A Reversed-Phase High-Performance Liquid Chromatographic

Rapid detection of the addition of soybean proteins to cheese and other dairy products by reversed-phase perfusion chromatography. M. C. García , M. ...
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Anal. Chem. 2000, 72, 1814-1818

A Reversed-Phase High-Performance Liquid Chromatographic Method for the Determination of Soya Bean Proteins in Bovine Milks Martin Kruså, Mercedes Torre, and Ma Luisa Marina*

Departamento de Quı´mica Analı´tica, Facultad de Ciencias, Universidad de Alcala´ , 28871 Alcala´ de Henares, Madrid, Spain

A reversed-phase high-performance liquid chromatographic method was designed for the quantitation of soya bean proteins in bovine milks. The method consisted of a linear binary gradient, acetonitrile-water-0.1% trifluoroacetic acid, at a flow rate of 1 mL/min and a temperature of 50 °C which resulted in a separation time for soya bean proteins of 11 min. Calibration by the external standard method using a soya bean protein isolate as standard was employed, and the method was validated by evaluating precision, accuracy, and robustness. This method was shown to be useful for the analysis of soya bean proteins in bovine milks spiked with soya bean protein isolate; soya bean protein concentrations of ∼13 µg/g of bovine milk could be detected by using the optimized method. The results obtained for some of the bovine milks were compared with those obtained by the method of standard additions. Products derived from soya bean now constitute an important source of high-quality vegetable proteins.1,2 In fact, soya bean is a low-cost leguminose that has good nutritional properties since it contains proteins, vitamins, minerals, and phosphorus. Furthermore, soya bean has a low fat content and possible beneficial effects on health.3-5 All these qualities make it an interesting alternative to the consumption of animal products and soya bean proteins have been added to a great number of products to enhance the quality of nonvegetable foods, such as dairy, bakery, and fish and meat products.6-10 However, the addition of soya bean proteins to bovine milks is illegal in many countries, so the development of analytical methods to detect soya bean proteins in dairy products is of great importance. Few methods for this * Corresponding author: (fax) 34-91-885-49-71; (e-mail) [email protected]. (1) Steinke, F. H. New Protein Foods in Human Health: Nutrition, Prevention, and Therapy; CRC Press: Boca Raton, FL, 1992; pp 59-66. (2) Garcı´a, M. C.; Torre, M.; Marina, M. L.; Laborda, F. CRC Crit. Rev. Food Sci. Nutr. 1997, 37, 361-91. (3) Messina, M.; Barnes, S. J. Natl. Cancer Inst. 1991, 83, 541-6. (4) Messina, M. Chem. Ind. 1995, 11, 412-5. (5) Sirtori, C. R.; Lovati, M. R.; Manzoni, C.; Monetti, M.; Pazzucconi, F.; Gatti, E. J. Nutr. 1995, 125, 598S-605S. (6) Chronakis, I. S.; Kasapis, S. Food Hydrocolloids 1993, 7, 459-78. (7) Camire, M. E.; King, C. C. J. Food Sci. 1991, 56, 760-3. (8) Keeton, J. T. Meat Sci. 1994, 36, 261-76. (9) Kashlan, N. B.; Hassan, S. A.; Srivastava, V. P.; Mohana, N. A.; Shubber, K. M. Food Chem. 1991, 42, 57-64. (10) Yadav, V. D.; Jha, Y. K.; Garg, S. K.; Mital, B. K. Aust. J. Dairy Technol. 1994, 49, 34-8.

