The PTFE pre-separation capillary (160 mm length; 0.8 mm I.D.) was connected with PTFE analytical capillary (90 mm; 0.3 mm I.D.). The isotachopherograms were evaluated with the ITPPro 32 software supplied with analyzer (KasComp Ltd., Slovakia). Food samples were extracted using an orbital shaker (Chemland, Starogard Szczecin´ski, Poland) and next centrifuged by an MPW-351 laboratory centrifuge (RPM/RCF 18,000/24,088 g, angle 308, falcon tubes 50 mL) (MPW Med. Instruments, Warsaw, Poland).
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The standard solutions of amino acids were prepared in a 100 mL volumetric flask, brought to the mark with 0.1 M hydrochloric acid and analyzed as the cationic form. The amino acids in the standard solution and in cheese samples were identified by the relative step height (RSH) parameter (Jastrze˛bska et al., 2013). Subsequently, in order to verify the stability of the RSH measurements, the five working mixtures (M1–M5), containing all tested amino acids in different concentrations, were analyzed. Concentrations of His, Lys, Arg, Orn, Phe and Tyr in the mixtures were as follows: 20, 30, 40, 50, 60, 70 mg L 1 (M1); 70, 60, 50, 40, 30, 20 mg L 1 (M2); 50, 40, 20, 60, 70, 30 mg L 1 (M3); 20, 20, 20, 70, 70, 70 mg L 1 (M4) and 70, 70, 70, 20, 20, 20 mg L 1 (M5), respectively. The obtained RSH values are expressed as an average value of all measurements confidence interval (p = 95%). Precision of RSH 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 tested mixtures of AAs three times per day. The intralaboratory reproducibility was determined by triple analysis of the amino acids mixtures solutions during 5 consecutive days. The calibration curves were constructed using eight standard solutions of tested amino acids in the concentration range: 10– 100 mg L 1, which were prepared in 10 mL volumetric flasks by appropriate dilution of the stock solutions of Orn, Lys, His, Arg, Phe and Tyr (100 mg L 1) in 0.1 M hydrochloric acid. Each concentration level was determined in triplicate. Accuracy was determined using recovery test based on the analysis of mixtures (M1–M5) containing all amino acids. Each mixture of AAs standard solutions was determined in triplicate and the obtained recovery data are presented as the mean value from all measurements. To reduce matrix effects standard additions as a second calibration method was applied. The analytical calibration procedure was performed on each cheese blank sample spiked at six equispaced concentration levels of all tested amino acids. Then, blank and spiked cheese samples were extracted, analyzed by the ITP method and the analytical results were calculated using the measured signal and plotted versus concentration of the added AAs standards (Miller and Miller, 2000). 2.3. Cheese samples Eight brands of cheeses produced in Poland, and distributed by main suppliers on the Polish market, were purchased from different local stores and tested. Samples included hard cheese: Emmental (27%, w/w fat, 29%, w/w protein – declaration of manufacturer, DM), Parmesan (32%, w/w fat, 41%, w/w protein – DM), Gouda (26%, w/w fat and 25%, w/w protein – DM); semi-hard cheese: Limburg (21%, w/w fat, 20%, w/w protein – DM); soft cheese: surface mould-ripened: Brie (31%, w/w fat, 17%, w/w protein – DM), and blue cheese: Bleu Gourmet (42%, w/w fat, 13%, w/w protein – DM); sheep milk blue cheese: Rokfort (31%, w/w fat, 18%, w/w protein – DM), and smoked sheep’s cheese: Oscypek (name under the EU protected Designation of Origin geographical indication). All samples (0.5–1 kg) were kept frozen until analysis.
2.2. Conditions of ITP analysis 2.4. Optimization of the sample preparation methodology The isotachophoretic analysis of FAAs was performed with two electrolyte systems: (I) Leading Electrolyte (LE): 0.01 M potassium acetate + 2% hydroxyethylcellulose (HEC) with 0.1 M acetic acid to pH = 6 and Terminating Electrolyte (TE): 0.01 M b-alanine (BALA) and (II) LE: 0.02 M a,a,a-tris-(hydroxymethyl)-aminomethane (TRIS) + 1% HEC with 0.01 M hydrochloric acid to pH = 8 and TE: 0.01 M valine with 0.1 M barium hydroxide to pH = 10, driving current 200 mA of the preseparation capillary and 100 mA for the analytical capillary.
