ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
sample compartment conditions.
ACKNOWLEDGMENT The authors express their thanks to D. Tucker for doing some of the preliminary R T P experiments when a t the Department of Chemistry, Imperial College, London, F. C. Nachod for the donation of the samples of pure naphthyridines studied, K. Skeff Net0 for the loan of the photomultiplier power supply and the channel photon counting instrument, and L. Styer Caldas for linguistic advice.
LITERATURE CITED G. Y. Lesher, E. J. Froelich, M. D. Gruett, and J. H. Bailey, J. Med. /?arm. Chem., 5, 1063 (1962). E. W. McChesney, E. J. Froelich, G. Y. Lesher, A. V. R. Crain, and D. Rossi, Toxicol. Appl. fharmacol., 6, 292 (1964). R. S. Browning and E. L. Pratt, J . Assoc. Off. Anal. Chem., 53,464
(1970).
E. F. Salim and I. S. Shupe. J . Pharm. Sci., 55, 1289 (1966). G.Dondi and M. Di Marco. Boll. Chim. Farm., 105,491 (1966). M. J. J. V. da Silva and M. T. C. Nogueira, Rev. f o r t . Farm., 15,290 (1966). V. G. Zubenko and I. A. Shcherba, Farm. Zh. (Kiev), 30,28 (1975); Chem. Abstr.. 83. 330624 (1975). Gy. Milch, I. Aninger, and K. Kaloy, h o c . Conf. Appl. fhys. Chem. 2nd,
1665
(10) M. Roth. J . Chromatogr., 30,276 (1967). (11) E. M. Schulman and C. Walling, Science, 178, 53 (1972). (12) E. M. Schulman and C. Walling, J . Phys. Chem., 77, 902 (1973). (13) R. M. A. Von Wandruszka and R. J. Hurtubise, Anal. Chem., 48, 1784 (1976). (14) R. M. A. Von Wandruszka and R. J. Hurtubise, Anal. Chem., 49, 2164 (1977). (15) R. M. A. Von Wandruszka and R. J. Hurtubise, Anal. Chim. Acta., 93, 331 (1977). (16) P. G. Seybold and W. White, Anal. Chem., 47, 1199 (1975). (17) T. Vo Dinh, E. Lue Yen and J. D. Winefordner, Talanta, 24, 146 (1977). (18) I. M. Jakovljevic, Anal. Chem., 49, 2048 (1977). (19) R. A. Paynter, S.L. Wellons, and J. D. Winefordner, Anal. Chem., 46, 736 (1974). (20) S.L. Wellons, R. A. Paynter, and J. D. Winefordner, Spectrochlm. Acta, Part A , 30, 2133 (1974). (21)T. Vo Dinh. E. Lue Yen, and J. D. Winefordner, Anal. Chem., 46, 1186 (1976). (22) T. Vo Dinh, G. L. Walden, and J. D. Winefordner, Anal. Chem., 49, 1126 (1977). (23) S. Wellons, W.D. Dissertation, University of Florida. Gainesvilk, Fh., (1974). (24) G.F. Kirkbright and C. G. de Lima, Ana/yst(London), 99,338 (1974). (25) C. A. Parker, "Photoluminescence of Solutions," Elsevier Publishing Co., Amsterdam, 1968. (26) E. M. Schulrnan and R. T. Parker, J . Phys. Chem., 81, 1932 (1977). (27) N. Detzer and B. Huber, Tetrahedron, 31, 1937 (1975). (28) R. J. Henry "Clinical Chemistry Principles and Technics", Hoeber-Harper International Reprint, New York, N.Y., 1964 (29) "MP-Systems 1000, Operations and Applications", McKee-Pedersen Instruments, 3rd ed., Danville, Calif., 1968,1-16,
I, 397 (1971). R. Staroscik and J. Sulkowska, Acta Pol. Pharm., 30,499 (1973);Chem. Abstr., 81, 1524372 (1975).
RECEIVED for review May 8, 1978. Accepted July 14, 1978.
