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1243
ANALYST, SEPTEMBER 1990, VOL. 115
Enzymic Determination of Ammonia in Food by Flow Injection
Published on 01 January 1990. Downloaded by University of Michigan Library on 23/10/2014 05:44:28.
Lucia Canale-Gutierrez, Angel Maquieira and Rosa Puchades Department of Chemistry, Polytechnic University of Valencia, 46022-Valencia, Spain
Ammonia in food samples was determined by its reaction in an immobilised enzyme reactor containing glutamate dehydrogenase (GIDH) in a flow injection system, by measuring the decrease in the absorbance of ultraviolet radiation by reduced nicotinamide adenine dinucleotide (NADH). There was a linear relationship ( r = 0.9995) between peak height and ammonia concentration over the range 0.05-0.6 mM. The detection limit was 0.005 mM for an injection volume of 19 PI. Sampling frequency was 60 h-1 and the precision was better than 1.09% for 11 successive assays. The interference effect of urea and ascorbic acid at concentrations greater than 100 m g per 100 g of product should be taken into account. The interference caused by glycine, creatinine and amino acids is negligible. Onlya 20% loss in the activity of the GlDH column was observed after 500 determinations during a 3-month period. Keywords: Ammonia; food; enzyme; flow injection
The presence of high levels of ammonia in food such as meat or fish, is an indication of the freshness of a product.1-4 Ammonia has also been found to have a role in the organoleptic properties of foods.5-7 The level of NH4+ in meat derivatives depends on the thermal treatment, additives, temperature and storage time.8 The determination of ammonia in foods poses an analytical problem which has to be solved because of the importance of this determination in food technology. The classical methods for the determination of ammonia are based on modifications of the Kjeldahl method and yield values that are higher than expected, the reason being the hydrolysis of the amino groups of the proteins.9 Other more simple methods, such as the use of ion-selective electrodes, determine the volatile amines together with NH3, thus specificity is lost.1O The use of dissolved enzymes is a valid alternative as a high specificity is obtained, however, it results in a high cost per analysis. The advantages of immobilised enzymes (higher stability, longer life span, lower analytical cost and higher selectivity) over dissolved enzymes make them suitable for routine analysis.11 The coupling of an immobilised enzyme reactor (IMER) with flow injection (FI) provides increased rapidity, precision and convenience compared with classical enzymic procedures12 and decreases the cost per assay. A number of immobilised enzymes have been coupled with an FI methodology for the analysis of foods.13 16 The enzyme glutamate dchydrogenase (GIDH) catalyses the following reaction:
Sigma (St. Louis, MO, USA). The pore size and particle size of the CPG were 242A and 100-200 mesh, respectively. Reduced nicotinamide adenine dinucleotide, grade I, 100% ; L-glutamate dehydrogenase E.C. 1.4.1.3, ex-bovine liver, 3000 U; and adenosine 5'-diphosphate disodium salt were purchased from Boehringer Mannheim (Barcelona, Spain). Glutaraldehyde solution, 25% , for electron microscopy, was obtained from Taab Laboratory Equipment (Aldermaston, Berkshire, UK). All other chemicals were commercially available and of analytical-reagent grade. Stock solutions of 0.01 and 0.1 M NH4CI were prepared by dissolving the appropriate, weighed amount of solid in distilled, de-ionised water. Working solutions were prepared just prior to use by appropriate dilution of the stock solutions in 0.1 M phosphate buffer of p H 8.0 with 1 mM ethylenediaminetetraacetic acid (EDTA); N A D H was prepared each week in 1% m/v sodium hydrogen carbonate. All solutions were prepared with distilled, de-ionised water; water quality was controlled by ion chromatography (Dionex, CA, USA).
Procedure
Enzyme immobilisation The activated glass was prepared as follows: the porous glass was washed in boiling 5% v/v nitric acid, silanised with 3-aminopropyltriethoxysilaneto give the alkylamino glass, and treated with 2.5% v/v glutaraldehyde in an aqueous system.17 It was then added to the GlDH dissolved in 3 ml of GlDH 0.1 M phosphate buffer (pH 7.0). The solution was kept at 4 "C 2-Oxoglutarate + NH4+ + N A D H for 2.5 h, and then washed with cold distilled water and cold L-glutamic acid + NAD+ + H 2 0 phosphate buffer to remove any unlinked enzyme. The enzyme-coated glass beads were packed into a 7.5 cm long where NAD+ = nicotinamide adenine dinucleotide and glass column (1.2 mm i.d.), capped with polyNADH = the reduced form of NAD. The NAD+ generated is tetrafluoroethylene (PTFE) end fittings and stored, filled with monitored in a flow-through spectrophotometric detector by phosphate buffer, at 4 "C. The void volume of the enzyme measuring the decrease in the absorbance of the NADH. reactor was 8 mm3. The purpose of this research is to develop an FI method for The protein concentration of the enzyme was determined by the determination of ammonia in foods, using GlDH immobia spectrophotometric method (Bradford protein assay, Biolised on controlled porosity glass (CPG) and a flow-through Rad Labs., Richmond, CA, USA) both before and after the spectrophotometric detector, thus combining the high speciimmobilisation reaction. The coupling yield was then calcuficity of an enzymic method with the advantages of an FI lated as the percentage disappearance of the amount of method, i.e., rapidity, precision, simplicity, cost saving and a protein initially added to the reaction mixture. Immobilisation minimum of sample treatment. resulted in the attachment of 95.4% of the protein initially present. The activity of the immobilised enzyme was deterExperimental mined according to Korenbrot et al. 18 The specific activity per Reagents mg of immobilised enzyme was 12% of that of the soluble 3-Aminopropyltriethoxysilane, 99% ; L-amino acids, L - A A ~ ~ ;enzyme and the specific acitivity of immobilised enzyme present in the reactor was 130 U. and controlled pore glass, CPG 240-200, were obtained from
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1244
ANALYST, SEPTEMBER 1990, VOL. 115
G.++r.&+-h
Sample
P
W
Published on 01 January 1990. Downloaded by University of Michigan Library on 23/10/2014 05:44:28.