Organic solvents (methanol and ethanol) and mineral acids of different concentrations: perchloric acid (1–7%), trichloroacetic acid (1–7%), sulphuric acid (5–15%), hydrochloric acid (0.05, 0.1, 0.5 and 1.0 M) (Gorostiza et al., 2004; Kabelova´ et al., 2009; Kvasnicˇka and Kra´tka´, 2006; Pinho et al., 2001; Subramanian et al., 2011) were used for the amino acids extraction. The obtained results were compared regarding the extracting efficiency by the recovery study. The influence of the extraction parameters on the
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ITP separation quality was studied. Extraction with organic solvents was carried out at 20–40 8C for 60 min, and with mineral acids at 20–70 8C for 60 min, except hydrochloric acid (20–70 8C, 20–60 min). The experiments were performed with the cheese samples: Emmental, Limburg and Rokfort representing hard, soft and blue cheeses, respectively. These samples were spiked with selected amino acids: aliphatic (Lys), heterocyclic (His) and aromatic (Phe). The amino acids (50 mg of Lys, His and Phe per 100 g sample) were added before extraction. Every time, five laboratory samples of each cheese (1 – blank sample, 2–4 – cheese samples spiked with single amino acid, and 5 – cheese samples spiked with all AAs) were prepared and analyzed. Prior to analysis, samples were homogenized twice at room temperature in food grinder (plate holes 3 mm diameter). Homogenized samples (15 0.0001 g) were extracted with the extraction reagent (three times – 15 mL), centrifuged and filtrated. Clear supernatants were pooled in a 50 mL flask and made up to the mark with redistilled water. Five samples of each cheese were prepared and analyzed in triplicate. 3. Results and discussion 3.1. Amino acids standard solutions analyses The typical isotachopherograms of amino acids standard solutions mixtures are presented in Fig. 1, and illustrate satisfactory separation of Lys, Orn, Arg and His for system I and Tyr, Phe and His for system II, confirmed by average values of the relative step height parameters presented in Table 1. Reasonable repeatability of RSH measurements for the electrolyte system I (CV from 0.53% to 0.91%) and for the electrolyte system II (from 1.54% to 2.54%) was obtained. The intralaboratory reproducibility of RSH values revealed CV values: 1.29–2.17% for system I and 2.17–3.28% for system II. The regression parameters of calibration curves for each AA determined by both electrolyte systems are listed in Table 2, indicated that for all analyzed AAs the linearity of L(C) dependence was satisfactory (R2 0.9990). The calculated detection (0.75–3.13 mg L 1) and quantification (2.51–10.43 mg L 1) limits confirm the range of linearity concentration for AAs analysis, whereas the mean recovery values for standard AAs solutions (97–100%) suggest satisfactory accuracy. For comparison, the presented R2 values were similar to those obtained by isotachophoretic (Jastrze˛bska et al., 2013; Kvasnicˇka, 1999; Kvasnicˇka and Voldrich, 2000; Kvasnicˇka and Kra´tka´, 2006), electrophoretic (Izco et al., 2002), and chromatographic methods (Jia et al., 2011; RubioBarroso et al., 2006) for amino acids analysis. It should be noted, that His was analyzed using both tested systems. However, for the electrolyte system II the obtained data (R2, accuracy, DL and QL) were more satisfactory. For this reason, this electrolyte composition was chosen for His quantification in cheese samples. 3.2. Optimization of the cheese samples preparation methodology Previously, we reported the histidine extraction methodology from meat and fish products using methanol (Jastrze˛bska et al., 2013). However, in the case of cheese samples, the extraction with methanol resulted in recoveries from 80 to 90% (mean recovery = 87%, CV = 7.21%). Moreover, the voltage during the isotachophoretic separation was too high and the current reduction caused elongation of the sample separation. When ethanol was applied the obtained supernatants were clear and recoveries ranged 89–96% (mean recovery = 94% and CV = 4.37%), however, separation time was longer (more than 40 min). Furthermore, in the case of extraction with solvents, the RSH values of amino acids were not stable, what excluded these reagents from further research. More
Fig. 1. The isotachopherogram of amino acids mixture, (a) system I (Leading Electrolyte, (LE): 0.01 M potassium acetate + 2% hydroxyethylcellulose + 0.1 M acetic acid; Terminating Electrolyte, (TE): 0.01 M b-alanine) and (b) system II (LE: 0.02 M a,aa-tris-(hydroxymethyl)-aminomethane + 1% hydroxyethylcellulose + 0.01 M hydrochloric acid; TE: 0.01 M Valine + 0.01 M barium hydroxide to pH = 10).
