Validation of Enzytec™ Liquid Combi Sucrose/D-Glucose for Enzymatic Determination of Sucrose and D-Glucose in Selected Foods and Beverages: First Action 2024.05

Abstract

Background

Sucrose is produced in the greatest quantity of all industrially produced organic substances and can be used in almost all processed foods. Glucose is the most abundant monosaccharide and contained in many foodstuffs naturally or added as an ingredient.

Objective

To validate the performance of the Enzytec™ Liquid Combi Sucrose/D-Glucose test kit for the determination of sucrose and D-glucose in juices, chocolate, breakfast cereals, ice cream, sweetened condensed milk, wine, beer, and soft drinks.

Methods

The kit contains all reagents in a ready-to-use format and is suitable for automation. Sucrose is cleaved by β-fructosidase. The resulting D-glucose from sucrose and glucose originally in the sample are phosphorylated and react in a NADH-generating reaction afterwards. β-Fructosidase is not present in the glucose system. The amount of NADH produced is equivalent to the amounts of sucrose and D-glucose and is measured at 340 nm within 30 min.

Results

The linear measurement range for a 100 µL test volume is from 17 to 2500 mg/L sucrose and 4 to 2000 mg/L D-glucose. Trueness was checked by using four Certified Reference Materials (CRM) and resulted in recoveries from 96 to 105%. Spiking of juices, ice cream, sweetened condensed milk, and shandy resulted in recoveries between 98 and 106% for both systems. Intermediate precision was 3% or lower for sucrose and glucose. For automation, three applications with different test volumes were validated. Linearity is given from 3.8 to 9500 mg/L for sucrose and 2.4 up to 10000 mg/L for glucose.

Conclusion

The method is robust and accurate for manual and automated applications. The method was approved as an AOAC Official Method of Analysis℠.

Highlights

The ready-to-use components of the test kit have a shelf of at least 29 months.

Out of the naturally occurring types of sugar, only a few are used on a large scale as sweeteners for human consumption. In addition to sucrose, glucose (from starch sugar and starch syrup), invert sugar (equimolar mixture of glucose and fructose), maltose, lactose, and fructose play a role (1).

Sucrose is extremely widespread in nature, namely in green plants, leaves, stems, fruits, seeds and also in roots and rhizomes. The two most important raw materials for the production of sucrose are sugar cane and sugar beet (2). In addition, molasses is produced during the extraction of cane and beet sugar. Molasses is used, for example, to produce alcohol and bakery yeast, and as an additive to compound feed (3). Sucrose is the most economically important type of sugar and is produced in the greatest quantity of all industrially produced organic substances (1). In the food industry, sucrose can be used in almost all processed foods. D-glucose is found in most plant products (4). In food, it is present in significant quantities in juice, honey, wine and beer, and in a range of solid foodstuffs such as bread, pastries, and chocolate (5).

Boehringer/Roche decided to discontinue the so-called Yellow Line test kits. The new Enzytec™ Liquid Sucrose/D-Glucose was created as a replacement for the Roche test kit. It was decided to refurbish enzymatic analysis per se. Ready-to-use reagents with a long shelf life and the possibility for automated use were the most important requirements from the market. In particular for the juice sector, automated analysis of sucrose and glucose is necessary.

Single-Laboratory Validation Study Design

The study design was developed to characterize the manual format using 4 mL cuvettes. These sections will contain all necessary performance characteristics as required by AOAC Standard Method Performance Requirement (SMPR^®) 2018.001 (6) for the reported sugar components and are based on a former validation study for ethanol in kombucha and other beverages, which led to OMA 2017.07 Final Action (7, 8). One part of the test kit is identical to AOAC OMA 2024.03 (Enzytec Liquid D-Glucose) and some performance characteristics are only cited here to prevent an overflow of information.

Linearity

The linearity check over a range from 5 to 3000 mg/L sucrose (in water) and 4 to 2000 mg/L D-glucose using three different test kit lots with two replicates in each lot. This experiment was also performed for the automated analyzer in a similar way.