Repetitive Determinations of Amylase, Maltose, Sucrose, and Lactose by Sample Injection in Closed Flow-Through Systems D. P. Nikolelis' and Horacio A. Mottola" Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74074
Conditions have been developed for the repetitive determination of amylase and some disaccharides by coupling enzyme-catalyzed reactions yielding glucose as a product with the glucose oxidase catalyzed oxidation of this sugar. Determinations have been performed by sample injection into a continuously circulated reagent mixture and monitoring of oxygen depletion with a three-electrode amperometrlc system. Maltose, sucrose, and lactose in the range of 50 to 500 mg/100 mL, 10 to 250 mg/100 mL, and 25 to 250 mg/100 mL, respectively, and amylase in the range of 50 to 500 units/100 mL have been determined with relative errors and relative standard deviations (population) of about 2 % The maximum determination rate is 240 injections/h for maltose, 700 injectionslh for sucrose and lactose; and 120 injectiondh for amylase at room temperature. Applications to real samples (a variety of food products, human blood serum, and serum calibration references and controls) are reported.
.
The usefulness of repetitive determinations using injection of the sample containing the sought-for species into a continuously circulated reagent mixture and based on the general reaction scheme: %substrate)
+ HzO + O2
E
Product(s1 + H 2 0 2
+
(1)
P e r m a n e n t address, L a b o r a t o r y of A n a l y t i c a l Chemistry, U n i v e r s i t y of Athens, Athens, Greece. 0003-2700/78/0350-1665$01 .OO/O
in which E is an appropriate enzyme, has been recently demonstrated ( I ) . The work reported here was designed to exploit the use of reaction 1 (with glucose as substrate and glucose oxidase as enzyme) as an "indicator reaction" for the determination of other chemical species entering into enzyme-catalyzed reactions producing glucose as a product. Detection is based on the amperometric measurement of oxygen decrease according to the reaction of Equation 1 by means of a three-electrode nonmembrane system reported previously (2). This paper describes the determination of maltose. sucrose, and lactose by their enzyme-catalyzed hydrolysis t o glucose and the estimation of the enzyme amylase by its catalytic effect on the hydrolysis of starch to glucose. In situations found in industrial process and quality control as well as in clinical determinations, the use of closed flow-through systems, such as the one used in the reported studies, affords decreased operating cost per determination by decreasing the number of manipulations, shortening the time for determination, and providing a better utilization of reagent solutions. Maltose levels are seldom measured as a clinical test but as interest in maltose metabolism increases, its determination in biological fluids will gain in relevance (3). On the other hand, disaccharides are regularly determined in several food products ( 4 ) . The determination of a-amylase in serum is of help in the diagnosis of acute pancreatitis, occlusion of the pancreas, and parotitis ( 5 , 6 ) . The sample injection procedures reported here have been applied to real samples: corn syrup and malt syrup for maltose, a variety of food products for sucrose, milk products for lactose, and human blood serum @ 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
for amylase. Recovery studies have also been performed for maltose in modified infant milk and for both maltose and amylase in serum calibration references and controls. Determination of amylase by the technique reported here was compared with a standard saccharogenic procedure (6);the maltose, sucrose, and lactose determinations in food products were also compared with parallel determinations using the Somogyi reagent after separation by column (adsorption) chromatography (7), the Lane and Eynon reagent before and after inversion (7) and the phenol-sulfuric acid method, respectively (8).
EXPERIMENTAL Apparatus. An experimental setup basically identical to the one shown in Figure 1of reference 1has been used for the different determinations. The recording and potentiostat units, however, were self-contained in a Beckman Electroscan 30. The flow rate from reservoir to detection zone was 0.17 mL/s and 0.07 mL/s for maltose and amylase determination, respectively. The same flow rate was 0.20 mL/s in sucrose and lactose determination. As in reference 1,the potential applied to the working electrode was -0.60 V vs. the SCE. Daily washing of the Pt working electrode in warm, 10% v/v, nitric acid, and thorough rinsing with deionized water is recommended. Reagents and Solutions. Glucose oxidase was purified Type I1 from Aspergillus niger; invertase was grade I11 from baker's yeast, and amylase was Type 11-A of bacterial origin. All were supplied by Sigma Chemical Company (St. Louis, Mo.). pGalactosidase (lactase) was purified from E. coli and supplied by P-L