results of recovery obtained for cheese samples after solvent extraction are presented in Supplementary material (Table S.1) In the case of perchloric and trichloroacetic acids, the extracts after centrifugation were turbid and not suitable for analysis. Extraction
Table 1 The relative step height (RSH) values for standard solution of tested amino acids. Amino acid
Relative step height XRSH
Within-day CVRSH (%)
Between-days CVRSH (%)
Electrolyte system I LE: 0.01 M CH3COOK + 2% HEC + 0.1 M CH3COOH to pH = 6; TE: 0.01 M BALA Lys 0.220 0.004 0.79 1.85 Orn 0.291 0.004 0.53 2.09 Arg 0.403 0.007 0.91 2.17 His 0.510 0.006 0.60 1.29 Electrolyte system II LE: 0.02 M TRIS + 1% HEC + 0.01 M HCl; TE: 0.01 M Valine + 0.1 M Ba(OH)2 to pH = 10 Lys + Arg + Orn 0.390 0.024 2.54 3.12 Tyr 0.451 0.002 1.67 2.17 Phe 0.520 0.003 1.54 2.87 His 0.670 0.005 1.87 3.25 Note: HEC – hydroxyethylcellulose; BALA – b-alanine; TRIS – a,aa-tris(hydroxymethyl)-aminomethane; CV – coefficient of variation (%), X – average value with confidence limit (tn 1 s/n(1/2); p = 95%). Each sample was analyzed in triplicate.
A. Jastrze˛bska et al. / Journal of Food Composition and Analysis 40 (2015) 136–142 Table 2 Linear regression calibration parameters of selected amino acids determination by capillary isotachophoretic method (linearity range: 10–100 mg L 1). Amino acids
a
Lys Orn Arg His (system I) His (system II) Tyr Phe
0.330 0.405 0.248 0.273 0.248 0.217 0.089
R2
b 0.246 3.536 1.259 0.1152 3.136 1.07 0.194
0.9999 0.9990 0.9990 0.9986 0.9991 0.9996 0.9999
DL (mg L
QL (%)
Recoverya
2.51 7.31 7.84 10.43 8.30 6.69 6.80
99 99 100 97 100 100 98
1
)
0.75 2.19 2.34 3.13 2.49 2.01 2.04
Note: a – slope of regression line; b – intercept of regression line, R2 – coefficient of determination, DL – Detection Limit calculated as; (y + 3 sy/x)/b, where y – the intercept of the calibration line, sy/x – standard deviation in the y-direction of the calibration line; QL – Quantification Limit calculated as (10 sy/x/b). Each concentration level was analyzed in triplicate. Recovery was calculated as mean value of (obtained concentration/theoretical concentration) 100%. a Analysis of standard solutions mixtures (M1–M5) containing all amino acids.