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purpose are currently available. Gel permeation chromatography (GPC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF) have been used for the determination of soya bean protein content in melted cheese.11 For soya bean protein determination in bovine milk, SDS-PAGE and fast protein liquid chromatography (FPLC) have been used.12 Both SDS-PAGE and FPLC techniques were adequate to detect soya bean proteins in mixtures of milks, but the analysis time obtained when using FPLC (25 min for each sample run) presented an important advantage with respect to SDS-PAGE techniques. The aim of this work was to develop a rapid chromatographic method to quantitate soya bean proteins in bovine milks. Since reversed-phase high-performance liquid chromatography (RPHPLC) has been shown to be useful to analyze soya bean proteins13-17 and to achieve the simultaneous separation of soya bean and bovine whey proteins,18 this technique was employed in this work in order to achieve the development and validation of an analytical method for the determination of soya bean proteins in bovine milks. EXPERIMENTAL SECTION Chemicals and Samples. HPLC grade acetonitrile (ACN) (Lab-scan, Dublin, Ireland), trifluoroacetic acid 99.5 atom % D (TFA) (Sigma, St. Louis, MO), and HPLC grade water (Milli-Q system; Millipore, Bedford, MA) were used in the preparation of mobile phases. The soya bean protein isolate (SPI) used for the spiking of the bovine milks and the calibration by the external standard method was obtained from ICN (Aurora, OH). The protein content of the SPI, determined by Kjeldahl analysis (seven replicates) was 92.99% (relative standard deviation (RSD) 2.86%).19 Powdered and liquid bovine milks, with different fat content and from different processing, were purchased from local markets in (11) Cattaneo, T. M. P.; Feroldi, A.; Toppino, P. M.; Olieman, C. Neth. Milk Dairy J. 1994, 40, 225-34. (12) Hewedy, M. M.; Smith, C. J. Food Hydrocolloids 1989, 3, 399-405. (13) Peterson, R. E.; Wolf, W. J. J. Chromatogr. 1988, 444, 263-8. (14) Peterson, R. E.; Wolf, W. J. Cereal Chem. 1992, 69, 101-4. (15) Oomah, B. D.; Voldeng, H.; Fregeau-Reid, J. A. Plant Foods Hum. Nutr. 1994, 45, 251-63. (16) Ashoor, S. H.; Stiles, P. G. J. Chromatogr. 1987, 393, 321-8. (17) Garcı´a, M. C.; Torre, M.; Laborda, F.; Marina, M. L. J. Chromatogr., A 1997, 758, 75-83. (18) Garcı´a, M. C.; Marina, M. L.; Torre, M. Anal. Chem. 1997, 69, 2217-20. (19) Garcı´a, M. C.; Torre, M.; Marina, M. L. J. Chromatogr. Sci. 1998, 36, 52734. 10.1021/ac990776m CCC: $19.00

© 2000 American Chemical Society Published on Web 03/18/2000

Alcala´ de Henares, Madrid, Spain. The following procedure for the preparation of the SPI solutions for the external calibration curve was used: different and known amounts of SPI were weighed, dissolved in Milli-Q water, and shaken. The mixtures were then sonicated for 3 min and centrifuged at 3000 rpm for 5 min (P-Selecta centrifuge; J. P. Selecta, Barcelona, Spain). A small volume (∼1 mL) of each the supernatant was introduced into a vial for injection in the chromatographic system. The preparation of the SPI spiked milk samples was as follows: (i) the milk to be used was diluted, in known proportion, with Milli-Q water and filtered through 0.2-µm pore size filters (Gelman Sciences, Ann Arbor, MI); (ii) SPI was dissolved in Milli-Q water and shaken; (iii) the SPI solution and the filtered milk were then mixed in proportions giving the appropriate concentrations of milk and SPI (see Results and Discussion), and the resulting solution was sonicated for 3 min and centrifuged at 3000 rpm for 5 min. Finally, a small volume (∼1 mL) of the supernatant was introduced into a vial for injection. High-Performance Liquid Chromatography. A HewlettPackard liquid chromatograph (Hewlett-Packard, Pittsburgh, PA) was used consisting of an automatic degasser system, quaternary pump, and a thermostated column compartment, all of the 1100 series; the automatic injector system and the variable-wavelength detector used were of the 1050 series. The chromatograph was coupled to a HP Vectra pentium MMX computer with a HPchemstation data acquisition and treatment program. The detection wavelength was set at 254 nm. The injection volume was 20 µL. A PLRP-S column (150 × 4.6 mm i.d.) from Polymer Laboratories Ltd. (Church Strutton, U.K), packed with polystyrene divinylbenzene beads (300 Å, 8 µm particle size) was used. The column dead time (2.00 min) and efficiency (18 888 plates/m) were determined by injecting phenol with 7:1 ACN-water as mobile phase. The chromatographic method was performed at a flow rate of 1 mL/min and 50 °C, using the following linear binary gradient: 20% B for 1 min, 20-32.2% B in 8.32 min, 32.2-100% B in 1.18 min, 100% B for 0.5 min and, to reequilibrate the column to starting conditions, 100-20% B in 2 and 3.5 min at 20% B. The mobile phases used were as follows: phase A, 0.1% TFA in Milli-Q water and phase B, 0.1% TFA in ACN. The ACN was filtered through a 0.45-µm nylon filter (MSI, Westboro, MA) before use. Calibration. Calibration was performed by the external standard method with aqueous solutions of SPI over the range 0.5-3.5 mg/mL. The SPI content of spiked bovine milks was determined by interpolating in the standard curve. All solutions were injected three times except in the studies of repeatability, where eight injections were made of the same SPI solution, and in the study of reproducibility where duplicate injections were made of each of eight individually prepared SPI spiked bovine milk solutions. The standard additions method was also applied. With this purpose, a sample solution consisting of a 4% defatted milk solution spiked with a known amount of SPI (0.51 mg/g) was prepared. From this sample solution, four equal volumes were taken to prepare four solutions to which known amounts of SPI over the range 0-2.65 mg/g were added. Each SPI spiked bovine milk solution was prepared in duplicate and injected three times into the chromatographic system. Data Treatment. In all the analyses, the integrated areas of certain peaks of SPI were added and these total areas were plotted