Fig. 1. Schematic diagram of the flow system for the determination of ammonia: P, pump; V, injection valve; IMER, immobilised enzymic reactor; D, detector; and W, waste
Flow system A schematic diagram of the flow system is shown in Fig. 1. A four-channel peristaltic pump, Minipuls I11 (Gilson, Worthington, O H , USA), a Rheodyne Model 5041 sample injection valve (Rheodyne, Cotati, CA, USA) and PTFE tube (0.5 mm i.d.) were used for this work. The carrier, 0.1 M phosphate buffer of pH 8.0, containing 1 mM EDTA, 0.2 mM NADH and 0.01 M oxoglutarate was pumped at a flow-rate of 1.0 ml min-1. The 19-pl sample was directly aspirated into the system and passed through the immobilised GlDH column. The decrease in the absorbance intensity caused by the disappearance of NADH was measured at 340 nm with a Spectronic 2000 ultraviolet - visible spectrophotometer (Bausch & Lomb, Rochester, NY, USA), equipped with an 18-pl flow cell (Hellma, Jamaica, NY, USA; 178.12 QS) and a Hewlett-Packard (Avondale, PA, USA) H P 3392A integrator. The flow system was operated at room temperature (20 "C). The peak height (arbitrary units) was used for the quantification of the ammonia.
Sample preparation Food samples (cheese and baked ham) were extracted using a modification of the method of Parris and Foglia.19 The modification consisted of sonicating for 10 min after the aqueous extraction step in order to disperse any clots and break any cells present, thus liberating their contents. Study of interferences Independent assays were performed with different aminated substances that were present in the analysed foods. Different concentrations of these interferents were added to 5 p.p.m. NH4+ standard solutions and these were then injected into the FI system both with and without the enzymic reactor, to assess if the interference was due to the enzymic reaction or to the inherent characteristics of the interfering agent at the monitoring wavelength. The interfering substances tested were the following: a mixture of glycine, phenylalanine, arginine, proline, glutamic acid, serine and asparagine (the major amino acids existing in ham) each at a concentration of 5 mg per 100 g of product; asparagine and glycine at concentrations from 10 to 100 mg per 100 g of product; urea and creatinine from 10 to 250 mg per 100 g of product; and ascorbic acid from 10 to 200 mg per 100 g of product.
Results and Discussion The conditions for the determination of ammonia were optimised by studying the effect of various parameters such as pH, the composition and concentration of the buffer, NADH and oxoglutarate concentration, alternative use of adenosine 5' -diphosphate activator and FI variables. Standard solutions of 100 p.p.m. NH4+ (the normal value found in meatsg) were used in all of the optimisation stages. The pH of the carrier must be carefully chosen to ensure maximum sensitivity and stability. At pH 9.0 the destruction of NAD+ was rapid20 and below pH 7.0 NADH was unstable.21 The effect of pH between 8.0 and 9.0 on ammonia measurements was examined by adding 0.1 M KOH to the
7.8
8.6
8.2
9.0
PH
Effect of pH on the response to 100 p.p.m. of NH4+
Fig. 2.
0
0.1
0.2
0.4
NADH/mM
Fig. 3. Effect of NADH concentration on the response to 100 p.p.m. of NH4+
.-$ 0.6 C 3
L.