with sulphuric acid resulted in clear solutions and the RSH values were stable but isotachophoretic separation revealed unsatisfactory repeatability (CV = 9.17%), and accuracy (mean recovery = 93%, min recovery = 81% and max recovery = 96%). The exemplary results of recovery for cheese samples extracted with 5% sulphuric acid are presented in Supplementary material (Table S.1). The best results of extraction and optimal isotachophoretic separation conditions (voltage, time, stability of RSH value, and zone length measurement) were obtained when hydrochloric acid solution (0.05–1.0 M) was used as the extraction reagents; the results about the recovery values using this reagent in different conditions for the extractions are listed in Table 3. Full recovery
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results obtained for individual amino acids are presented in Supplementary material (Table S.2). In a first step, the influence of the concentration of the used hydrochloric acid was evaluated by the recovery values (mean, min, max) of the extraction at 20 8C for 60 min (Table 3), and all results are presented in Supplementary material (Table S.2). The differences between the mean recoveries and CV values for 0.1 M and 0.5 M solution were very small. However, maximum recovery (109%) obtained for 0.5 M hydrochloric acid suggests that combination of these parameters (concentration, temperature and time of extraction) was not appropriate for more complicated samples (Supplementary material Table S.2). Similar results were observed for 1 M hydrochloric acid where recovery varied from 95% (for samples spiked with Phe) to 125% for cheese samples spiked with all AAs (Table 3 and Table S.2). It may suggest the release of amino acids from protein. When 0.05 M hydrochloric acid was used the recovery decreased, hence a 0.1 M solution was chosen for the AAs extraction. In the next stage, the impact of the temperature on the recovery was tested in experiments of extraction using 0.1 M hydrochloric acid at 60 min. The mean recovery values for AAs extracted at 50 8C and 60 8C were almost identical. However, the CV was above 5% for 60 8C, while mean value of CV was below 3.2% at 50 8C. At 70 8C, the hydrolysis process onset was observed, resulting in high recovery (120%) and CV values (8.24%). On the other hand, lower recoveries than 95% were obtained using temperatures of extraction below 50 8C, so these temperatures were not suitable for cheese sample extraction. The last step of cheese samples preparation optimization was the choice of extraction time, this was evaluated in experiments using 0.1 M hydrochloric acid and 50 8C in the extraction. We proposed triple extraction, where time of a single
Table 3 Recovery values obtained for optimization of the cheese sample preparation methodology using hydrochloric acid as the extraction reagent. CV (%)b
Min recoveryc (%)
Max recoveryd (%)
2.35 2.89 3.01 2.79
87 94 93 95
96 99 109 125
0.1 M HCl, time 60 min 20 8C 93 30 8C 94 40 8C 95 50 8C 99 60 8C 99 70 8C 120 0.1 M HCl, temp. 50 8C
3.04 2.96 3.24 3.19 5.75 8.24
89 91 93 93 94 98
94 97 99 99 105 135
20 min 30 min 40 min 50 min 60 min 90 min
4.15 2.86 3.14 2.90 4.01 5.17
87 94 94 95 94 95
96 102 102 103 103 115
Mean recoverya (%) Temp. 20 8C, 0.05 M 0.1 M 0.5 M 1.0 M
time 60 min 92 98 99 110
93 99 99 99 99 105
a Mean recovery value calculated as average value for analysis of cheese samples (Emmental, Limburg and Rokfort) spiked with Lys, Him, Phe and mixture of Lys, Him and Phe (number of blank samples = 9; number of spiked cheese samples = 36). b CV values were calculated for all cheese samples used in recovery studies (number of blank samples = 3 for all kind of cheese; number of spiked cheese samples = 36). c Minimal average recovery obtained for analysis of single amino acid or mixture (number of spiked cheese samples = 9). d Maximal average recovery obtained for analysis of single amino acid or mixture (number of spiked cheese samples = 9); CV – coefficient of variation (%), Recovery = [((X2 X0)/X1) 100%], where: X0 – concentration found in the blank sample; X1 – added amount of AA; X2 – concentration of AA found in the spiked cheese sample.
Fig. 2. The isotachopherogram of cheese sample (Sheep cheese Oscypek), where: (a) system I, 1 – unknown; 2 – Lys; 3 – Orn; 4 – Arg; 5 – His; 6 and 7 – unknown and (b) system II, 1 – (Lys + Orn + Arg); 2 – Tyr; 3– Phe; 4 – His; 5 and 6 – unknown.