Figure 1. Chromatogram of aqueous solutions of (a) soya bean protein isolate, 2.93 mg/g; (b) defatted bovine milk solution, 40 mg/ g; and (c) bovine milk solution (40 mg of milk/g of solution) spiked with SPI (2.45 mg of SPI/g of solution).Chromatographic conditions: flow rate, 1 mL/min; gradient, 20% B for 1 min, 20-42% B in 15 min, 42-46% B in 4 min, and 46-100% B in 0.5 min; mobile phase A, 0.1% TFA in water; mobile phase B, 0.1% TFA in ACN; injection volume, 20 µL; detection at 254 nm. Peaks identification: 1-4, soya bean proteins; 1′, bovine milk.

against the injected concentrations of SPI. The linearity in this relationship was obtained by least-squares regression analysis carried out with a univariate linear calibration program.20 The linear model was validated by means of the analysis of residuals and the analysis of variance. RESULTS AND DISCUSSION Optimization of the Chromatographic Method. To achieve the separation of soya bean proteins from the components of bovine milk, a gradient previously used by our research team to separate soya bean proteins from bovine whey proteins18 was first tried. This linear binary gradient, consisting of six steps (20% B for 1 min, 20-42% B in 15 min, 42-46% B in 4 min, 46-100% B in 0.5 min, 100% B for 0.5 min, and 100-20% B in 0.5 min; mobile phase A, 0.1% TFA in water; mobile phase B, 0.1% TFA in ACN), was applied to the separation of soya bean proteins from an aqueous solution of SPI. Figure 1a shows the resulting chromatogram in which four peaks with retention times ranging from 2.6 to 7.5 min appeared. The same gradient was applied to a diluted (20) Blanco, M.; Boque´, R.; Cela, R.; Coello, J.; Maspoch, S.; Ortiz, M. C.; Riba, J.; Ruiz, F. X.; Sarabia, L. A.; Toma´s, X. Avances en Quimiometrı´a Pra´ ctica; Servicio de Publicacio´n e Intercambio Cientı´fico: Santiago de Compostela, Spain, 1994.