!F
. F
0.4
m
r i .-0, a,
0.2 Y
0
Q a,
0
10
20
30
40
Oxog Iuta rate/m M
Fig. 4. Effect of oxoglutarate concentration on the response to 100 p.p.m. of NH4+
carrier (Fig. 2). A gradual decrease in the analytical signal was observed with increasing pH. A pH of 8.0 was, therefore, used for subsequent experiments to ensure greater sensitivity and NADH stability. The study of buffer composition was performed with the following two buffers: phosphate22 and triethanolamine chlorohydrateg at pH 8.0. The analytical signal was higher when phosphate buffer was used. The 0.1 M potassium phosphate buffer pH 8.0 was retained for all experiments. The effect of NADH concentration on the response of the system is shown in Fig. 3; the signal increased with NADH concentration, hence, 0.2 mM NADH was subsequently chosen; at higher concentrations a decrease in the sensitivity of the method was observed. This conclusion is in agreement with that of Tabata et aZ.,23 who used the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) . The oxoglutarate concentration has no influence on the results
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1245
ANALYST, SEPTEMBER 1990, VOL. 115 Table 1. Effect of the interfering agents on the determination of ammonia
P
5 0.9
1
Published on 01 January 1990. Downloaded by University of Michigan Library on 23/10/2014 05:44:28.
Intcrfcrcnce,%
Interfering agent Amino acids (various) . . Asparagine . . . . . .
. . . . . .
Glycinc Urea
. . . . . . . .
Creatinine
. . . . . .
. . . .
Ascorbicacid
Concentration of interfering agent@ per 100 g o f sample 5 (each) 7 10 60 100 10 60 100 10 60 100 250 10 60 100 250 10 100 200
With enzymic reactor 0.2 0.2 0.2 1.2 0.9 -0.6 1.9 3.6 0.3 1.2 0.2 3.0 0.8 0.5 0.5 0.3 1.o 4.8 5.3
Without enzymic reactor 0.9 -0.9 -1.0 -2.4 -3.1 -0.3 -0.6 -0.8 0.7 0.0 6.8 8.4 0.0 1.6 2.8 9.8 2.7 6.6 9.1
Table 2. Recoveries obtained with the modified extraction method in ham samples
g
2 0.3
: I
Y
a
01 0.8
Total found 5.028 4.861
Originally prcsent 4.727 4.727
Amount rccovered 0.301 0.134
Fig. 6.
1
2.0
.-E
Effect of flow-rate on analytical signal
1.0
C 3
L-
E 0.8
4.-
+?
2 0.6 .-0-l 1 a, Y
m 0.4
a 0.2 I
1
0
Recovery, % 90.96 100.40
I
1.6
Flow-rate/ml min-'
Concentration of NH4+/mM NHI+ addcd/mM 0.333 0.133
I
1.2
Fig. 7.
0.4
1
0.8 NH4+/mM
I
1.2
Calibration graph for ammonia
1.o c v) I-
c
System Performance
3
Lc .-
0.8
+? c
r : . 0.6 1 Y (0
a 0.4 I 0
I
I
I
50
100
150
200
Injection vo Iu me/pt
Fig. 5. NHI+
Effect of injection volume on the response to 100 p.p.m. of
when concentrations greater than 0.01 M (Fig. 4) are used, therefore, this value was chosen as optimum. When adenosine 5'-diphosphate (ADP), disodium salt (from 500 to 4000 p ~ ) , was added to the carrier stream as an enzymic activator as described by Bruce et a1.,'4 no significant difference in the analytical signal was observed. Under these conditions (0.1 M phosphate buffer of pH 8.0, 1 mM EDTA, 0.2 mM NADH and 0.01 M oxoglutaratc) sample injection volumes from 19 to 143.4 pl were investigated. The peak height increased markedly with injection volume (Fig. 5). Therefore, a 19-pl sample injection volume was selected for higher sensitivity and sampling frequency and was used in all subsequent runs. The flow parameters were optimised by varying the flow-rate of the carrier. Increasing the flow-rate from 0.85 to 2.50 ml min-1 does not influence the signal (Fig. 6). The choice of a flow-rate of 1.0 ml min-1 was a compromise between the sample output rate and the elimination of over-pressures in the system.
Standard solutions of ammonia were prepared in a 0.1 M phosphate buffer of pH 8.0 and 1 mM EDTA, and injected in triplicate. Under the working conditions previously stated, the calibration graph (Fig. 7) was linear between 0.05 and 0.60 mM ammonia, with a correlation coefficient . ( Y ) of 0.9995. The reproducibility was established by repeated determinations using 0.25 mM NH4+. The relative standard deviation of the peak heights was 1.09% ( n = 11) with a sampling frequency of 60 h-1. When the enzymic reactor was stored at 4"C, a 20% loss of the initial activity occurred after 500 determinations, over a 3-month period. Interferences The effect of the interferents on the results for the determination of ammonia is shown in Table 1. The interferences observed are spectral in most instances and they can be reduced to a minimum by the use of the enzymic reactor. The interference found is less than 1% in nearly every instance and, therefore, negligible as this is similar to the precision of the method. Only the interference caused by urea (at concentrations greater than 100 mg per 100 g of sample) and ascorbic acid (from 100 to 200 mg per 100 g of sample) should be taken into account. The urea interference could be because of the instability of NAD+ at high p H values. In addition, the presence of urea drives the equilibrium of the NADH/NAD+ reaction to the right. The interference caused by ascorbic acid is probably due to its reducing capacity, which causes a non-specific consumption of NADH. Amino acids interfere at concentrations greater than 100 mg per 100 g of sample, which do not occur in normal samples.