LOD and LOQ (Estimates)

According to DIN 32645:2008–11 (based on DIN ISO 11843–2:2008–06) concentrations ranging from 5 mg/L up to 35 mg/L sucrose (in water) and 4 to 50 mg/L D-glucose were analyzed using three independent test kit lots (n = 2 replicates per lot).

LOQ

Derived out of data from a precision profile. This experiment was also performed for the automated analyzer in a similar way.

Precision Profile

Data were calculated from the linearity data set (aqueous sucrose and D-glucose solutions). The calculated RSD values were derived from two replicates using three independent test kit lots each.

Interferences

Different sugars, sugar substitutes, artificial sweeteners, sugar alcohols, sulfite, and important organic acids were tested in presence of sucrose and D-glucose.

Trueness

Four certified reference materials (NIST SRM 3233 Breakfast Cereals; SRM 1869 Infant/Adult Nutritional Formula II; LGC 7016 Chocolate Confectionary; NIST SRM 3282 Cranberry Juice) were analyzed using six test portions in one or three lots by one analyst.

Recovery

Five different matrixes (orange juice, carrot juice, ice cream, sweetened condensed milk, and shandy) were spiked to half of their endogenous concentrations or higher with the exception of carrot juice, which was spiked to less than the half innate value; six test portions were extracted and analyzed using one lot by one analyst. This experiment was also performed for the automated analyzer in a similar way for the wine matrix.

Repeatability

An orange juice and an ice cream were used over a period of 3 days by one analyst with six replicates per extract and day in one lot; repeatability was also determined during characterization of intermediate precision. This experiment was also performed for the automated analyzer in a similar way.

Inter-Lot Precision

Aqueous sucrose solutions with concentrations of 50, 750, and 1500 mg/L and extracts of NIST SRM 3233 and LGC 7016 were analyzed with six replicates per extract on 1 day by one analyst using three different test kit lots.

Intermediate Precision

Three aqueous sucrose solutions were used directly for measurement and analyzed using three test kit lots by three different analysts on three different days with six replicates per measurement.

Robustness

Incubation temperature was varied between 18 and 37°C. Incubation times before measuring absorption A1 or A2 at 340 nm were varied between 5 and 15 min. NIST SRM 3233 Breakfast Cereals and LGC 7016 Chocolate Confectionary with concentrations above the measurement range were checked for dilutability within the measurement range.

Stability

All three test kit lots were stored at 2 to 8°C for up to 30 months and tested at regular intervals. One lot was used to determine the stability of an open kit stability in parallel.

AOAC Official Method^SM 2024.05

Sucrose and D-Glucose in Selected Foods and Beverages

Enzymatic Determination by Enzytec™ Liquid Combi Sucrose/D-Glucose Manual and Automated Applications

First Action 2024

[Applicable for the quantitative measurement of sucrose and D-glucose in wines, fruit and vegetable juices, ice cream, chocolate, breakfast cereals, sweetened condensed milk, and beer. D-mannose and D-fructose interfere at 6.25 g/L and 12.5 g/L or more, respectively. Raffinose interferes at 8.34 g/L or more in the sample solution. Sulfite interferes at 0.5 g/L or higher. In case the ratio of glucose to sucrose is greater than 7.5, the glucose content must be reduced/removed before measuring sucrose, otherwise the precision of the measurement of sucrose will be impaired. Short freeze–thaw cycles will destroy the functionality of the enzymatic test system.]

Caution: See material safety data sheet and instructions for use available at https://eifu.r-biopharm.com/food/US/food.

A. Principle

The Enzytec Liquid Combi test kit contains two different sets of reagents. One, consisting of reagents 1 and 2, is for the determination of sucrose and free D-glucose as a sum of apparent sucrose (also called the sucrose/D-glucose system) and a second one, consisting of reagents 3 and 4, is for the measurement of free D-glucose (also called the D-glucose system). The sucrose content of a test sample is determined from the results of these two measurements by calculation.