Biochemicals, Inc. (Milwaukee, Wis.). The glucose oxidase used for pre-incubation to remove glucose in serum, corn, and malt syrup samples was purified grade (from Aspergillus niger) supplied by ICN Pharmaceuticals (Cleveland, Ohio). Deionized water was found satisfactory for solutions preparation. Typical reservoir solutions contained acetate buffer of pH 5.00 (0.10 M total acetate concentration) and 270 U/mL of glucose oxidase for maltose determination, and "Tris" or phosphate buffer of pH 7.00 (0.10 M in total Tris or phosphate) and 350 U/mL of glucose oxidase for sucrose or lactose; for amylase determination, the reservoir solution consisted of a phosphate buffer (pH 7.00,O.lO M total phosphate concentration), 0.35 M in NaCl, 4 g/L of soluble starch as substrate, and 360 U/mL of glucose oxidase. One unit of glucose oxidase corresponded to that amount of enzyme needed to oxidize 1.0 pmol of glucose per minute at pH 5.1 and at 35 "C. The glucose oxidase in the circulated reservoir solution contained 11units of catalase impurity and 0.026 unit of maltase impurity per unit of glucose oxidase. One unit of maltase corresponds to the amount of enzyme that will convert 1.0 pmol of maltase to 2.0 pmol of D-glucose per minute a t pH 6.00 and at 25 "C. The catalytic effect of the maltase impurity lasts for about 25 days if the reservoir solution is stored at +4 "C when not in use. One unit of catalase is the amount of enzyme decomposing 1.0 rmol of H202per minute at pH 7.0 and 25 OC, while the H202concentration falls from 10.3 to 9.2 pmol/mL of reacting mixture. For invertase, one unit hydrolyzes 1.0 pmol of sucrose to invert sugar per minute at pH 4.50 and 55 "C. The lactase unit is defined as the amount of enzyme that hydrolyzes 1.0 pmol of lactose to glucose and galactose per minute at pH 7.2 and 37 "C. The unit definition for amylase: 1 unit liberates 1.0 mg of maltose from starch in 3 min at pH 6.9 and 20 "C. Determination of all enzyme units was performed following recommended procedures (6,9-13). Standard solutions for maltose were prepared in water; standard sucrose solutions in an acetate buffer of pH 4.20 (0.10 M in total acetate; pH adjusted with 5 M NaOH); and the standard lactose solutions in an imidazole buffer of pH 6.50 (0.10 M in total imidazole, pH adjusted with 12 M HC1). Food products were homogenized, and carefully weighed samples were dissolved in and diluted with the same buffer used in the preparation of the standard solutions. For the determination of sucrose in vegetables, nuts, and cereals, the sugars were extracted with ethanol and diluted with the acetate buffer. Working curves for the determination of maltose and amylase in human blood serum samples were constructed by use of Versato1 reference solutions (General Diagnostics, Morris Plains, N.J.) to which known amounts of the sought-for species were added.
W U
ey
I 1
I00
200
300
kaz
TIME ( 5 E C O N D 5 )
Figure 1. Effect of incubation time on peak height. Concentration of sucrose = 100 mg/100 mL. Other conditions as described in Procedure
Procedures. Determination procedures paralleled the one reported in reference 1except that a sample volume of 20 pL was used per injection. For sucrose determination, 20-pL samples were pre-incubated with 20 U of invertase for a period of 5 min and at 55 "C. Incubation time shorter than 5 min will result in incomplete hydrolysis and decreased peak height (Figure 1). In the case of lactose, each 20-kL sample was mixed with 80 U of lactase/ determination and injected without delay; alternatively one can use 30 U of lactase per determination but inject after 120 s of incubation, which gives the same sensitivity as using 80 U of enzyme/determination and direct injection. For sucrose determination in food products, if the sample contains glucose, the sample was diluted so that the final glucose concentration was below 63 mg/100 mL and the blank reading was subtracted from the peak reading obtained after preincubation. Interfering glucose in serum and food samples was removed by incubation of a 20-pL sample with 2.8 units of purified glucose oxidase per determination, for 5 to 10 min at room temperature prior to injection (3). RESULTS AND DISCUSSION Coupling of chemical reactions is a common practice in kinetic-based determinations, including those involving enzymes (14). The method illustrated here involves signal measurements under dynamic conditions in a system approaching equilibrium and belongs to the area of kinetic-based determinations using catalyzed reactions and is also within the province of the so-called "flow injection analysis" (15). T h e indicator reaction refers t o the reaction in which the monitored species participates; in this particular case, dissolved oxygen is the monitored species and reaction 1 is the indicator reaction. Pertinent kinetic considerations for this reaction, after sample injection into a streaming solution and into a mixing coil, and the detection (amperometric) of the monitored species have been presented in a previous paper (1). T h e reactions coupled with the indicator reaction and preceding it were, for amylase determination: a-amylase Starch + nHzO (n 1 - 3c) Malto-oligosaccharides (2)
