Fluorometric Determination of Alkaline ... - ACS Publications

Article pubs.acs.org/JAFC

Fluorometric Determination of Alkaline Phosphatase Activity in Food Using Magnetoliposomes as On-flow Microcontainer Devices Vanessa Román-Pizarro, Juan Manuel Fernández-Romero, and Agustina Gómez-Hens* Department of Analytical Chemistry, Institute of Fine Chemistry and Nanochemistry (IUQFN UCO), Campus of Rabanales, Marie Curie Building (Annex) University of Córdoba, E-14071 Córdoba, Spain ABSTRACT: Liposomes containing magnetic gold nanoparticles (AuNPs) and an enzymatic substrate (4-methylumbelliferylphosphate) have been used as on-flow microcontainers for reagent preconcentration in a flow injection method for the determination of alkaline phosphatase (ALP) activity. The dynamic range of the calibration graph was 6.4 × 10−3−0.25 U L−1 ALP, and the detection limit was 1.9 × 10−3 U L−1. The precision, expressed as relative standard deviation (RSD%), was in the range of 0.7−2.4%. The overall method showed a sampling frequency of 10 h−1. The method was applied to the determination of ALP in milk samples with recovery values ranging between 87.5 and 104.6%. The residual ALP activity in milk samples subjected to temperature treatments was also determined. The results obtained in the analysis of all milk samples were compared with those obtained by applying a previously described flow injection method. KEYWORDS: magnetic gold nanoparticle liposome hybrids, on-flow microcontainers, alkaline phosphatase activity, flow injection, fluorometric detection, food analysis

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INTRODUCTION The capability of liposomes to encapsulate different materials, together with their biocompatible and biodegradable properties, allows their use as delivery systems, which protect sensitive compounds against degradation and control their release. These features have given rise to the application of liposomes in the food industry to deliver nutrients and food additives.1 Several studies have been carried out on the bioavailability of natural polyphenols, such as salidroside,2 curcumin,3 and tea polyphenols,4 encapsulated into liposomes for the production of functional foods. Similarly, the potential application of liposomes as carriers for the natural carotenoid lutein has been described.5 Also, the ability of liposomes to incorporate food antimicrobials, such as nisin, has been investigated to improve the microbiological stabilization of food products.6 Another recent research area in the food industry is the development of food products containing therapeutic macromolecules such as proteins. For instance, the stability of alkaline phosphatase (ALP) encapsulated into alginate-loaded liposomes when exposed to simulated gastric pH has been studied for this purpose.7 Alginate was used to improve the stability of liposomes. Also, the usefulness of alginate and chitosan coated on the surface of liposomes has been described to stabilize liposomal structure.8 Liposomes have been described as useful tools for analytical purposes involving chromatography, capillary electrophoresis, immunoassays, sensors, and microfluidic systems.9,10 Chromogenic and fluorescent dyes, electrochemical active molecules, nucleic acids, oligonucleotides, peptides, and proteins, including enzymes, have been entrapped in liposomes to improve the features, mainly sensitivity and selectivity, of a large number of analytical methods. However, the analytical application of NPs entrapped in liposomes has been scarcely described. An automatic method for biotin determination in food samples has been developed using AuNP-biotinylated liposome hybrids © 2014 American Chemical Society

as enhancement reagents in a competitive assay with resonance light scattering detection.11 Functionalized magnetic nanoparticles (MNPs) have been described for separation and preconcentration purposes,12−14 in which magnetic fields are applied to control the motion and properties of these NPs. Among the different procedures reported to protect MNPs from oxidation and corrosion, gold is considered a useful option for the shell owing to its chemical stability and facility in surface modification using usually thiol chemistry.15,16 Liposomes entrapping MNPs, usually named magnetoliposomes (MLs), have been described as drug delivery systems, which can be guided and localized to the therapeutic site of interest by external magnetic field gradients.17 Also, the use of MLs as efficient magnetic resonance imaging contrast agents has been widely discussed.18−21 However, the use of MLs as analytical reagents has not been described, to the best of our knowledge. The objective of this work has been to study the usefulness of MNPs entrapped in liposomes for the automatic determination of the enzymatic activity using a flow system and ALP as model analyte to check the applicability of this approach. The method has been applied to determine ALP in milk samples because ALP activity is usually accepted for the rapid validation of milk product pasteurization.22 Hydrophobic MNPs, obtained by modifying the surface of Fe3O4@AuNPs with 1-dodecanethiol (DT), were used to improve the encapsulation yield in the liposomes, in which the enzymatic substrate 4-methylumbelliferyl-phosphate (MUP) was also entrapped. These MLs act as on-flow microcontainers Received: Revised: Accepted: Published: 1819