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1246
ANALYST, SEPTEMBER 1990, VOL. I15
Application to Food Samples
2.
In order to apply this method to the determination of ammonia in cheese and baked ham, the extracts of the samples were diluted, taking into account the average concentration of ammonia in meats9 and cheeses,6 as well as the linear range of the method. The matrix effect can be considered negligible in the analysed samples, as the differences between the slopes of the calibration graphs run with internal and extcrnal references were very small, being 0.19 and 0.21% for ham and cheese, respectively. This, therefore, avoids the necessity to work with an internal reference, thus making the analysis easier. The recoveries obtained with samples of ham using the modified Parris and Foglia extraction method (Table 2) range between ca. 91 and 100%, which is acceptable for these types of samples that contain low levels of ammonia.
3. 4.
Conclusions The determination of ammonia in foods can be automated by the use of F1; 60 analyses per hour can be carried out. Linearity, recovery and precision parameters obtained confirm the reliability of the results achieved with this technique. Substances that, according to their labile amino groups, potentially liberate ammonia, such as urea, creatinine and amino acids, d o not exert any positive influence. The specificity of the enzymic method facilitates the determination of ammonia, in the presence of other nitrogenous substances common in foods, without previous distillation. Non-specific reducing agents, such as ascorbic acid, do not exert an effect as they do not consume NADH owing to the reduced analysis times involved in FI systems. As a consequence, the use of NADPH25 is not necessary. The FI - immobilised enzyme combination has proved to be excellent for the determination of ammonia in foods, the use of enzyme activators being unnecessary. The authors gratefully acknowledge financial support from the Comision lnterministerial de Ciencia y Technologia (CICYT); Project 285/86.
References 1.
Voets, J. P., Br. Poult. Sci., 1973, 14, 17
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 1s. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Koval’chuck, G. K., Patsera, A . D., and Glcizcr, I. G., Rybn. Kho7. (MOSCOW),1969, 45, 85. Waters. M. E., J . Milk Food Technol., 1970, 33, 319. Cantoni, C.. Bianchi, M. A . , and Renon, P., Arch. Vef. Ital., 1969, 20, 355. Brooks, G. M., Diss. Abstr. Int., B , 1973, 34, 2079. Kapoor, C. M., Singh, J., and Rao. R. V., Indian J . Anim. Re.\.. 1972, 6, 71. Bodini, L. F., and Guicciardi, M., Lutte, 1969, 43, 711. Sorman, L., and Stoselova. E.. Zbornik Pruc. Chernikotechnol. Fak. SVST. 1972, 299. Bergmeyer, H . U., and Beutler, H., in Bcrgmcycr. H. U., Editor, “Methods of Enzymatic Analysis,” Vcrlag Chcmie, Wcinhcim, 1981, p. 454. Mcycrhoff, M. E., and Robins, R . H., A n d Clzem., 1980. 52, 2383. Osborn, J. A., and Yacynych, A. M., Anal. Chim. Acta, 1986, 183. 287. Valcarcel, M . , and Luque de Castro, M. D.. “Flow-injection Analysis: Principles and Applications,” Ellis Horwood, Chichcstcr, 1987. Masoom, M., and Townshend, A., Anal. Chim. Acta, 1985, 171, 185. Yao, T., and Wasa, T., Anal. Chim. Acta. 1985, 175, 301. Ruz, J . , Lazaro, F., and Luque de Castro, M. D., .I. Autom. Chem., 1988, 10, 15. Olsson, B., Salbou. B., and Johansson, G., Anal. Chirn. A m , 1986, 179,203. Masoom. M.. and Townshend, A . , Anal. Chim. Actu, 1984, 166, 111. Korenbrot, J. I., Perry. R., and Copenhagen, D. R.. Anal. Biocliern., 1987, 161, 187. Parris, N . , and Foglia, T. A., J. Agric. Food Chem., 1983, 31, 887. Masoom, M . , and Townshend, A , , A n d . Chim. Actu, 1986, 185, 49. Guilbault, G. G., “Analytical Uses of Immobilized Enzyme\,” Marcel Dekker, New York. 1984. Puchades. R., Lemieux, L, and Simard, R. E . , J . Food Sci., 1989, 54, 423. Tabata, M., Kido, T., Totani, M., and Murachi, T., Anal. Biochem., 1983, 134. 44. Brucc, A . W . , Leindecker. C. M . , and Freier, E. F., Clin. Chem., 1978, 24. 782. Humphries, B. A., Melnychuk, M., Donegan, E . J . , and Snee, R., Clin. Chem., 1979, 25, 26.