• Sucrose/D-glucose system.—Sucrose is hydrolyzed with H₂O in the presence of β-fructosidase to D-glucose and D-fructose (Figure 2024.05A). The resulting D-glucose (and free D-glucose that was present in the sample before the β-fructosidase treatment) is phosphorylated in the presence of ATP and a hexokinase to D-glucose-6-phosphate (G-6-P). D-glucose-6-phosphate dehydrogenase (G-6-P-DH) oxidizes the G-6-P formed with NAD⁺ to D-gluconate-6-phosphate and NADH/H⁺.

  The amount of NADH formed in this reaction is equivalent to the total amount of D-glucose. The NADH is measured by the increase in absorbance at 340 nm. The result is calculated as mg/L (kg) of apparent sucrose.

• D-glucose system.— Free D-glucose is phosphorylated in the presence of ATP and a hexokinase to G-6-P (Figure 2024.05A). G-6-P-DH oxidizes the G-6-P formed with NAD⁺ to D-gluconate-6-phosphate and NADH/H⁺. The amount of NADH formed in this reaction is equivalent to the amount of D-glucose. NADH is measured by the increase in absorbance at 340 nm. The result is calculated as mg/L (kg) of glucose.

B. Chemicals and Reagents

Items (a)–(d) are available as a test kit (Enzytec Liquid Combi Sucrose/D-Glucose, Cat. No. E8185; R-Biopharm AG, Darmstadt, Germany). Refer to kit label for expiry at 2–8°C (36–46°F) and for date of production.

• Reagent 1.—1 × 50 mL (β-fructosidase, NAD, and ATP in buffer).

• Reagent 2.—1 × 12.5 mL (hexokinase and Glu-6-P-DH in buffer).

• Reagent 3.—1 × 50 mL (NAD and ATP in buffer).

• Reagent 4.—1 × 12.5 mL (hexokinase and Glu-6-P-DH in buffer).

  Remark: Reagents 1 and 2 are also available as Enzytec Liquid Sucrose/D-Glucose (Cat. No. E8180), while reagents 3 and 4 are available as Enzytec Liquid D-Glucose (Cat. No. E8140), which was approved as AOAC OMA 2024.03. The composition and production of these reagents is identical to E8185.

• Enzytec Multi-sugar standard high.—10000 mg/L sucrose and 10000 mg/L D-glucose, Cat. No. E8445 (R-Biopharm).

• Enzytec Multi-sugar standard low.—500 mg/L sucrose and 500 mg/L D-glucose, Cat. No. E8440 (R-Biopharm).

  Required but not provided with the test kit:

• Distilled water.

• Potassium hydroxide, 1 M.—Solubilized in water; do not store in glass vials and keep plastic containers closed to prevent reaction with carbon dioxide.

• Polyvinylpolypyrrolidone (PVPP).—e.g., Anafin Soft P, ZEFÜG GmbH & Co. KG, Bingen, Germany.

• Carrez Solution I.—85 mM; 3.60 g K₄[Fe(CN)₆] ⋅ 3 H₂O/100 mL).

• Carrez Solution II.—250 mM; 7.20 g ZnSO₄ ⋅ 7 H₂O/100 mL).

• Sodium hydroxide, 0.1 M.—Solubilized in water; do not store in glass vials and keep plastic containers closed to prevent reaction with carbon dioxide.

C. Apparatus

The specified apparatus has been tested. Equivalent apparatus may be used.

• Analytical balance.—Entris 623i-1S, Sartorius Lab Instruments, Goettingen, Germany.

• pH meter.—FiveEasy™ pH/mV bench meter, Mettler-Toledo, Giessen, Germany.

• Beakers.

• Fluted paper filters.

• Magnetic stirrer.—Cimarec™ Poly 15, Fisher Scientific, Schwerte, Germany.

• Graduated flasks.—50 mL, 100 mL, and 200 mL.

• Reaction tubes.—2 mL; re-closable.