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process was varied from 20 to 90 min. The recovery values as a function of the extraction time (30–60 min.) were very small, hence 30 min was selected for the AAs analysis. Summarizing the discussion, the best sample preparation conditions were triple 30 min extractions with 0.1 M hydrochloric acid at 50 8C. It should be emphasized that 0.1 M hydrochloric acid is often used for food samples extraction before amino acids analysis. Flo´rez et al. (2006) discussed evaluation of FAAs and biogenic amines concentration in cheese inoculated with Penicillium roqueforti spores, and applied this reagent for the cheese sample preparation. Amino acids and biogenic amines were analyzed in beer, cheese and sausage samples by the liquid chromatography method coupled with quadrupole time-of-flight mass spectrometry after extraction with 0.1 M hydrochloric acid and derivatization with dansyl chloride (Jia et al., 2011).
In our study, the obtained mean recovery (99%) and CV values (<3%) for the proposed cheese samples extraction and isotachophoretic analysis indicate that this methodology exhibits satisfactory accuracy and precision. For comparison, Ko˝ ro¨s et al. (2008) discussed acidic extraction of AAs and biogenic amines from cheese samples applying hydrochloric, trichloroacetic, sulfosalicylic and perchloric acids. The authors concluded that maximum extraction yield, regarding their efficiency in terms of the amino acids and amines analysis, was obtained with 1 M perchloric acid after 10 min and double protein residue centrifugation. Moreover, methodology revealed average recovery of AAs = 101.5% and relative standard deviation (RSD) = 3.2%. Analysis of free amino acids and biogenic amines in cheese samples by RP-HPLC was described by Pinho et al. (2001). Perchloric acid (0.2 M) was used for sample preparation and proposed methodology included
Table 4 Results of tested free amino acids determination in cheese samples by capillary isotachophoretic method. Sample
Amino-acids
Calibration curve X (mg 100 g
1
)
Standard addition method CV (%)
X (mg 100 g
1
)
CV (%)
F-Test
t-Test
Fcrit = 6.39
tcrit = 2.77
Sheep cheese Oscypeka
His Phe Lys Arg Orn Tyr
42.3 2.2 157.2 2.9 163.0 4.0 29.1 2.3 12.4 1.0 22.3 0.9
4.27 1.48 1.96 6.40 6.68 3.49
50.1 1.1 162.0 2.4 171.7 3.0 31.4 1.3 15.2 0.7 25.3 1.3
1.83 1.20 1.40 3.42 3.68 4.15
3.82 1.42 1.77 2.95 2.06 1.82
0.94 0.21 0.09 1.15 0.48 2.31
Parmesana
His Phe Lys Arg Orn Tyr
78.1 1.3 267.1 5.2 129.2 5.0 30.1 1.7 below QL below QL
1.33 1.56 3.10 4.64 – –
81.1 0.9 269.0 1.0 134.9 4.8 33.4 0.9 6.0 0.3 8.4 0.6
0.87 0.31 2.89 2.21 4.99 5.82
2.12 24.7 1.05 3.60 – –
0.64 1.59 2.01 0.34 – –
Emmentala
His Phe Lys Arg Orn Tyr
66.1 3.0 232.0 7.5 51.3 0.9 246.1 8.0 nd 12.5 0.7
3.66 2.60 1.75 2.64 – 4.57
72.1 1.2 249.0 1.5 61.4 1.3 253.7 4.5 – 20.3 1.1
2.60 0.48 1.70 1.43 – 4.51
6.22 25.5 1.97 3.17 – 2.61
1.88 0.77 2.41 2.07 – 2.15
Goudaa
His Phe Lys Arg Orn Tyr
75.5 3.0 224.4 10.3 125.1 1.3 16.5 0.5 nd below QL
3.18 3.70 1.02 3.19 – –
85.6 3.0 234.0 8.1 127.5 3.1 16.6 0.5 – 9.1 0.4
2.78 2.79 1.99 2.23 – 4.00
1.01 1.61 3.13 1.22 – –
0.57 1.99 1.85 2.53 – –
Limburga
His Phe Lys Arg Orn Tyr
952.0 13.9 1555.1 15.4 73.5 2.4 32.8 1.8 nd 12.9 0.9
1.17 0.80 2.67 4.47 – 5.50
965.0 15.2 1575.1 7.1 81.5 1.3 41.4 1.5 – 16.2 0.5
1.27 0.36 1.25 3.05 – 2.33
1.19 4.75 3.69 1.36 – 3.64
2.29 2.88 3.69 1.54 – 0.30
Bleu Gourmeta
His Phe Lys Arg Orn Tyr
22.4 1.0 1910.0 35.3 200.4 4.4 24.8 1.7 nd below QL
3.78 1.49 1.77 5.34 – –
24.9 0.6 1917.0 6.0 208.0 3.4 30.5 1.0 – 11.6 0.4
1.79 0.25 1.33 2.85 – 5.80
3.44 34.8 1.62 2.54 – –
2.47 0.55 0.75 0.49 – –
Briea
His Phe Lys Arg Orn Tyr
353.7 12.9 452.4 12.3 109.3 6.2 29.5 1.6 nd 27.5 1.2