Paper 0100601G Received February 9th, 1990 Accepted May I7th, 1990
1243
ANALYST, SEPTEMBER 1990, VOL. 115
Enzymic Determination of Ammonia in Food by Flow Injection
Published on 01 January 1990. Downloaded by University of Michigan Library on 23/10/2014 05:44:28.
Lucia Canale-Gutierrez, Angel Maquieira and Rosa Puchades Department of Chemistry, Polytechnic University of Valencia, 46022-Valencia, Spain
Ammonia in food samples was determined by its reaction in an immobilised enzyme reactor containing glutamate dehydrogenase (GIDH) in a flow injection system, by measuring the decrease in the absorbance of ultraviolet radiation by reduced nicotinamide adenine dinucleotide (NADH). There was a linear relationship ( r = 0.9995) between peak height and ammonia concentration over the range 0.05-0.6 mM. The detection limit was 0.005 mM for an injection volume of 19 PI. Sampling frequency was 60 h-1 and the precision was better than 1.09% for 11 successive assays. The interference effect of urea and ascorbic acid at concentrations greater than 100 m g per 100 g of product should be taken into account. The interference caused by glycine, creatinine and amino acids is negligible. Onlya 20% loss in the activity of the GlDH column was observed after 500 determinations during a 3-month period. Keywords: Ammonia; food; enzyme; flow injection
The presence of high levels of ammonia in food such as meat or fish, is an indication of the freshness of a product.1-4 Ammonia has also been found to have a role in the organoleptic properties of foods.5-7 The level of NH4+ in meat derivatives depends on the thermal treatment, additives, temperature and storage time.8 The determination of ammonia in foods poses an analytical problem which has to be solved because of the importance of this determination in food technology. The classical methods for the determination of ammonia are based on modifications of the Kjeldahl method and yield values that are higher than expected, the reason being the hydrolysis of the amino groups of the proteins.9 Other more simple methods, such as the use of ion-selective electrodes, determine the volatile amines together with NH3, thus specificity is lost.1O The use of dissolved enzymes is a valid alternative as a high specificity is obtained, however, it results in a high cost per analysis. The advantages of immobilised enzymes (higher stability, longer life span, lower analytical cost and higher selectivity) over dissolved enzymes make them suitable for routine analysis.11 The coupling of an immobilised enzyme reactor (IMER) with flow injection (FI) provides increased rapidity, precision and convenience compared with classical enzymic procedures12 and decreases the cost per assay. A number of immobilised enzymes have been coupled with an FI methodology for the analysis of foods.13 16 The enzyme glutamate dchydrogenase (GIDH) catalyses the following reaction:
Sigma (St. Louis, MO, USA). The pore size and particle size of the CPG were 242A and 100-200 mesh, respectively. Reduced nicotinamide adenine dinucleotide, grade I, 100% ; L-glutamate dehydrogenase E.C. 1.4.1.3, ex-bovine liver, 3000 U; and adenosine 5'-diphosphate disodium salt were purchased from Boehringer Mannheim (Barcelona, Spain). Glutaraldehyde solution, 25% , for electron microscopy, was obtained from Taab Laboratory Equipment (Aldermaston, Berkshire, UK). All other chemicals were commercially available and of analytical-reagent grade. Stock solutions of 0.01 and 0.1 M NH4CI were prepared by dissolving the appropriate, weighed amount of solid in distilled, de-ionised water. Working solutions were prepared just prior to use by appropriate dilution of the stock solutions in 0.1 M phosphate buffer of p H 8.0 with 1 mM ethylenediaminetetraacetic acid (EDTA); N A D H was prepared each week in 1% m/v sodium hydrogen carbonate. All solutions were prepared with distilled, de-ionised water; water quality was controlled by ion chromatography (Dionex, CA, USA).
Procedure
Enzyme immobilisation The activated glass was prepared as follows: the porous glass was washed in boiling 5% v/v nitric acid, silanised with 3-aminopropyltriethoxysilaneto give the alkylamino glass, and treated with 2.5% v/v glutaraldehyde in an aqueous system.17 It was then added to the GlDH dissolved in 3 ml of GlDH 0.1 M phosphate buffer (pH 7.0). The solution was kept at 4 "C 2-Oxoglutarate + NH4+ + N A D H for 2.5 h, and then washed with cold distilled water and cold L-glutamic acid + NAD+ + H 2 0 phosphate buffer to remove any unlinked enzyme. The enzyme-coated glass beads were packed into a 7.5 cm long where NAD+ = nicotinamide adenine dinucleotide and glass column (1.2 mm i.d.), capped with polyNADH = the reduced form of NAD. The NAD+ generated is tetrafluoroethylene (PTFE) end fittings and stored, filled with monitored in a flow-through spectrophotometric detector by phosphate buffer, at 4 "C. The void volume of the enzyme measuring the decrease in the absorbance of the NADH. reactor was 8 mm3. The purpose of this research is to develop an FI method for The protein concentration of the enzyme was determined by the determination of ammonia in foods, using GlDH immobia spectrophotometric method (Bradford protein assay, Biolised on controlled porosity glass (CPG) and a flow-through Rad Labs., Richmond, CA, USA) both before and after the spectrophotometric detector, thus combining the high speciimmobilisation reaction. The coupling yield was then calcuficity of an enzymic method with the advantages of an FI lated as the percentage disappearance of the amount of method, i.e., rapidity, precision, simplicity, cost saving and a protein initially added to the reaction mixture. Immobilisation minimum of sample treatment. resulted in the attachment of 95.4% of the protein initially present. The activity of the immobilised enzyme was deterExperimental mined according to Korenbrot et al. 18 The specific activity per Reagents mg of immobilised enzyme was 12% of that of the soluble 3-Aminopropyltriethoxysilane, 99% ; L-amino acids, L - A A ~ ~ ;enzyme and the specific acitivity of immobilised enzyme present in the reactor was 130 U. and controlled pore glass, CPG 240-200, were obtained from
View Article Online
1244
ANALYST, SEPTEMBER 1990, VOL. 115
G.++r.&+-h
Sample
P
W
Published on 01 January 1990. Downloaded by University of Michigan Library on 23/10/2014 05:44:28.