• Syringe filters.—Minisart^® High Flow syringe filter, type 16532 K, Sartorius.

• Disposable plastic cuvettes.—1 cm light path.

• Micropipettors.—20–200 and 100–1000 μL.

• Multi-stepper pipette.—To dispense 2 mL aliquots of reagent 1/reagent 3 and 500 µL reagent 2/reagent 4 for manual pipetting.

• Spectrophotometer capable of reading at 340 nm.—Cary 60, UV-Vis spectrophotometer, Agilent Technologies, Waldbronn, Germany, or equivalent. For manual application.

• Analyzer.—Pictus 500 (Diatron, Budapest, Hungary) or equivalent. For automated application.

• Vortex mixer.

• Ultrasonic bath.—Sonorex Super RK 31, Bandelin, Berlin, Germany.

• Centrifuge.—Mikrozentrifuge 5427 R, Eppendorf SE, Hamburg, Germany.

D. General Preparation

Store the kit at 2–8°C (36–46°F). Let all kit components come to room temperature between 20 and 25°C (68–77°F) before use. Do not freeze any of the kit components.

Use separate tips for each test and control solution to avoid cross-contamination and pre-flush the tip before pipetting.

Use a multi-stepper pipette for addition of the three reagents. Use a single tip for each of these components.

Components and procedures of the test kit have been standardized for use as descibed here. Do not interchange components between kits of different batches (lot numbers).

Store samples in a cold and dry room protected from light. Ensure that no cross-contamination takes place.

E. Sample Preparation

Use clear, colorless, and pH-neutral liquid samples directly, or after dilution between 17 and 2500 mg/L apparent sucrose and 4 to 2000 mg/L glucose. Increase the sample volume if concentrations close to the LOQ are expected, e.g., for red wine (see also G[i]).

For turbid samples, filter (fluted paper filter or syringe filter) or centrifuge the sample in a reaction tube (recommended 3000g for at least 5 min) until a clear filtrate or supernatant is obtained.

Degas samples containing carbon dioxide with the aid of an ultrasound burst.

Adjust the pH value of strongly acidic samples (wines and juices) by adding 1 M KOH to a known test sample volume until the pH value is between 6.5 and 7.5 (using the pH meter); shake or stir between additions; bring to a known volume and dilute further if necessary with distilled water.

Use PVPP in the case of juices or wines with a strong dark color that are measured undiluted: Add 0.1 g PVPP to 10 mL of juice or wine, stir/shake for 1 min, and filter (fluted paper filter or syringe filter) or centrifuge using reaction tubes.

Crush or homogenize solid or semi-solid samples. Weigh a sufficient quantity of sample in a volumetric flask (considering the measuring range), extract with water; make up to the mark and filter if necessary using fluted paper filters, syringe filters, or centrifugation in reaction tubes. Additionally, use Carrez clarification if necessary [see below in this section].

For fat-containing samples like chocolate, weigh a sufficient quantity (considering the measuring range) into a volumetric flask, extract with hot water (approximately 70°C), and heat in a water bath at 60–65°C for 20 min. Cool to allow the fat to separate, make up to the mark with water (fat above the mark); place the volumetric flask in an ice bath for 15 min and filter (fluted paper filter or syringe filter); and use a clear filtrate for analysis.

Alternatively, use Carrez clarification after extraction: Weight an appropriate amount of sample into a 100 mL beaker and add 60 mL distilled water. In the case of liquid samples, pipette the sample into a 100 mL beaker pre-filled with 60 mL disilled. water. Afterwards, add 5 mL Carrez-solution I and 5 mL Carrez-solution II to the flask. Mix after each addition. Adjust the pH with 0.1 M NaOH to between 7.5 and 8.5, using with pH meter. Transfer into a 100 mL volumetric flask, make the flask up to the mark, mix, and filter using fluted paper filters or syringe filters.