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Table 1. Calibration of Soya Bean Proteins by RP-HPLC Using the External Standard Method and SPI as Standarda,b working conc rangec (mg/g)

slope ( confidence interval

intercept ( confidence interval

standard error

r2 e

0.51-3.07(5)d 0.82-3.24(4) 0.50-3.60(5) 0.50-3.49(3) 0.49-3.55(3) 0.50-3.48(4) 0.69-3.14(3) 0.72-3.10(3) 0.70-3.43(3)

73 ( 1 71 ( 2 70 ( 0 69 ( 2 68 ( 3 69 ( 2 69 ( 0 69 ( 4 70 ( 3

0.13 ( 1.84 2.57 ( 4.33 1.96 ( 1.06 3.09 ( 3.74 3.92 ( 7.26 2.55 ( 3.53 0.00 ( 0.65 1.94 ( 8.48 1.76 ( 7.74

0.5971 0.8141 0.3699 0.2679 0.5587 0.7927 0.0321 0.5258 0.5165

1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

a Experimental conditions: flow rate, 1 mL/min; gradient, 20% B for 1 min, 20-32.2% B in 8.32 min, 32.2-100% B in 1.18 min, 100% B for 0.5 min, 100-20% B in 2 min, 20% B for 3.5 min; mobile phase A, 0.1% TFA in water; mobile phase B, 0.1% TFA in ACN; temperature, 50 °C; injection volume, 20 µL; detection at 254 nm. b Calibration lines obtained in nine different days within a three-week period. c Aqueous solutions of the soya bean protein isolate. d Number of points considered for the regression. e Squared correlation coefficient.

solution of defatted bovine milk (Figure 1b) and a diluted solution of defatted bovine milk spiked with SPI (Figure 1c). By comparing the three chromatograms of Figure 1, it can be observed that the first eluting peak of SPI overlaps with a peak of the bovine milk while three of the four SPI peaks were, however, completely separated from the bovine milk peaks. Thus, the integration of all four peaks of SPI for the quantitative analysis would result in an overestimation of the SPI concentration. To avoid this, all further work was carried out by measuring peak area of peaks 2-4 in Figure 1a and c. On the other side, since all SPI peaks eluted during the first 8 min, the gradient was shortened in order to decrease analysis time without impairing the chromatographic separation of soya bean peaks. Then the final gradient was as follows: 20% B for 1min, 20-32.2% B in 8.32 min, 32.2-100% B in 1.18 min, and 100% B for 0.5 min (see Experimental Section). Analytical Parameters of the Quantitative Chromatographic Method. In order to use the RP-HPLC method to quantitate soya bean proteins in SPI spiked bovine milks, the overall system was validated by evaluating its linearity, precision, accuracy, and robustness. Table 1 compiles the linear working concentration range and the values of the slope, intercept, standard error, and correlation coefficient for the equations of the linear calibration plots by using the external standard method. The plots were obtained in nine different days within a three-week period. Excellent linear correlations (r2 >0.999) were found between the total peak area measured for the last three soya bean protein peaks (peaks 2-4) and the concentration of SPI by least-squares regression analysis. The slope of the straight line showed good reproducibility interdays (RSD ∼2%), which accounted for a good robustness of the method. In all cases, the intercept did not differ significantly from zero (t-test, P < 0.05). In addition, detection limits, calculated from the calibration curve as the concentration of soya bean protein giving a signal equal to the intercept plus three times the standard error of the straight line, were ∼20 µg of soya bean protein/g of solution. Precision, expressed as RSD (%), of the RP-HPLC method was evaluated using the SPI and a SPI spiked defatted milk. Results obtained in this study are given in Table 2. Repeatability, in peak area and concentration, for eight injections of a 1.00 mg/g of solution of SPI, was better than 1%. Besides, a reproducibility interdays for SPI, calculated as the RSD in area for the injection of a SPI solution of 0.50 mg/g in four different days over a 10-day period, as good as 0.5% was obtained. In addition, Table 2 shows that the reproducibility, in 1816 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

Table 2. Precision, Expressed as Relative Standard Deviation (RSD, %), for the Results in Peak Area and in Concentration Corresponding to the Injection of a Soya Bean Protein Isolate and a SPI Spiked Defatted Milk Analyzed by RP-HPLCa