2.94 2.20 4.56 4.55 – 3.91
379.2 10.7 457.1 11.8 119.1 3.1 36.4 1.1 – 30.4 1.0
2.27 2.08 2.09 2.51 – 2.67
1.46 1.09 3.98 2.20 – 1.79
1.75 1.12 3.97 0.51 – 1.74
Rokforta
His Phe Lys Arg Orn Tyr
502.6 24.1 169.0 9.7 59.3 3.6 47.1 2.1 nd 19.2 0.8
3.86 4.60 4.83 3.58 – 3.65
512.2 11.8 171.0 4.9 65.5 3.5 52.4 1.9 – 21.2 0.7
1.86 2.32 4.32 3.02 – 2.85
4.13 3.85 1.04 1.15 – 1.29
0.88 0.69 2.59 0.86 – 1.09
Note: X – average value [mg 100 g 1] with confidence limit (tn 1 s/n1/2); p = 95%); CV – coefficient of variation (%); nd – not detected. a For each type of cheese five samples were extracted and each samples were analyzed in triplicate.
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30 min homogenization in ultrasonic bath and centrifugation. The obtained average repeatability was less than 4%, whereas recovery was above 90%. The recoveries for AAs in cheese samples after triple 30 min extraction with 0.6 M perchloric acid and UHPLC analysis reported by Fiechter et al. (2013) varied from 83% (for arginine) to 120% (for gamma-aminobutyric acid) with repeatability in the range: 1.0–4.5%. Izco et al. (2002) proposed capillary electrophoresis for analysis of organic acids, lactose and some of the amino acids in cheese samples and in other dairy products. Sample preparation included stirring for 15 min with buffer consisting of 4.5 mM sulphuric acid and boric acid. Studies of the recoveries for Asp, Tyr, Leu, Phe, and Trp, determined by capillary electrophoresis were: 98.9%, 104.0%, 99.6%, 102.4% and 97.8%, respectively. The ITP method was proposed by Kvasnicˇka and Kra´tka´ (2006) for the analysis of L-theanine in green tea and supplements and reported recovery was 99%. Kvasnicˇka (1999) achieved 95% recovery of 3-methylhistidine for meat and meat products analysis, while precision of the method, expressed as RSD value, was 1.5%. In our previous paper we proposed ITP for histidine analysis in meat and fish samples, and the average recovery was 98% with the coefficient of variation varying from 0.65% to 5.75% (Jastrze˛bska et al., 2013). Summarizing the above discussion it can be concluded, that our methodology of sample preparation reveals a good performance (linearity, precision and recovery) in order to quantify AAs in cheeses. No pre-purification and cleanup steps are required before analysis. Since no significant matrix effects were found for the cheese samples, the proposed methodology of sample preparation might be applicable for the ITP detection of amino acids in cheeses. 3.3. Application of the proposed methodology for AAs analysis in cheese samples The typical isotachopherograms for cheese samples are presented in Fig. 2. The FAAs quantification in cheese samples was performed by the calibration curves and standard additions methods (Table 4). The FSnedecor test and paired t-test were employed to compare the precision and accuracy of the used methods (Miller and Miller, 2000). As expected, tested samples varied substantially in amino acids contents, which can be related to manufacturing procedures and an intrinsically different degree of proteolysis. The highest total level of AAs was obtained for Limburg type cheese (calibration curve: 2626.3 mg 100 g 1 and standard additions method: 2679.2 mg 100 g 1). Similar levels of tested six AAs were achieved for blue cheese (Gourmet). The latter is in good agreement with results obtained by Kabelova´ et al. (2009). On the contrary, low amino acids concentrations were found in sheep cheese (426.3 mg 100 g 1 for calibration curves and 455.7 mg 100 g 1 for standard additions method) and Gouda samples (441.5 mg 100 g 1 and 472.8 mg 100 g 1, respectively). Results listed in Table 4 indicates that majority AAs were Phe, Lys, His and