Fig. 1. Schematic diagram of the flow system for the determination of ammonia: P, pump; V, injection valve; IMER, immobilised enzymic reactor; D, detector; and W, waste
Flow system A schematic diagram of the flow system is shown in Fig. 1. A four-channel peristaltic pump, Minipuls I11 (Gilson, Worthington, O H , USA), a Rheodyne Model 5041 sample injection valve (Rheodyne, Cotati, CA, USA) and PTFE tube (0.5 mm i.d.) were used for this work. The carrier, 0.1 M phosphate buffer of pH 8.0, containing 1 mM EDTA, 0.2 mM NADH and 0.01 M oxoglutarate was pumped at a flow-rate of 1.0 ml min-1. The 19-pl sample was directly aspirated into the system and passed through the immobilised GlDH column. The decrease in the absorbance intensity caused by the disappearance of NADH was measured at 340 nm with a Spectronic 2000 ultraviolet - visible spectrophotometer (Bausch & Lomb, Rochester, NY, USA), equipped with an 18-pl flow cell (Hellma, Jamaica, NY, USA; 178.12 QS) and a Hewlett-Packard (Avondale, PA, USA) H P 3392A integrator. The flow system was operated at room temperature (20 "C). The peak height (arbitrary units) was used for the quantification of the ammonia.
Sample preparation Food samples (cheese and baked ham) were extracted using a modification of the method of Parris and Foglia.19 The modification consisted of sonicating for 10 min after the aqueous extraction step in order to disperse any clots and break any cells present, thus liberating their contents. Study of interferences Independent assays were performed with different aminated substances that were present in the analysed foods. Different concentrations of these interferents were added to 5 p.p.m. NH4+ standard solutions and these were then injected into the FI system both with and without the enzymic reactor, to assess if the interference was due to the enzymic reaction or to the inherent characteristics of the interfering agent at the monitoring wavelength. The interfering substances tested were the following: a mixture of glycine, phenylalanine, arginine, proline, glutamic acid, serine and asparagine (the major amino acids existing in ham) each at a concentration of 5 mg per 100 g of product; asparagine and glycine at concentrations from 10 to 100 mg per 100 g of product; urea and creatinine from 10 to 250 mg per 100 g of product; and ascorbic acid from 10 to 200 mg per 100 g of product.
Results and Discussion The conditions for the determination of ammonia were optimised by studying the effect of various parameters such as pH, the composition and concentration of the buffer, NADH and oxoglutarate concentration, alternative use of adenosine 5' -diphosphate activator and FI variables. Standard solutions of 100 p.p.m. NH4+ (the normal value found in meatsg) were used in all of the optimisation stages. The pH of the carrier must be carefully chosen to ensure maximum sensitivity and stability. At pH 9.0 the destruction of NAD+ was rapid20 and below pH 7.0 NADH was unstable.21 The effect of pH between 8.0 and 9.0 on ammonia measurements was examined by adding 0.1 M KOH to the
7.8
8.6
8.2
9.0
PH
Effect of pH on the response to 100 p.p.m. of NH4+
Fig. 2.
0
0.1
0.2
0.4
NADH/mM
Fig. 3. Effect of NADH concentration on the response to 100 p.p.m. of NH4+
.-$ 0.6 C 3
L.