SPI repeatability (n ) 8)b reproducibility, in peak area, interdays (n ) 4)c SPI spiked defatted bovine milk reproducibility, in 1 day (n ) 8)d reproducibility, between 2 dayse

peak area

concn

0.99 0.53

0.97

1.79

1.82 6.24

a Experimental conditions as in Table 1. b Number of injections of a SPI solution of 1.00 mg/g. c Analysis performed in four different days over a 10-day period (each sample was injected three times); concentration of SPI (mean value ( SD), 0.50 ( 0.01 mg/g. d Analysis of eight individually prepared samples (each one injected two times); concentration of added SPI (mean value ( SD), 2.05 ( 0.01 mg/g; milk concentration in samples (mean value ( SD), 39.80 ( 0.48 mg/g. e Two samples were injected with 13 days between (each sample was injected three times); concentration of added SPI (mean value ( SD), 2.34 ( 0.16 mg/g; milk concentration in samples (mean value ( SD), 39.84 ( 0.23 mg/g.

peak area and concentration, for a SPI spiked milk (eight individually prepared samples, each one injected two times) was better than 2%, and the reproducibility interdays for a SPI spiked milk solution injected two different days within a 13-day period was 6.2%. Quantitative Analysis of Soya Bean Proteins in SPI Spiked Bovine Milks. Recoveries (%) obtained for SPI found in some bovine spiked milks are given in Table 3. The added SPI concentration in milks ranged from 0.55 to 3.1 mg/g. Mean recoveries obtained for all milks were ∼93%. The lowest recoveries were found for the defatted milks for which solutions containing 8 and 15% milk were analyzed. This fact is not surprising since a higher milk concentration naturally produces greater matrix effects. The highest recovery and a very low standard deviation in the determinations were found in the powdered milk. The whole milks (except for the whole milk in which the fat was replaced with vegetal fat) gave high average recoveries but also the highest standard deviations. From this study, it was possible to estimate the detection limit of soya bean proteins in bovine milks. For example, for the pasteurized defatted milk 1, the detection limit was estimated at 13 µg of soya bean protein/g of defatted milk.

Table 3. Soya Bean Recovery for Some SPI Spiked Bovine Milks Found by the RP-HPLC Method Using the Calibration by the External Standard Methoda concn of milk in the sample (mg/g)

concn of SPI added (mg/g)

SPI concn found by the RP-HPLC method (mg/g)

recovery (%)

mean recovery (% ( SD)

fresh whole

39.25 ( 0.21 39.86 ( 0.49

pasteurized semidefatted

39.76 ( 0.16

pasteurized defatted

39.93 ( 1.07

0.506 ( 0.019 2.556 ( 0.007 0.637 ( 0.012 3.041 ( 0.015 0.738 ( 0.002 2.936 ( 0.004 0.629 ( 0.006 1.425 ( 0.009 2.081 ( 0.005 2.921 ( 0.005 0.650 ( 0.005 2.633 ( 0.014 0.879 ( 0.001 1.563 ( 0.024 2.220 ( 0.017 2.646 ( 0.007 0.741 ( 0.004 1.711 ( 0.007 2.750 ( 0.017 0.657 ( 0.012 1.728 ( 0.004 2.821 ( 0.012

96.4 89.8 89.1 98.1 91.5 97.3 91.2 92.7 93.5 92.9 87.6 88.2 88.9 90.8 89.5 87.6 92.2 92.8 92.2 94.5 95.8 95.7

93.1 ( 4.7

pasteurized whole

0.563 2.729 0.715 3.099 0.806 3.018 0.689 1.537 2.227 3.143 0.743 2.984 0.988 1.722 2.481 3.019 0.804 1.845 2.982 0.695 1.804 2.974

bovine milk sample

79.33 ( 0.50 148.5 ( 1.2

pasteurized wholeb

39.97 ( 0.08

powdered defatted

3.70 ( 0.06c

93.6 ( 6.4 94.4 ( 4.1 92.6 ( 1.0

87.9 ( 0.5 89.2 ( 1.3

92.4 ( 0.3 95.3 ( 0.7

a Experimental conditions as in Table 1. b Liquid whole milk in which the animal fat has been replaced with vegetal fat. c Amount milk powder, equivalent to ∼40 mg/g of prepared milk.