+ + -
Maltose
H20
maltase
2 D-Glucose
(3)
For maltose determination, reaction 3 is directly coupled with the indicator reaction; for sucrose and lactose determination, reactions 4 and 5 below are the ones so coupled:
+ H20 Lactose + H 2 0
Sucrose
invertase
lactase
+ fructose D-Glucose + galactose D-Glucose
(4)
(5)
T h e @-galactosidase used in the present work had a very high lactase activity (almost 100%);lactase preparations from
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
1667
Table I. Effect of Temperature on Peak Height and Rate of Determination amylasea maltoseu lactosea temp. of reservoir, C i/hourb h, P A i/hour h, P A i/hour h, P A 25 120 4.67 240 5.83 700 2.10 30 150 5.25 270 6.83 700 3.23 35 270 6.42 400 7.00 700 5.66 40 330 7.00 480 7.58 700 8.73 45 700 10.35 Effect of Temperature on Incubation Time in Sucrose Determination temp. of incubationC minimum incubation time, sd peak height, @ A 23 700 5.17 30 600 5.36 40 500 5.56 50 400 5.75 300 5.75 55 a Injection of 20-pL sample containing 250 U/100 mL of amylase or 250 mg/lOO mL of maltose, or 100 mg/100 mL of lactose. i/hour: number of injections per hour; h : peak height. Injection made at room temperature. Sucrose: 100 mg/100 mL. Longer incubation times d o not affect peak height. other sources are available but being more expensive increase the cost per analysis. Christian et al. (3,16) have determined maltose and lactose using the same set of reactions but using, basically, the “Beckman Glucose Analyzer” commercial system which monitors oxygen with an electrode of the membrane, Clark-type. Their determination rates are, however, lower than those for the method reported here. Reaction 4 has been previously used for the determination of sucrose (19,but coupled with a different indicator reaction. In this case a 24-h preincubation time was recommended. Guilbault et al. (18, 19) utilized the same set of reactions for the determination of disaccharides but monitoring, fluorometrically, the HzOzproduced. Although the fluorometric method offers better sensitivity, automation and rapid assay are recognized improvements of the method proposed here. Reactions 2 and 3 have been used by Guilbault e t al. (20) for amylase determination, but also coupling with a different indicator reaction. Advantages of the proposed implementation are simplicity, reduced cost per determination, and meaningful statistical treatment of data afforded by large determination rates. Recirculation of the glucose oxidase reagent and the advantage of its sufficiently high maltase impurity help in decreasing the cost per determination in comparison with other proposed procedures. The kinetic characteristics of the transient signal-based determinations ( I , 21) allow rapid repetition of the cycle. It is of interest to note that, a t room temperature, the determination rates for amylase and maltose do not parallel the one reported for glucose determination ( I ) . Increasing the temperature of the reservoir solution, as shown in Table I, increases the determination rate (by decreasing the time for return to baseline) as well as the sensitivity of the method if quantitative data are derived from the height of the transient peak. Table I also shows the effect of temperature during incubation. Considering that an increase of temperature decreases the incubation time, a temperature of 55 OC is recommended for sucrose determination. As should be expected, the temperature of incubation does not affect peak height or determination rate. The applications reported here were performed a t room temperature since the reactions involved are competitive and the sensitivity is sufficient without introducing the complicating factor of temperature control. The temperature effect, studied by changing the temperature of the reservoir, is the result of factors such as oxygen saturation, sensitivity of the voltammetric sensing, and, for comparison purposes, primarily the effect of temperature on
I
2.5rA
LO
20
30
40
Tirne.s
Figure 2. Change in oxygen level (from saturation) with time at zero flow rate. Curve a: 100 mL buffer (pH 7.00) solution containing 350 U/mL of glucose oxidase 1.0 mL water. Curve b: same as curve a -t 0.010 g of starch to produce an amount of glucose equivalent to that for curve d; 1 mL of amylase solution (25 U) injected at time zero. Curve c: 100 mL buffer (pH 5.00) containing 350 U/mL of glucose oxidase; 1 mL of maltose :jolution (0.0050 g) injected at time zero. The maltose added was calculated to produce an equivalent amount of glucose to that added for the experiment of curve d. Curve d: same as curve a; 1 mL of glucose solution (0.0010 g) injected at time zero. Temperature 23 O C
+