October 30, 2013 February 4, 2014 February 5, 2014 February 5, 2014 dx.doi.org/10.1021/jf5004804 | J. Agric. Food Chem. 2014, 62, 1819−1825

Journal of Agricultural and Food Chemistry

Article

previously reported,27 which was modified to introduce the DT. Briefly, 25 mL of an aqueous solution containing 0.8 M iron(III) chloride, 0.4 M iron(II) chloride, and 40 mM hydrochloric acid was dropwise added into 250 mL of 1.5 M sodium hydroxide solution under vigorous stirring using a nonmagnetic stirrer. Black-brown Fe3O4NPs were immediately formed, which were separated using a commercial neodymium magnet (NdFeB) and washed three times with deionized water. The Fe3O4 NPs were collected by centrifugation at 5352.80g for 15 min and dispersed again in 100 mL of deionized water. The NPs concentration of this solution was about 100 mM,18 which was stored refrigerated at 4 °C for further use. The preparation of Fe3O4@AuNPs-DT was carried out by adding 2.2 mL of HAuCl4·3 H2O (2.0 mg mL−1) to 22.5 mL of deionized water and heated to boiling for 5 min. Then, 2 mL of the above prepared Fe3O4 NPs solution was added to the reaction mixture followed by the addition of 0.5 mL of sodium citrate (80 mM) and 5 mL of DT, and the mixture was vigorously mixed under stirring. The color of the solution gradually changed from brown to burgundy. The reaction mixture was boiled under stirring for 40 min. TEM images were obtained by dropping the resulting Fe3O4@ AuNPs-DT on a carbon-coated copper grid and setting a completely dried drop by the oven. XPS spectra were also obtained to identify the presence of a particular element and distinguish the different forms in the NPs. Also, SEM-EDX measurements were made to determine the elemental composition of the NPs. Preparation, Separation, and Characterization of Magnetoliposomes. Liposomes containing MNPs and the enzyme substrate MUP were prepared using the rapid solvent evaporation (RSE) method.28 Briefly, 100 μL of a lipid mixture containing DSPC, DOPE, and CH (75:12.5:12.5) was diluted to 1.0 mL with a chloroform/ methanol (880:120) mixture and was put into a 50 mL round-bottom flask. Then, a mixture containing 3 mL of an about 7 mM Fe3O4@ AuNPs-DT suspension, 2.5 mL of 40 mM MUP solution, and 3.5 mL of deionized water was carefully added along the flask wall. The organic solvent was removed in the rotary evaporator at 40 °C and 300 rpm under vacuum pressure. After 20 min, a pale brown opalescent fluid was obtained, which contained a wide variety of liposomes. The liposome population was homogenized by using two physical treatments, mechanical shaking and sonication. A volume (10 mL) of the liposome suspension was shaken in a shaking incubator at 40 °C, which is close to the transition temperature, and at 250 rpm for 30 min, stopping each 10 min for 2 min. After this treatment, the liposomes were resized by sonication. This liposome suspension was partially submerged in a bath sonicator and treated for a period of 10 min, stopping each 2 min for 10 s. The suspension changed in its appearance from milky to a clear suspension. The separation of filled liposomes from empty liposomes, phospholipid residues, and untrapped magnetic NPs was carried out using conventional centrifugation at 20073g for 30 min. Filled liposomes, which showed a brown appearance, were located in the intermediate zone, whereas empty liposomes and untrapped NPs appeared in the top and bottom zones, respectively. After separation, filled liposomes were washed four times with deionized water. These liposomes were stable for at least 30 days. Procedure for the Determination of ALP Activity. Figure 1 depicts the hydrodynamic arrangement used for the determination of ALP activity (A) and the process time schedule (B). A suspension of MLs containing MUP, prepared in Tris buffer solution (50 mM, pH 9.8), was injected through the injection valve IV1 at a flow rate of 0.4 mL min−1. The liposomes were retained before the detector, using an electromagnet device, for a time (retention time, Δt1) of 200 s. A solution containing Triton X-100 and ALP standard or diluted milk sample solution, with an ALP activity in the range of the calibration graph, was then injected using the injection valve IV2. The surfactant provided the liposome lyse and the release of the enzyme substrate, which was hydrolyzed by ALP, obtaining a transitory fluorescence signal that was monitored at λex 365 nm and λem 449 nm, using a 10/5 slit ratio and a detector gain of 600 V. As Figure 1B shows, the system takes about 120 s (release time, Δt2) to achieve the baseline. Each standard or sample solution was assayed in triplicate.