F. Determination

For a test volume of 100 µL in the manual application, the linear range is 17 to 2500 mg/L sucrose and 4 to 2000 mg/L D-glucose. The LOD values are 5.0 mg/L and 1.4 mg/L for sucrose and D-glucose, respectively. For the automated applications, the measurement ranges for sucrose are from 15 to 1900 mg/L (basic), 75 to 9500 mg/L (high range), and 3.8 to 190 mg/L (sensitive). For the automated applications, the measurement ranges for D-glucose are 18 to 1900 mg/L (Basic), 75 to 10000 mg/L (High range), and 2.4 to 190 mg/L (Sensitive).

• Manual assay in 4 mL cuvettes.—

  1. It is recommended to use control samples like references or standard solutions [e.g., referenced in B(e) and (f)].

  2. Pipette the test or control solutions with a variable micropipette and the reagents 1, 2, 3, and 4 with a multi-stepper pipette to ensure good mixing.

  3. Insert a sufficient number of cuvettes in a holder for a single determination of all test or control solutions. Record the test and control positions.

  4. With each measurement, it is necessary to determine a reagent blank (RB) using distilled water instead of test or control solution.

  5. Follow the pipetting scheme (see  Table 2024.05A):

     Table 2024.05A.

     Pipetting scheme for the manual assay

     System	Sucrose/glucose		Glucose
     Pipette into cuvette	Blank	Sample	Blank	Sample
     Reagent 1	2000 µL	2000 µL	—	—
     Reagent 3	—	—	2000 µL	2000 µL
     Dist. water	100 µL	—	100 µL	—
     Sample	—	100 µL	—	100 µL
     	Mix with disposable plastic spatula and incubate for 15 min at temperatures between 20 and 37°C		Mix with disposable plastic spatula and incubate for 3 min at temperatures between 20 and 37 °C
     	Read absorbance A1 at 340 nm
     Reagent 2	500 µL	500 µL	—	—
     Reagent 4	—	—	500 µL	500 µL
     	Mix and incubate for 15 min at temperatures between 20 and 37°C		Mix and incubate for 15 min at temperatures between 20 and 37°C
     	Read absorbance A2 at 340 nm

  6. In the case of higher test volumes (up to 1000 µL), the volumes for all reagents remain unchanged; remember to use the modified calculation as described in section G; check the pH value of the sample and neutralize the pH in case of any doubt: test solutions must be clear.

• Automation on a Pictus 500 device.—See  Table 2024.05B.

  1. Calibration is required when using the Pictus device.—To give a user maximum flexibility, three different applications with different measurement concentration ranges, depending on a test volume of 2, 10, or 100 µL, are provided. Volumes of reagent 1 and reagent 3 are reduced to 200 µL and reagent 2 and reagent 4 to 50 µL and are not changed for the three different applications.

  2. To ensure that these calibrations are valid over a longer period of, for example, 1 week, aqueous control solutions (see  B[e] and [f]) have to be analyzed with every run. In case these control solutions are not within specifications (see  H[b]), a re-calibration must be done.

  3. Sucrose/D-glucose system.—

     • (a) High-range application.—Two-point linear calibration with 0 mg/L (water) and 7500 mg/L of apparent sucrose (use Enzytec Multi-sugar standard high, B[e], and use calculation given in G[e]).

     • (b) Basic-range application.—Two-point linear calibration with 0 mg/L (water) and 1500 mg/L of apparent sucrose (use Enzytec Multi-sugar standard high, B[e], and dilute with water; use calculation given in G[e]).

     • (c) Sensitive-range application.—Four-point calibration with 0, 15, 45, and 150 mg/L of apparent sucrose (use Enzytec Multi-sugar standard low, B[f], and dilute with water; use calculation given in G[e]).

  4. D-glucose system.—

     • (a) High-range application.—Two-point linear calibration with 0 mg/L (water) and 10000 mg/L D-glucose (use Enzytec Multi-sugar standard high, B[e]).

     • (b) Basic-range application.—Two-point linear calibration with 0 mg/L (water) and 1500 mg/L D-glucose (use Enzytec Multi-sugar standard high, B[e], and dilute with water).