the enzymatic reactions involved. This is illustrated in Figure 2, which shows rate profiles of oxygen depletion at zero flow rate. Because of the lower catalytic activity of amylase and the inhibitory action of amides (e.g., urea) in human blood serum (22),the slower release of glucose helps permit a large number of injections without considerably affecting the baseline level. This fortunate situation does not occur with other enzymes; accumulation of glucose oxidase, for instance, after about 30 injections (1.2 units per injection) produces a serious drift of the baseline and prevents further use of the recirculated substrate solution for enzyme activity determination (23). Removal of the enzyme by immobilization on hygrophobic supports, just after leaving the detection flow-cell, however, makes such a determination possible beyond 30 injections (23). Recirculation of invertase was tested, but reaction 4 was found to be very slow for our purpose. Moreover, the optimum
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
Table 11. Determination of Disaccharides and Amylase in Aqueous Solutions maltose, sucrose, lactose, mg/100 mLf mg/100 mL mg/100 mL taken foundazd taken founda' taken founda,c 50.0 100 200 300 400 500
50.5 99.5 205 310 399 485
10.0 25.0 50.0 100 250
9.95 24.7 50.7 98.0 251
25.0 50.0 100 200 250
amylase, units/lOO mL taken founda,e
25.2 49.0 102 203 246
50.0 100 200 300 400
49.5 101 197 311 408 494
500
From interpolation into working curve. Average of three determinations. Average relative % error: 1.8. Average relative %error: 1.1. Average relative %error: 1.6. e Average relative % error: 1.5. Limit of Detection: (calculated as the average value for the blank + 3 x standard deviation for the blank): maltose, 20.4 mg/lOO mL; sucrose, 6.59 mg/100 mL; lactose, 15.3 mg/lOO mL; amylase, 35.7 U/100 mL. Sensitivity: (calculated as the slope of the calibration curve of peak height vs. concentration): maltose, 0.0204 pA mg-l.100 mL; sucrose, 0.058 JLAmg-*.100 mL; lactose, 0.019 JLAmg-l.100 mL; amylase, 0.0131 pA U-'.lOO mL. a
p H for invertase is 4.2, but a t this pH considerable blank readings were observed and a pH of 5.00 was found to be the lowest a t which the method could be applied. At this pH, however, only small transient signals were obtained, even when as much as 800 U of enzyme/mL was used. Since (3galactosidase loses activity rather rapidly with time (24), its recirculation is not recommended. Also, when this enzyme is present in the reservoir, cloudiness develops with time and eventually obstruction of the transporting tubing was observed. Deserving comment is the fact that the use of 7 times the amount of glucose oxidase activity used previously for direct glucose determination ( I ) apparently shifts the tautomeric equilibrium by fast destruction of the /3 anomer and allows development of transient peaks even though the direct product of disaccharide hydrolysis is the a anomer, which is not acted upon by the enzyme. T h e flow rate in the loop branch going from the reservoir to the detection cell (controlled by gravity) critically affects both signal height and signal duration. The flows recommended represent a compromise between sensitivity and rate of determination. Figure 3 shows the effect of glucose oxidase on peak height. Operating conditions were chosen so that the enzyme activity used in the determinations lay in the plateau region for each enzyme. The effect of invertase and of /3galactosidase activity on peak height followed the trend illustrated in Figure 3 for glucose oxidase. Results obtained with 100 mg of sucrose/100 mL showed that peak height is maximum a t about 25 U of invertase/mL and remains constant with increasing amounts of enzyme. In the case of &galactosidase, the increase in peak height levels off after 80 U of enzyme/mL (100 mg of lactose monohydrate/100 mL). The pH values a t which maximum response to maltose and amylase occurs were found to be about 5 and 7 , respectively. The optimum pH values for incubation of sucrose and lactose were found about 4.2 and 6.6, respectively. All optimum pHs found are in agreement with recommended values (3, 5 , 16, 25). For amylase determination, higher concentrations of NaCl [added as an enzyme activator ( 5 , 2 0 ) ] and starch than 0.35 M and 4.000 g/L, respectively, did not increase the peak height of the transient signal. Effect of Foreign Species and Studies in Aqueous Solutions. Interferences related to the monitoring of the indicator reaction have been discussed elsewhere ( 1 ) . The specificity of the enzymes involved have also been discussed previously (18, 19). In the determination of maltose or amylase, any glucose present must be destroyed by preincubation as described earlier. Glucose also interferes in sucrose and lactose determination, if its concentration is above 63 mg/100 mL. Concentrations of glucose below 63 mg/100 mL can be ac-
7 00[
itl
I
9
100
300
5 L L C O 5 E 3 X 3F5-C R C - 1 i P - V
ie0
780
(J/YL>