!F
. F
0.4
m
r i .-0, a,
0.2 Y
0
Q a,
0
10
20
30
40
Oxog Iuta rate/m M
Fig. 4. Effect of oxoglutarate concentration on the response to 100 p.p.m. of NH4+
carrier (Fig. 2). A gradual decrease in the analytical signal was observed with increasing pH. A pH of 8.0 was, therefore, used for subsequent experiments to ensure greater sensitivity and NADH stability. The study of buffer composition was performed with the following two buffers: phosphate22 and triethanolamine chlorohydrateg at pH 8.0. The analytical signal was higher when phosphate buffer was used. The 0.1 M potassium phosphate buffer pH 8.0 was retained for all experiments. The effect of NADH concentration on the response of the system is shown in Fig. 3; the signal increased with NADH concentration, hence, 0.2 mM NADH was subsequently chosen; at higher concentrations a decrease in the sensitivity of the method was observed. This conclusion is in agreement with that of Tabata et aZ.,23 who used the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) . The oxoglutarate concentration has no influence on the results
View Article Online
1245
ANALYST, SEPTEMBER 1990, VOL. 115 Table 1. Effect of the interfering agents on the determination of ammonia
P
5 0.9
1
Published on 01 January 1990. Downloaded by University of Michigan Library on 23/10/2014 05:44:28.
Intcrfcrcnce,%
Interfering agent Amino acids (various) . . Asparagine . . . . . .
. . . . . .
Glycinc Urea
. . . . . . . .
Creatinine
. . . . . .
. . . .
Ascorbicacid
Concentration of interfering agent@ per 100 g o f sample 5 (each) 7 10 60 100 10 60 100 10 60 100 250 10 60 100 250 10 100 200
With enzymic reactor 0.2 0.2 0.2 1.2 0.9 -0.6 1.9 3.6 0.3 1.2 0.2 3.0 0.8 0.5 0.5 0.3 1.o 4.8 5.3
Without enzymic reactor 0.9 -0.9 -1.0 -2.4 -3.1 -0.3 -0.6 -0.8 0.7 0.0 6.8 8.4 0.0 1.6 2.8 9.8 2.7 6.6 9.1
Table 2. Recoveries obtained with the modified extraction method in ham samples
g
2 0.3
: I
Y
a
01 0.8
Total found 5.028 4.861
Originally prcsent 4.727 4.727
Amount rccovered 0.301 0.134
Fig. 6.
1
2.0
.-E
Effect of flow-rate on analytical signal
1.0
C 3
L-
E 0.8
4.-
+?
2 0.6 .-0-l 1 a, Y
m 0.4
a 0.2 I
1
0
Recovery, % 90.96 100.40
I
1.6
Flow-rate/ml min-'
Concentration of NH4+/mM NHI+ addcd/mM 0.333 0.133
I
1.2
Fig. 7.
0.4
1
0.8 NH4+/mM
I
1.2
Calibration graph for ammonia
1.o c v) I-
c
System Performance
3
Lc .-
0.8
+? c
r : . 0.6 1 Y (0
a 0.4 I 0
I
I
I
50
100
150
200
Injection vo Iu me/pt
Fig. 5. NHI+
Effect of injection volume on the response to 100 p.p.m. of
when concentrations greater than 0.01 M (Fig. 4) are used, therefore, this value was chosen as optimum. When adenosine 5'-diphosphate (ADP), disodium salt (from 500 to 4000 p ~ ) , was added to the carrier stream as an enzymic activator as described by Bruce et a1.,'4 no significant difference in the analytical signal was observed. Under these conditions (0.1 M phosphate buffer of pH 8.0, 1 mM EDTA, 0.2 mM NADH and 0.01 M oxoglutaratc) sample injection volumes from 19 to 143.4 pl were investigated. The peak height increased markedly with injection volume (Fig. 5). Therefore, a 19-pl sample injection volume was selected for higher sensitivity and sampling frequency and was used in all subsequent runs. The flow parameters were optimised by varying the flow-rate of the carrier. Increasing the flow-rate from 0.85 to 2.50 ml min-1 does not influence the signal (Fig. 6). The choice of a flow-rate of 1.0 ml min-1 was a compromise between the sample output rate and the elimination of over-pressures in the system.
Standard solutions of ammonia were prepared in a 0.1 M phosphate buffer of pH 8.0 and 1 mM EDTA, and injected in triplicate. Under the working conditions previously stated, the calibration graph (Fig. 7) was linear between 0.05 and 0.60 mM ammonia, with a correlation coefficient . ( Y ) of 0.9995. The reproducibility was established by repeated determinations using 0.25 mM NH4+. The relative standard deviation of the peak heights was 1.09% ( n = 11) with a sampling frequency of 60 h-1. When the enzymic reactor was stored at 4"C, a 20% loss of the initial activity occurred after 500 determinations, over a 3-month period. Interferences The effect of the interferents on the results for the determination of ammonia is shown in Table 1. The interferences observed are spectral in most instances and they can be reduced to a minimum by the use of the enzymic reactor. The interference found is less than 1% in nearly every instance and, therefore, negligible as this is similar to the precision of the method. Only the interference caused by urea (at concentrations greater than 100 mg per 100 g of sample) and ascorbic acid (from 100 to 200 mg per 100 g of sample) should be taken into account. The urea interference could be because of the instability of NAD+ at high p H values. In addition, the presence of urea drives the equilibrium of the NADH/NAD+ reaction to the right. The interference caused by ascorbic acid is probably due to its reducing capacity, which causes a non-specific consumption of NADH. Amino acids interfere at concentrations greater than 100 mg per 100 g of sample, which do not occur in normal samples.