Arg in the analyzed samples. It is evident, that Phe and Lys are in prevailing amounts in Bleu Gourmet cheese, His in Limburg cheese and Arg in Emmental cheese. Tyr was detected in five samples at similar levels (12.4–27.5 mg 100 g 1), whereas Orn only in Oscypek and Parmesan. Kabelova´ et al. (2009) studied FAAs content in similar surface mould-ripened cheese (President) and determined: 20 mg 100 g 1 of His, 150 mg 100 g 1 of Arg, 40 mg 100 g 1 of Tyr, 350 mg 100 g 1 of Lys and 280 mg 100 g 1 of Phe. The results presented by Ko˝ ro¨s et al. (2008) suggested that the prevailing AAs in cheese samples were phenylalanine, lysine, aspartic and glutamic acid. Different results of amino acids concentration in cheese samples reported in the literature (Kabelova´ et al., 2009; Izco et al., 2002; Jia et al., 2011), and obtained in this work arise from
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the fact that FAAs composition and concentration generally depend on the milk source, manufacturing technology, the presence of natural bacteria strains and other sources of enzymes, and ripening conditions and duration. The CV values obtained for calibration curves and standard additions methods (Table 4) were below 7%, what indicates satisfactory precision for food analysis. For comparison, Prest et al. (2004) proposed the ITP method for AAs analysis and obtained repeatability and reproducibility from 1.6% to 7.6% and from 1.4% to 7.1%, respectively. The t-test indicates that there are no significant differences between the average concentrations of tested amino acids in cheese samples, assayed by studied calibration methods. The calculated F-test values were above the theoretical ones only in three cases (Table 4), indicating that there was no significant difference of precision between the methods. However, the standard additions method seems to be more appropriate for AAS analysis in cheese samples, because allows the quantification of lower concentrations as can be seen in Table 4. As example, the amount of Orn and Tyr could be quantified by the standard additions method, while the values were below the QL when the calibration curve was used for quantification. In summary, the utility of the described methodology was confirmed by the analysis of six amino acids in samples from different types of cheeses. In addition, satisfactory separation and analysis, and no matrix effect indicate that the proposed method of sample preparation and AAs quantification could be useful for analysis of many amino acids in different food samples. 4. Conclusion The optimization of a cheese sample preparation methodology for determination of selected amino acids by capillary isotachophoresis method was developed. The accuracy, precision and simplicity of separation and quantification of these compounds were satisfactory. The best results in cheese samples were obtained for triple extraction (30 min each) with 0.1 M HCl at 50 8C. Acceptable linearity, repeatability and accuracy in combination with simplicity of amino acids analysis make the proposed method a good alternative to chromatographic techniques. Moreover, the proposed methodology of sample preparation followed by ITP analysis is simpler in comparison to HPLC due to the direct injection of samples without their derivatization. The results obtained in our work are particularly important because free amino acids are commonly accepted as naturally occurring substances, and their analysis in food samples is a widely discussed current analytical problem. Acknowledgement The study was supported by the Polish Ministry of Science and Higher Education: grant No. NN 312 465640 (4656/B/P01/2011/40).
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