Flgure 3. Effect of glucose oxidase activity on peak height. A: 250 mg maltose/ 100 mL; B: 250 U of amylase/ 100 mL; C: 100 mg of sucrosellO0 mL; D: 100 mg of lactose/100 mL. Other conditions
as under Procedure counted for by determining glucose separately and subtracting its contribution to the peak height. Starch interferes in the maltose determination because of the amylase impurity present in the glucose oxidase. The starch interference can be eliminated as suggested by Christian et al. (3). Maltose, dextrins, and starch interfere in sucrose determinations because of the maltase impurity in glucose oxidase: this interference can be completely eliminated, however, by use of the Tris buffer which is known to inhibit maltase activity (17). If maltose is present in samples in which amylase is to be determined, it must be estimated prior to the enzyme determination to compensate for its contribution to the peak height. Results for the determination of disaccharides and amylase in aqueous solutions are reported in Table 11. Deviation from working curve linearity occurs at concentrations larger than the higher ones listed in Table 11, probably because at high concentrations pseudo-first-order conditions do not apply to the indicator reaction. Relative standard deviations (30 determinations) were in the order of 1.8 to 1.9% and the overall time for return t o baseline (room temperature) was 15 s for maltose, 5 s for sucrose and lactose, and 30 s for amylase. These return times include the delay time due to injection. Determination of Disaccharides in Food Products and Recovery Studies for Maltose in Serum Calibration References and Controls. A summary of the application of the reported procedure to disaccharides determination in some food products is shown in Tables 111, IV, and V. Results obtained by application of the Somogyi reagent after chromatographic separation (7) for maltose, the Lane and Eynon
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
Table 111. Determination of Maltose in Food Products by the Proposed Method and by Use of the Somogyi Reagent after Chromatographic Separation (7)' sample injection procedure Somogyi working curve standard addition determination interpolation corn syrup 23.8 21.6 22.2 (brand I) corn syrup 23.7 24.6 23.9 (brand 11) corn syrup 20.6 21.0 20.3 (brand 111) malt syrup 60.1 61.3 61.3 a Results in g maltose/lOO g sample. All reported values are average of at least 3 determinations. Maltose levels were determined from a calibration curve prepared with maltose standards containing 50 mg/100 mL of starch or by applying the standard addition method (blank correction taken in account).
Table VI. Recovery of Maltose Added to Modified Infant Milk and Versatol maltose, mg/100 mL recovered from infant milk Versatol added 50.0 100 200 300 400 500
Table V. Determination of Lactose in Some Food Products by the Proposed Method and the Phenol-Sulfuric Acid Method (8)' phenolsample sulfuric sampleb injectionC acidd skim milk (powder) 52.8 5 1.3 51.7 4.05 2 0.14 3.96 ice cream condensed milk 15.8 = 0.2 15.4 (sweetened) skim milk (liquid) 4.93 3 0.15 5.02 buttermilk (liquid) 4.91 I0.14 5.01 (genuine and cultured) homogenized milk 4.69 = 0.08 4.80 yogurt 3.85 = 0.07 3.78 ' Results in g of lactose/lOO g of sample. Sample dissolved in imidazole buffer of pH 6.50. All reported values are average of at least 6 determinations. Lactose levels were determined from a calibration curve prepared with lactose standards in imidazole buffer of pH 6.50. Average of three determinations. reagent (before and after inversion) for sucrose (7), and the phenol-sulfuric acid method (8)for lactose, are also included in Tables 111, IV, and V showing satisfactory agreement with
49.5 101 197 310 397 490
recovery, % infant milk Versatol
50.0 97.5 204 295 408 490
average
99.0 100 101 97.5 98.5 102 103 98.3 99.2 102 98.0 98.0 -~ 99.8 99.6
Table VII. Determination of Amylase Added to Versatol (Serum Calibration Reference and Control)u amylase, U/lOO mL taken found relative error, % 0.0 50.0 100 200 300 400 500
Table IV. Determination of Sucrose in Some Food Products by the Proposed Method and the Lane and Eynon Method ( 7 ) e sample injection, working curve Lane and sample type interpolationu Eynonb apple juiceC 4.12 i 0.09 4.21 orange juice (fresh)c 4.80 i. 0.06 4.71 ice creamC 18.6 i 0.2 18.2 condensed milk 43.2 ?: 0.7 42.9 (sweetened)c jellies (pectin)c 54.9 i 1 . 2 56.1 maple syrupC 60.6 r 1.4 61.8 green peas 5.62 i 0.11 5.52 (frozenld corn ( fresh)d 0.302 i 0.008 0.295 wheat flourd 0.191 ? 0.004 0.198 peanuts (fresh)d 4.94 i 0.12 4.64 a Reported values are average of at least 6 determinations. Reported values are average of 3 determinations. Product directly dissolved in acetate buffer of pH 4.20. Sugar content extracted with ethanol and diluted with buffer pH 4.20 (acetate) (17). e Results in g of sucrose/ 100 g of sample. Sucrose levels were determined by reference t o a calibration curve prepared with sucrose standards in acetate buffer of pH 4.20.