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ANALYST, SEPTEMBER 1990, VOL. I15
Application to Food Samples
2.
In order to apply this method to the determination of ammonia in cheese and baked ham, the extracts of the samples were diluted, taking into account the average concentration of ammonia in meats9 and cheeses,6 as well as the linear range of the method. The matrix effect can be considered negligible in the analysed samples, as the differences between the slopes of the calibration graphs run with internal and extcrnal references were very small, being 0.19 and 0.21% for ham and cheese, respectively. This, therefore, avoids the necessity to work with an internal reference, thus making the analysis easier. The recoveries obtained with samples of ham using the modified Parris and Foglia extraction method (Table 2) range between ca. 91 and 100%, which is acceptable for these types of samples that contain low levels of ammonia.
3. 4.
Conclusions The determination of ammonia in foods can be automated by the use of F1; 60 analyses per hour can be carried out. Linearity, recovery and precision parameters obtained confirm the reliability of the results achieved with this technique. Substances that, according to their labile amino groups, potentially liberate ammonia, such as urea, creatinine and amino acids, d o not exert any positive influence. The specificity of the enzymic method facilitates the determination of ammonia, in the presence of other nitrogenous substances common in foods, without previous distillation. Non-specific reducing agents, such as ascorbic acid, do not exert an effect as they do not consume NADH owing to the reduced analysis times involved in FI systems. As a consequence, the use of NADPH25 is not necessary. The FI - immobilised enzyme combination has proved to be excellent for the determination of ammonia in foods, the use of enzyme activators being unnecessary. The authors gratefully acknowledge financial support from the Comision lnterministerial de Ciencia y Technologia (CICYT); Project 285/86.
References 1.
Voets, J. P., Br. Poult. Sci., 1973, 14, 17
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Koval’chuck, G. K., Patsera, A . D., and Glcizcr, I. G., Rybn. Kho7. (MOSCOW),1969, 45, 85. Waters. M. E., J . Milk Food Technol., 1970, 33, 319. Cantoni, C.. Bianchi, M. A . , and Renon, P., Arch. Vef. Ital., 1969, 20, 355. Brooks, G. M., Diss. Abstr. Int., B , 1973, 34, 2079. Kapoor, C. M., Singh, J., and Rao. R. V., Indian J . Anim. Re.\.. 1972, 6, 71. Bodini, L. F., and Guicciardi, M., Lutte, 1969, 43, 711. Sorman, L., and Stoselova. E.. Zbornik Pruc. Chernikotechnol. Fak. SVST. 1972, 299. Bergmeyer, H . U., and Beutler, H., in Bcrgmcycr. H. U., Editor, “Methods of Enzymatic Analysis,” Vcrlag Chcmie, Wcinhcim, 1981, p. 454. Mcycrhoff, M. E., and Robins, R . H., A n d Clzem., 1980. 52, 2383. Osborn, J. A., and Yacynych, A. M., Anal. Chim. Acta, 1986, 183. 287. Valcarcel, M . , and Luque de Castro, M. D.. “Flow-injection Analysis: Principles and Applications,” Ellis Horwood, Chichcstcr, 1987. Masoom, M., and Townshend, A., Anal. Chim. Acta, 1985, 171, 185. Yao, T., and Wasa, T., Anal. Chim. Acta. 1985, 175, 301. Ruz, J . , Lazaro, F., and Luque de Castro, M. D., .I. Autom. Chem., 1988, 10, 15. Olsson, B., Salbou. B., and Johansson, G., Anal. Chirn. A m , 1986, 179,203. Masoom. M.. and Townshend, A . , Anal. Chim. Actu, 1984, 166, 111. Korenbrot, J. I., Perry. R., and Copenhagen, D. R.. Anal. Biocliern., 1987, 161, 187. Parris, N . , and Foglia, T. A., J. Agric. Food Chem., 1983, 31, 887. Masoom, M . , and Townshend, A , , A n d . Chim. Actu, 1986, 185, 49. Guilbault, G. G., “Analytical Uses of Immobilized Enzyme\,” Marcel Dekker, New York. 1984. Puchades. R., Lemieux, L, and Simard, R. E . , J . Food Sci., 1989, 54, 423. Tabata, M., Kido, T., Totani, M., and Murachi, T., Anal. Biochem., 1983, 134. 44. Brucc, A . W . , Leindecker. C. M . , and Freier, E. F., Clin. Chem., 1978, 24. 782. Humphries, B. A., Melnychuk, M., Donegan, E . J . , and Snee, R., Clin. Chem., 1979, 25, 26.
Paper 0100601G Received February 9th, 1990 Accepted May I7th, 1990