     • (c) Sensitive-range application.—Four-point calibration with 0, 4.5, 45, and 150 mg/L D-glucose (use Enzytec Multi-sugar standard low, B[f], and dilute with water).

  5. Determination.—

     Table 2024.05B.

     Pipetting scheme for the automated assay

     System	Sucrose/glucose		Glucose
     Pipette into cuvette	Blank	Sample	Blank	Sample
     Reagent 1	200 µL	200 µL	—	—
     Reagent 3	—	—	200 µL	200 µL
     Distilled water	2/10/100 µLa	—	2/10/100 µLa	—
     Calibratorb or sample	—	2/10/100 µLa	—	2/10/100 µLa
     	Incubate for 15 min at 37°C		Incubate for 2 min at 37°C
     	Read absorbance A1 at 340 nm
     Reagent 2	50 µL	50 µL	—	—
     Reagent 4	—	—	50 µL	50 µL
     	Incubate for 15 min at 37°C		Incubate for 10 min at 37°C
     	Read absorbance A2 at 340 nm

     a

     Volume depending on application: high range, basic range or sensitive range.

     b

     Running each calibrator in quadruplicate (four replicates) is recommended.

• Recommendations for other automated analyzers.—The ratio of 4:1 for reagent 1 and 2 (and, accordingly, reagent 3 and 4) should not be changed and the test volume should not be bigger than twice the volume of reagent 2. A calibration must be performed but is often stable for several days so that it does not need to be repeated on every day. Control solution(s) should be analyzed with every run to check the validity of the calibration. If these control solutions are not within specifications, a re-calibration must be performed.

G. Calculations

• Calculate ΔA for every test or control solution (manual).—

  $$\Delta \mathrm{A}={\left(\mathrm{A}2-\text{df} \mathrm{\times } \mathrm{A}1\right)}_{\text{test}\hspace{0.17em}\text{or}\hspace{0.17em}\text{control}}– {\left(\mathrm{A}2-\text{df} \mathrm{\times } \mathrm{A}1\right)}_{\text{reagent}\hspace{0.17em}\text{blank}}$$
  where df is a dilution factor calculated as follows:

  $$\begin{array}{c}100 \mu \mathrm{L}, \text{df}=\left(\text{sample} \text{volume}+\mathrm{R}1\right)/\left(\text{sample} \text{volume}+\mathrm{R}1+\mathrm{R}2\right)=0.808\\ 1000 \mu \mathrm{l}, \text{df}=\left(\text{sample} \text{volume}+\mathrm{R}1\right)/\left(\text{sample} \text{volume}+\mathrm{R}1+\mathrm{R}2\right)=0.857\end{array}$$
• Calculate concentrations for every test or control solution (manual).—

  $$\mathrm{c}=(\mathrm{V} \mathrm{\times } \text{MW}\mathrm{\times }\Delta \mathrm{A})/(\epsilon \mathrm{\times }\mathrm{d}\mathrm{\times }\mathrm{v})$$

  where V = final volume; MW = molecular weight of sucrose or D-glucose; ε = absorption coefficient of NADH at 340 nm; d = light path within cuvette; and v = test volume

• For sum of sucrose and D-glucose (apparent sucrose).—

  Apparent sucrose [mg/L] =

  (2.6 mL × 342.3 g × mole⁻¹ × ΔA)/

  (6.3 L × mmole⁻¹ × cm⁻¹ x 2 × 1 cm × 0.1 mL)

  or

  Apparent sucrose [mg/L] = 1413 × ΔA

• For D-glucose.—

  D-glucose [mg/L] =

  (2.6 mL × 180.16 g × mole⁻¹ × ΔA)/

  (6.3 L × mmole⁻¹ × cm⁻¹ × 1 cm × 0.1 mL)

  or

  D-glucose [mg/L] = 743.5 × ΔA

  1. If a test solution was diluted before measurement, this result has to be multiplied with the dilution factor.

• For sucrose calculation.—The result takes into account the ratio in molecular weight (factor 1.9) of the glucose subtracted from the apparent sucrose content:

  $$\text{Sucrose} \left[\text{mg}/\mathrm{L}\right]=\mathrm{c} \left[\text{mg}/\mathrm{L} \text{apparent} \text{sucrose}\right]–\mathrm{c}$$