that are concentrated just before the detector using an external electromagnet device. The introduction into the flow system of the surfactant Triton X-100, together with ALP, gives rise to the lysis of the liposomes, the “in-situ” development of the enzymatic reaction, and the fluorometric detection of the substrate product. The new method for ALP determination has been applied to the analysis of raw and commercial milk samples. The results have been compared with those obtained using a method based on the same enzymatic reaction, but involving the retention of the reaction product on a resin placed in the cell flow.23 Although several methods have been described for the determination of ALP activity in milk samples,24−26 the above-mentioned method was chosen because it also involves the use of MUP and a flow system, which enables an effective assessment of the MLs on the system. The usefulness of the method has been also checked by its application to study the milk pasteurization process by monitoring the degradation of ALP.

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MATERIALS AND METHODS

Apparatus and Instruments. A Cary Eclipse Varian spectrofluorometer (Walnut Creek, CA, USA; http://www.varianinc.com) was used. A desktop laboratory MPW-350R centrifuge (MPW Med. Instrument, Warsaw, Poland) with a cooled rotating chamber and equipped with an angle rotor HSL-11199 (45°, 12 × 12 × 1.5 mL, maximum speed = 18000 rpm, 24088 RCF, and rmin/max = 3.5/6.25) was used. A VorTemp 56 LA-S2056 shaking incubator (Labnet International, Woodbrige, NJ, USA) and an ultrasound bath were used. A Büchi rotavapor R-205 (Flawil, Switzerland) was used to evaporate the organic solvent and form the liposomes. The characterization of the synthesized nanomaterials was performed by conventional and both transmission (TEM) and scanning (SEM) electron microscopy. Images were acquired in the first case using a bright-field UIS2 optical system microscope (Olympus, Hamburg, Germany; http://www.olympus.de/ microscopy/, BX51, model U MDOB3) equipped with a DP20 digital camera, which provided high-precision images of 2 megapixels in UXGA (1600 × 1200), operating at 15 frames per second. TEM images were obtained using a CM-10 Philips microscope (Philips Research, Eindhoven, The Netherlands; http://www.research.philips. com) with 0.5 × 0.34 nm resolution and equipped with a digital megaview III camera. Copper grids (200C-FC) coated with a Formvar carbon film 200 mesh supplied by Aname (Madrid, Spain; http:// www.aname.es) were used as support in TEM experiments. The XPS measurements of Fe3O4@AuNPs-DT were carried out using a Specs Phoibos 150 MCD instrument equipped with a monochromatized AlK (12 kV). The morphology and elemental composition of these NPs were studied by SEM, using a microscope (JEOL JSM 6300) operating at 20 kV, which was also equipped with an energy dispersive detector (EDX). Reagents. All chemicals used were of analytical grade. 1,2-Diacylsn-glycerol-3-phosphatidylcholine (DSPC), 3-sn-phosphatidylethanolamine (DOPE) (type II-S from sheep brain), cholesterol (CH), tetrachloroauric acid (HAuCl4) trihydrate, 1-dodecanethiol (DT), Triton X-100, 4-methylumbelliferyl-phosphate disodium salt (MUP), alkaline phosphatase (ALP), and sodium hydroxide were purchased from Sigma-Aldrich (Steinheim, Germany). Trisodium citrate dihydrate, iron(II) chloride, and other common reagents were also purchased from Merck, and iron(III) chloride was from Panreac. A buffer solution containing 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris) (Merck) and adjusted to a pH of 9.8 was used. Methanol and chloroform were used for preparing the liposomes. Aqueous solutions were prepared using deionized water purified with a Milli-Q system (Millipore, Bedford, MA, USA). Synthesis and Characterization of the Nanomaterial. Hydrophobic NPs (Fe3O4@AuNPs-DT) were prepared using a procedure 1820

dx.doi.org/10.1021/jf5004804 | J. Agric. Food Chem. 2014, 62, 1819−1825


Article

ALP, the transient immobilization of the reaction product on a resin (Dowex-1 strongly basic anion exchange resin) packed in the flow cell, and the use of the fluorescence intensity as the analytical parameter. A solution containing EDTA (100 mM) and sodium chloride (150 mM) is used to remove the product of the reaction from the support. This method reaches a detection limit of 0.05 U L−1 ALP. Statistical Analysis. All experiments were replicated three times. Statistical analyses were performed with Statgraphics Plus 5.1 software (Statistical Graphics Corp.). The paired t test was used to compare the results obtained for the analysis of milk samples using both the new method and the previously reported method.23