1669
0.0 51.0 99.0 193 302 395 503
+ 2.0 -1.0 -3.5 + 0.7 -1.2 +0.6 1.5
average All results are average of a t least 3 determinations. Limit of detection (as average of blank + 3 standard deviation of blank): 36.7 mg/lOO mL. Sensitivity (as slope of calibration curve): 0.0125 PA mg-',100 mL. a
those obtained by the proposed procedure. T h e values obtained are within the range reported in the literature for disaccharides in the given food products ( 4 ) . T h e accuracy of the proposed method was further tested by means of recovery studies. Maltose recovery of amounts added in the range of 50 to 200 mg/100 mL was found to be from 93 to 108% with an average of 100.8%. Sucrose recovery in the range 10 to 100 mg/100 mL was found to be 96 to 105%, with an average of 99.2%. Lactose recovery (25 to 100 mg/100 mL range) varied from 96 t o 108%,with a n average of 101.0%. Since maltose is reportedly added to modified infant milk ( 3 ) ,recovery studies for maltose have also been performed in a commercial brand of infant milk; results are reported in Table VI. Table VI also presents data for recovery studies for maltose in Versatol, a serum calibration reference and control formulated so as to approximate normal adult levels for frequently tested serum constituents. Recovery S t u d i e s f o r Amylase f r o m Versatol a n d Determination of T h i s Enzyme in H u m a n Blood Serum. Recovery studies from Versatol are reported in Table VII. These results provided data for the preparation of a working curve used for the determination of amylase in hospital samples of human blood serum. Glucose was removed prior to injection as described earlier, in 100-pL samples of serum. Results obtained in human blood serum by the proposed method have been compared with those from parallel determinations using the well established saccharogenic procedure (6). Figure 4 graphically illustrates the comparison. The Pearson correlation coefficient for both methods was found to be 0.99 for 32 individual samples. The saccharogenic method requires 40 min per determination compared with the few seconds required by the sample injection procedure reported here. T h e accuracy of the method was also verified by means of recovery studies on three representative samples containing the fasting level of amylase by adding amylase in the range of 50-150 U/dL. The analytical recovery averaged 101.3% with extreme values of 95 and 108%.
1670
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
-=La -
L. B. Marshall, G. D. Christian, and A. Kuma, Analyst(London), 102, 424
I
(1977). M. G. Hardinge, J. B. Swarner, and H. Crooks, J . Am. Diet. Assoc., 46,
197 (1965).
YI
,
-."
- . 222
-i
'=CCd=TJEf.~,'
^J (L.
..-
_-
ZCL
2;
N. W. Tietz, Ed.. "Fundamentals of Clinical Chemistry", W. 8. Saunders, Philadelphia, Pa., 1970,pp 409, 809. M. W. Kanter, "Clinical Chemistry", The Bobbs-Merrill Co., Indianapolis, Ind., 1975,p 169. R. Lees, "Food Analysis: Analytical and Quality Control Methods for the Food Manufacturer and Buyer", 3rd ed., CRC Press, Cleveland, Ohio, 1975,pp 68 and 168. G. G. Birch and 0. M. Mwangelwa, J . Sci. Food Agric., 25, 1355 (1974). Sigma Chemical Co., Form No. 322, St. Louis, Mo. "Enzymatic Assay of a-Amyhse (E.C. 3.2.1lr',December 1971,Revised August 1977,Sigma Chemical Co., St. Louis, Mo. January 1978,Sigma Chemical Co., St. "aGlucosidase (E.C. 3.2.1.20)", Louis, Mo. "The Enzymatic Assay of Invertase", November 1977,Sigma Chemical Co., St. Louis, Mo. "Lactase", Sigma Chemical Co., St. Louis, Mo. G. G. Guilbault, "Handbook of Enzymatic Methods of Analysis", Marcel Dekker, New York, N.Y., 1977. E. H. Hansen, A. K. Ghose, and J. Ruzicka, Ana&st(London), 102, 714 (1977),and references therein. F. S. Cheng and G. D. Christian, Ana/yst(London), 102, 124 (1977). J. Cerning-Beroard, Cereal Chem., 52, 431 (1975). G G.Guilbauk, P. J. Brignac, Jr., and M . Juneau, Anal. Chem., 40, 1256
Figure 4. Comparison of results obtained by sample injection into a closed-flow-through system and by the saccharogenic method. Results represent determinations in 32 samples of human blood serum with amylase content mostly in the normal range. (Pearson's correlation coefficient: 0.99; slope obtained by linear regression: 0.973,intercept:
11968).