   

  $$\left[\text{mg}/\mathrm{L} \mathrm{D}-\text{glucose}\right] \mathrm{\times } 1.9$$

  Remark: For different test volumes up to 1000 µL, the formulas in G(c) and G(d) must be adapted.

• Excess of D-glucose compared to sucrose.—In case the ratio of glucose to sucrose is greater than 7.5, the glucose content must be reduced/removed before measuring sucrose (see application note for Enzytec Liquid Glucose Remover available by contacting [email protected]).

• Calculation in solid samples.—Content [sucrose or D-glucose] = (g ⁄ L sucrose or D-glucose test solution/test sample weight in g ⁄ L test solution)*100 [g/100 g]

• Calculations for automated analysis.—The Pictus 500 device will calculate a calibration function from the calibrators and use this function to calculate concentrations for unknown test and control solutions.

• Results close to LOQ.—If the result for apparent sucrose is between 30 und 50 mg/L for a test volume of 100 µL, repeat measurement with 200 µL, if lower than 50 mg/L, repeat measurement with 500 µl or 1000 µL test volume for better accuracy (trueness, precision).

  $$\begin{array}{c}\text{Measurement} \text{range} \text{for} 200 \mu \mathrm{L} \text{test} \text{volume:} 10 \text{mg}/\mathrm{L} \text{to} 1250 \text{mg}/\mathrm{L}\\ \text{Measurement} \text{range} \text{for} 1000 \mu \mathrm{L} \text{test} \text{volume:} 2 \text{mg}/\mathrm{L} \text{to} 250 \text{mg}/\mathrm{L}\end{array}$$

H. System Suitability Tests/Analytical Quality Control

• For each analytical run an aqueous control solution (e.g., Enzytec Multi-sugar standard low, 500 mg/L D-glucose and 500 mg/L sucrose; B[e] and [f]) should be analyzed. The apparent sucrose concentration of this solution is 500 mg/L + 500 mg/L × 1.9 = 1450 mg/L according to G(e). If available, use certified reference materials.

• Recoveries for an aqueous control solution should be within the 95 to 105% range.

• If results do not fall within this range.—

  1. Check data sets for any suspicious values, e.g., increased A1 values or unexpected high variation

  2. Check incubation temperature and time.

  3. Check pipettes for accuracy.

  4. Check photometer for correct wavelength.

Results and Discussion

General Remarks

The following sections contain the characterization of the manual format using 4 mL cuvettes. These sections will contain all necessary performance characteristics as required by AOAC SMPR 2018.001 (6) for the reported sugar components, which are based on a former validation study for ethanol in kombucha and other beverages that led to OMA 2017.07 Final Action (7, 8). Performance characteristics for the glucose system (reagents 3 and 4) were taken from AOAC OMA 2024.03 and are only cited here very briefly.

In addition, the Diatron Pictus 500 spectrophotometric auto-analyzer was taken as one example of an automated pipetting device that is used regularly in laboratories performing enzymatic analysis. Since some performance characteristics are independent on the way the test kit is used, each single performance characteristic was not repeated for both manual and automated applications.

Linearity/Measurement Range

Figure 1 shows the characterization of linearity of the sucrose/D-glucose system using mean data from three lots with three independent runs each for concentration ranging from 5 to 3000 mg/L for sucrose. The upper value for linearity is 2500 mg/L. All RSD values within the measurement range (17 to 2500 mg/L) are at 5% and below 2% for concentrations between 100 and 2500 mg/L.

Figure 1.