Table 4. Investigation of the Existence of Fixed and Relative Bias in the Determination of SPI in Spiked Bovine Milks (P < 0.05)a

bovine milk

milk concn in solns spiked with SPI (mg/g)

eq of the best straight line that fits the exptl points: measd SPI concn ) f(true SPI concn)

pasteurized wholeb pasteurized defatted pasteurized defatted powdered defatted

39.97 ( 0.08 39.93 ( 1.07 148.48 ( 1.17 3.70 ( 0.06c

y ) 0.92((0.07)x + 0.00((0.14) y ) 0.93((0.02)x - 0.01((0.05) y ) 0.87((0.09)x + 0.04((0.19) y ) 0.96((0.03)x + 0.01((0.07)

existence of bias (size, %) fixed relative no, 0 no, 0 no, 0 no, 0

yes, -7.8 yes, -6.5 yes, -12.8 yes, -3.9

a Analysis performed by making use of the statistical technique of regression analysis, in which the best straight line that fits the experimental points corresponding to SPI measured concentration as a function of the SPI true concentration in the spiked milks was obtained. The intercept and the slope of the equation of the least-squares method accounted for the fixed and relative bias, respectively. b Whole milk in which the animal fat has been replaced with vegetal fat. c Amount of powdered milk, equivalent to ∼40 mg/g of prepared milk.

The fixed and relative bias of the method was studied21 for some representative samples spiked with SPI: a pasteurized whole milk with vegetable fat included in its formulation, a pasteurized deffated milk (at two levels of milk concentration in the solution analyzed), and a powdered defatted milk. The results of this study, given in Table 4, suggested the existence of a negative relative bias, confirming that the method suffered from some matrix effects. As it could be expected, these matrix effects were more pronounced when the milk concentration in the analyzed solutions was increased (see results for the pasteurized defatted milk). According to these results, the method of standard additions was investigated for the determination of SPI in a spiked pasteurized defatted bovine milk. The results of this study are given in Table 5 and showed that with this calibration method an average recovery of 106% could be obtained. By comparing the method of standard additions and the method of the calibration by the external standard, it was obvious that the errors are approximately of the same size, since both are ∼6% for the recoveries of soya (21) Caulcutt, R.; Boddy, R. Statistics for Analytical Chemists; Chapman and Hall: London, 1983; pp 47-60.

Table 5. Application of the Method of Standard Additions for the Determination of Soya Bean Proteins in a Pasteurized Defatted Bovine Milk Spiked with SPI by RP-HPLCa-c

slope ( confidence range (P ) 0.05) intercept ( confidence range (P ) 0.05) standard error of the straight line r2 d SPI concn found (mg/g) recovery (%) mean recovery ( SD (%)

expt 1

expt 2

67.5 ( 4.0

67.2 ( 1.7

36.6 ( 6.6

35.5 ( 2.8

1.924 1.000 0.542 105.8

0.815 1.000 0.543 106 105.9 ( 0.1

a Experimental conditions as in Table 1. b Milk and SPI concns in solutions were, respectively, 40.6 and 0.51 mg/g. c Four different solutions were prepared in which different and known concentrations of SPI (ranging from 0 to 2.65 mg/g) were added. d Squared correlation coefficient.

bean proteins in SPI spiked bovine milks. The external standard method underestimated the amount of soya bean proteins in Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

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bovine milks, but when the method of the standard additions was used for calibration, this concentration was overestimated. Since the error obtained by both calibration methods is similar and the method of the external standard is much less time-consuming, this last method is recommended in this work for calibration with a soya bean protein isolate as external standard in order to determine soya bean proteins in bovine milks. CONCLUSIONS To control the presence of soya bean proteins in bovine milk, a RP-HPLC method was designed for their detection and quantitation in bovine milks spiked with a soya bean protein isolate. The sample preparation was fast and the analysis time was only 11 min, by using the binary linear gradient ACN-water-0.1% TFA. The robustness, precision, reproducibility, accuracy, and detection

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limit of the quantitative chromatographic method were evaluated and considered good enough for the method to be valid for routine quantitative analysis of soybean proteins in bovine milks. The proposed method allowed the detection of concentrations of soya bean protein as low as 13 µg of protein/g of bovine milk. ACKNOWLEDGMENT The authors thank the Comunidad Auto´noma de Madrid (Spain) for project 06G/047/96.

Received for review July 15, 1999. Accepted January 18, 2000. AC990776M