3.14 U/100 mL)
(1967).
ACKNOWLEDGMENT The authors are indebted to David Jackson (Oklahoma State University Student Hospital and Clinic) for samples of human blood serum. LITERATURE CITED (1) Ch-Michel Wolff and H. A. Mottola, Anal. Chem.. 50, 94 (1978). (2) Ch-Michel Wolff and H. A. Motfola, Anal. Chem., 49, 2118 (1977).
G. G. Guilbauk, M. H. Sadar, and K. Peres, Anal. Biochem., 31,91 (1969). G.G. Guilbault and E.B. Rietz. Clin. Chem. ( Winston-Salem, N.C.),22,
1702 (1976). H. A. Mottoia and A. Hanna, Anal. Chim. Acta, in press. G. C. Toralballa and M. Eitingon, Arch. Biochem. Biophys., 119, 519 I.Asfaha, Oklahoma State University, unpublished observations, 1978. "Worthington Enzyme Manual", L. A. Decker, Ed., Wwthington Biochemical Corp., Freehold, N.J., 1977, p 195. G. Reed, "Enzymes in Food Processing", 2nd ed., Academic Press, New York, N.Y., 1970, p 90.
RECEIVED for review April 10, 19'78. Accepted July 20, 1978. This work was supported by the National Science Foundation. One of the authors (D.P.N.) gratefully acknowledges a leave of absence and support from the University of Athens, Greece.
Unexpectedly Rapid Esterification of Nitrite Applied to the Determination of Nitrite in Water Marcus Keh-Chung Chao, Takeru Higuchi, and Larry A. Sternson" Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045
Nitrite is converted to an alkyl nitrite by passing an acidified solution through a bed of packed beads (XAD-2) coated with 1-decanoi, the latter acting both as a reactant and as an extractant. The reaction proceeds by rate limited diffusion of nitrous acid into the aikanoi phase where it is rapidly esterified. Esterification by this pathway is qulte rapid (obsd f,,2 = 11 s) and efficient. The product, decyl nitrite, is retained in the aikanoi phase [partition coefficient (C,,,H,,OH:H,O) = 2.5 X lo'] where it is resistant to hydrolysis because of the limlted solubility of water in the alkanoi phase. Under optimized conditions, 6 8 % of the nitrite present in the original water soiutlon was retained on the column as decyi nitrite. Decyi nitrite was eluted from the column with acetone and then converted to a highly colored azo dye by sequential reactlon with sulfanilamide and N-( 1-naphthyl)ethyienediamine. The chromophore was monitored spectrophotometrically. The detection limit for nitrite quantitatlon by this method was 5 X 10-e M (200 pg/mL).
Recently, a great deal of interest has been generated concerning potential health hazards of nitrites. Nitrites are 0003-2700/78/0350-1670$01 .OO/O
frequently used as preservatives in food products and their precursors are widely distributed in nature because of the use of nitrogen fertilizers. Nitrites have been regarded as potentially hazardous compounds ( I ) . They oxidize hemoglobin to methemoglobin which is unable to transport oxygen (2-4) and they react with amines and amides to form nitrosamines, which are potent carcinogens (5-9). In view of the ubiquitous presence of nitrites in the environment (IO),a sensitive method for trace level determination of nitrites is desirable. Although nitrites can be determined polarographically ( I I ) , the classical methods are colorimetric procedures. The nitrite is reacted with a primary aromatic amine (e.g., sulfanilamide) in acid solution to yield a diazonium salt which is coupled with an electron rich aromatic molecule [e.g., N-(1-naphthyl)ethylenediamine (NEDA)J to produce a highly colored azo compound which is measured spectrophotometrically (12,13). T h e detection limit for nitrite ion by this method using sulfanilamide and NEDA as reagents is lo4 M (13). Sensitivity has been improved (by a factor of 30) by employing a concentration step after formation of the azo dye. The azo compound has been concentrated by a series of extractions into increasingly smaller volumes of solvent ( 2 4 , 15); or by retention on a strong cation-exchange resin and G 1978 American Chemical Society