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RESULTS AND DISCUSSION Study of the Features of Hydrophobic Magnetic Nanoparticles. As indicated above, several methods have been described to protect the surface of Fe3O4 NPs against oxidation and corrosion and to improve their reactivity. The use of gold to coat magnetic NPs is frequently chosen because these core/ shell NPs show adequate magnetic properties and chemical stability, and their surface can be easily modified using the welldeveloped Au−S chemistry.15,16 After the synthesis of Fe3O4@ -AuNPs, their surface was covered with DT to obtain hydrophobic MNPs and facilitate their entrapment into the internal medium of the liposomes. These Fe3O4@AuNPs-DT, characterized by TEM (Figure 2A), exhibited spherical structures with an average diameter of 30 ± 10 nm. Also, it was checked that they can be easily separated from the solution when an external magnetic field is applied. The chemical composition of the hydrophobic magnetic NPs was studied using XPS and SEM-EDX. The XPS spectrum (Figure 2B) indicates the presence of Fe, Au, C, S, and O in the NPs. As can be seen in this figure, Fe(2p) electrons show binding energy (BE) at 725 and 712 eV, respectively, whereas the Au(4f) electrons BE correspond to 84 and 89 eV, respectively, which also indicate the presence of iron and gold in the Fe3O4@AuNPs-DT nanocomposites. In addition, the small peaks at 162 and 164 eV correspond to the S(2p3/2) and S(2p1/2) electrons, whereas the strongest peak obtained at 532 eV corresponds to the O(1s) electron. Figure 2C depicts the image acquired using SEM-EDX, the results of which indicate the presence of Fe, Au, and S in the

Figure 1. (A) Scheme of the flow injection system used for the determination of ALP activity. (B) Time schedule of the process. PP, peristaltic pump; IV1 and IV2, sample and reagent injection valves, respectively; EM, electromagnet device; FL, fluorescence detector; W, waste. The same enzymatic reaction was assayed using a flow injection system in the absence of liposomes, with the aim of comparing the features of both methods. For this purpose, a solution containing 10 mM MUP prepared in Tris buffer solution (50 mM, pH 9.8) was pumped at a flow rate of 0.4 mL min−1 and mixed with the same buffer solution in which the ALP solution was injected. A transitory fluorescence signal (λex 365 nm, λem 449 nm, 10/5 slit ratio, detector gain of 600 V) was obtained when the mixed reaction plug passed through the detector. The area of the peak was used as the analytical parameter. Analysis of Food Samples. Several raw and commercial milk samples were processed to determine ALP activity. The raw samples were treated with bronopol as preservative and stored at −15 °C until use. The samples were adequately diluted using 50 mM Tris buffer solution (pH 9.8) and analyzed as described above. Comparison Method. The results obtained by applying the new method to the analysis of milk samples were compared with those obtained using a flow-through method previously described.23 This method involves the hydrolysis of MUP (0.4 mM) in the presence of

Figure 2. (A) TEM image, (B) XPS and (C) SEM-EDX measurements obtained for Fe3O4@AuNPs-DT nanocomposites. 1821



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phobic MNPs27 and maximum encapsulation efficiency for the synthesis of the liposomes.28 The synthesis of Fe3O4@AuNPs was carried out using HAuCl4 and sodium citrate,16 and the surface of these NPs was modified with DT to improve their hydrophobicity and the efficiency of their encapsulation into liposomes. The experimental conditions used to obtain these NPs are summarized in Table 1. Liposomes containing hydrophobic MNPs and MUP were prepared using the RSE method and homogenized by shake/ ultrasound treatments. A method previously described,11 which was modified with respect to the type of phospholipids and the internalized material, was used to obtain these liposomes. The composition of the phospholipid mixture and the chloroform and methanol volume ratio are critical variables in the synthesis process. As Table 1 shows, the best results were obtained using a phospholipid mixture (100 μL) composed by DSPC, DOPE, and CH (75:12.5:12.5), which was diluted to 1 mL with a chloroform/methanol (880:120) mixture. This solution was mixed with a solution containing 3 mL of about 7 mM hydrophobic MNPs, 2.5 mL of 40 mM MUP, and 3.5 mL of deionized water. The influence of the temperature in the RSE method was studied in the range of 30−60 °C, which covered the transition temperature of the phospholipids used. The percentage of liposomes increased up to a temperature of 40 °C, remaining constant at higher temperature values. The study of the stability of the liposome suspension showed that it increased when the suspension was refrigerated at 4 °C. The purification of the liposome suspension, to separate filled liposomes from empty liposomes, phospholipid residues, and untrapped MNPs, was carried out using conventional centrifugation, in which the suspension (10 mL) was treated at different centrifugation speeds, between 2007.3g and 20073g at 4 °C, assaying also variable centrifugation times, between 15 and 30 min. A centrifugation of 20073g for 30 min provided the best results. Filled liposomes, which showed a brown appearance, were located in the intermediate zone, whereas empty liposomes and untrapped NPs appeared in the top and bottom zones, respectively. The liposomes were washed by 100 mL of deionized water four times. Variables Affecting the Enzymatic Reaction. The method was developed by using a flow injection system in which a Tris buffer solution was used as the carrier to transport, first, the MLs containing the enzymatic substrate and, second, the ALP and Triton X-100 mixture. Hydrodynamic, instrumental, and chemical variables were optimized, as shown in Table 2, which includes the ranges assayed and the values chosen for each variable. All of the assays were carried out at 25 °C to avoid degradation of the liposomes. The injection volumes chosen to introduce MLs (IV1) and ALP (IV2) solutions into the system were 1.3 mL and 150 μL, respectively, using a flow rate of 0.4 mL min−1. The influence of the MLs retention and release times on the system is shown in Figure 3A, selecting 200 and 120 s, respectively, as the optimal values. A pH of 9.8 was chosen, which was adjusted using 50 mM Tris buffer solution. Figure 3B shows the influence of Triton X-100 concentration on the analytical signal, in which it can be seen that a 10 mM concentration is suitable to obtain the maximum peak area value. The influence of the enzymatic substrate on the system was studied by assaying different MLs synthesized in the presence of solutions containing different MUP concentrations. As Figure 3C shows, a maximum signal

Fe3O4@AuNPs-DT nanocomposites. The EDX diagram also provides a semiquantitative chemical composition of 64:8:6 from the Fe/Au/S distribution that confirms the composition of the hydrophobic MNPs. The absorption spectrum obtained for these NPs showed the typical band at 530−550 nm corresponding to the surface plasmon resonance of AuNPs. Optimization of Variables. The variables affecting the synthesis of MLs containing MUP and those involved in the development of the method for ALP determination were studied using the univariate method. Tables 1 and 2 summarize the variables studied, the ranges assayed, and the values chosen. Table 1. Optimization of Variables Affecting the Magnetoliposomes Synthesis type of variable

variable

range studied

optimal value

hydrophobic MNP formation

HAuCl4, mM Fe3O4−NPs, mM sodium citrate, mM dodecanethiol, mL

0.23−2.27 5−25 1.5−6.2 1−10

1.13 15 3.1 5

liposome formation

phosphatidylcholine (DSPC), mM phosphatidylethanolamine (DOPE), mM cholesterol (CH), M deionized water, mL chloroform, μL methanol, μL MUP, mM hydrophobic MNPs, mM temperature, °C evaporation time, min rotatory speed, rpm

0.45−0.72

0.68

0.045−0.135

0.11

0.045−0.135 600−900 100−200 0.2−20 0.1−7 30−60 5−30 5−500

0.11 3.5 880 120 10 2.1 40 20 300

temperature, °C time, min

20−100 5−40

40 30

shaking treatment

Table 2. Optimization of Variables Affecting the Enzymatic Reaction type of variable

variable

range studied

optimal value

hydrodynamic

flow rate, mL min−1 sample injection, μL reagent injection, mL retention time, s release time, s

0.2−0.8 50−200 0.3−2.0 5−300 5−300

0.4 150 1.3 200 120

instrumental

λex, nm λem, nm excitation slit, nm emission slit, nm PMT gain, V

200−800 200−800 2.5−10 2.5−10 450−800

360 449 5 10 600

chemical

pH Tris buffer, mM Triton X-100, mM

8−12 20−200 5−150

9.8 50 10

Variables Affecting the Synthesis of Magnetoliposomes Containing the Enzymatic Substrate. The optimization study was carried out to obtain adequate structural features (size, shape, and magnetic efficiency) of the hydro1822



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Figure 3. Influence of experimental variables on the flow system: (A) retention time of the MLs by the magnet (1) integration time of the analytical signal (2); (B) Triton X-100 and (C) MUP concentrations. pH 9.8; [ALP] = 50 mU L−1; [MUP] = 2 mM in (A) and (B); [Triton X-100] = 3 mM in (A) and 10 mM in (C).

was obtained when the MUP concentration was 10 mM MUP, remaining constant for higher substrate concentrations. Features of the Method. The area of the peak obtained in the flow injection scheme depicted in Figure 1A was used as the analytical parameter to obtain the figures of merit of the method. The same system was studied in the absence of MLs with the aim of checking the positive effect of the use of MLs on the ALP determination. Table 3 summarizes the features of both approaches. As can be seen, the dynamic range of the calibration graph in the presence of MLs is 6.4 × 10−3−0.250 U L−1 ALP, and the limit of detection (LOD)29 is 1.9 × 10−3 U L−1, which is 58 times lower than that obtained in the absence of MLs (0.11 U L−1). Also, the LOD is 26 times lower that that reached by retaining the product of the enzymatic reaction in a resin placed in the flow cell (0.05 U L−1).23 Table 3 also shows the repeatability (n = 7, r = 3) and reproducibility (n = 5, r = 3) data, expressed as RSD%, obtained in the presence and absence of liposomes. As can be seen, the precision values are better in the first case. Application of the Method. The ALP activity in milk samples from farms and commercial local markets was determined using the new method, and the results were evaluated by comparison with the flow method involving the retention of the enzymatic reaction product above-described.23 The standard addition method was used to carry out the analysis. Table 4 lists the content found in the samples using both methods. The paired t test was applied to the results at a 5% significance level. It was found that there were not significant differences in the results provided by both methods, which confirms the practical utility of the new method to the analysis of these samples. A recovery study was carried out by

Table 3. Features of the Method FI method with liposomes

without liposomes

equation parameters a b r2 LOD, U L−1 linear range, U L−1

calibration graph

2.9 (±0.7) 1.10 (±0.01) 0.9930 1.9 × 10−3 6.4 × 10−3−0.25

0.64 (±0.03) 0.805 (±0.004) 0.9990 0.11 0.35−10

repeatabilitya (n = 7, r = 3) low level high level

2.4 0.7

8.7 0.9

reproducibilitya (n = 5, r = 3) low level 2.8 high level 1.2

8.8 3.6

RSD% values at 6.4 × 10−3 and 0.023 U L−1 ALP activity for low and high concentration levels, respectively, in the presence of liposomes, and at 0.35 and 4.8 U L−1 ALP activity for low and high concentration levels, respectively, in the absence of liposomes.

a

adding two different amounts of ALP to each sample and subtracting the results obtained from similarly treated unspiked samples. Table 4 shows the recovery percentages, which ranged from 87.5 to 104.6%. This internal validation also confirms the usefulness of the developed MLs method for the analysis of real samples. The utility of the new method was also checked for milk pasteurization verification, in which ALP is used as a thermal 1823



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Table 4. Application of the Method comparison method23

MLs method recovery %a

sample 1 2 3 4 5 6 7 8 9 10 a

type of milk raw raw raw raw raw raw raw raw pasteurized pasteurized

goat goat goat goat cow cow cow cow whole skimmed

dilution factor

[ALP], mU L−1

1000 1000 1000 1000 1000 1000 1000 1000 100 100

3995 4487 3934 2730 6211 5491 6058 5709

± ± ± ± ± ± ± ±

[ALP], mU L−1

42 10 25 49 41 20 9 3

3800 4400 3818 2284 6818 5291 6001 6110 18.2 11.6

± ± ± ± ± ± ± ± ± ±

3 8 6 1 4 17 24 20 0.5 0.5

first add

second add

88.2 89.2 100.1 94.0 90.9 94.0 95.8 96.1 100.0 88.7

100.4 90.8 89.7 104.1 104.1 90.9 90.9 87.5 104.6 99.6

First add, 10 mU L−1; second add, 50 mU L−1.

marker.22 U.S. and EU regulations specify that pasteurized dairy products may contain

Fluorometric Determination of Alkaline ... - ACS Publications

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