Linearity of the system between 5 and 3000 mg/L for sucrose from data derived from three lots with three replicates and a test volume of 100 µL; the regression equation was calculated without the data marked as open circles.

The upper limit of linearity of the D-glucose system is 2000 mg/L D-glucose and was confirmed after a storage time of 24 months at 2 to 8°C (see AOAC OMA 2024.03).

This upper limitation of linearity is valid for samples that contain mostly sucrose or additional glucose in comparable amounts. If the mass ratio between glucose and sucrose increases by more than a factor of 7.5, a reduced recovery of sucrose is observed for the sucrose/D-glucose system (Figure 2). For a reliable and precise measurement of sucrose for ratios greater than 7.5, most of the glucose has to be destroyed by using Enzytec Liquid Glucose Remover (application note available on request).

Figure 2.

Effect of the mass ratio between D-glucose and sucrose on recovery of 100 mg/L sucrose derived from six replicates for each ratio.

To check whether at a D-glucose to sucrose ratio of 7.5 the LOQ of 17 mg/L is still valid, sucrose solutions with concentrations ranging from 10 to 50 mg/L were measured in the presence of a 7.5-fold D-glucose concentration. To calculate the real sucrose value from the apparent sucrose value, the D-glucose concentration had to be measured.

The results in Figure 3 clearly show that at a concentration of 20 mg/L sucrose or more, the recovery is close to 100% and the RSD value is about 10%. The LOQ for a sucrose solution without D-glucose was calculated to be 17 mg/L.

Figure 3.

Characterization of recovery for sucrose in the presence of D-glucose at a ratio of 1:7.5; mean recovery and RSD value from six replicates are shown; the RSD for 10 mg/L could not be calculated because the mean concentration was zero.

An experiment to characterize the whole measurement range when using a higher test volume was not performed for sucrose/D-glucose in the manual application but was performed for the automated version (high-range application; see Automation on a Pictus 500 spectrophotometric analyzer).

Estimation of LOD

The LOD was determined for the sucrose/D-glucose system according to DIN 32645 (comparable to DIN ISO 11843–2). A detailed description of the calculation can be found in AOAC OMA 2017.05 (7). Each measurement was performed with an aqueous sucrose control solution that was diluted to concentrations between 5 and 35 mg/L (ten different equidistant concentrations; the test volume is 100 µL; see  Figure 1). This set of dilutions was tested three times independently using three lots resulting in LOD values of 1.91, 4.97, and 3.23 mg/L, respectively. According to these results, the LOD is 5 mg/L sucrose.

The D-glucose system showed LOD values ranging between 0.7 and 1.4 mg/L D-glucose (see AOAC OMA 2024.03 for Enzytec Liquid D-Glucose).

Estimation of LOQ

The LOQ was also determined for the sucrose/D-glucose system according to DIN 32645 (comparable to DIN ISO 11843–2). Each measurement was tested with ten different aqueous solutions with concentrations between 5 and 35 mg/L sucrose. This set of dilutions was tested three times independently with each lot resulting in LOQ values of 3.46, 8.97, and 5.83 mg/L, respectively.

This led to the assumption that the LOQ is around 9 mg/L. Data in Figure 4 clearly show that this level does not fulfil a maximum RSD around 10%. This criterion is met at 17 mg/L or higher for sucrose. A comparable experiment was performed for the D-glucose system and revealed an LOQ of 4 mg/L glucose derived from a precision profile. Larger sample volumes were tested for the automated application (see Automation on a Pictus 500 spectrophotometric analyzer).

Figure 4.

Measured mean (mg/L sucrose) values for aqueous sucrose solutions with concentrations between 5 and 35 mg/L for three independent measurements of three lots (closed circles) and a test volume of 100 µL; the RSD, %, is also given (open triangles).

Selectivity Study: Interference

Table 1 shows that raffinose interferes at concentrations higher than 8.34 g/L, while mannose and fructose react at concentrations higher than 6.25 g/L and 12.5 g/L, respectively.

Table 1.

Interference towards mannose, raffinose, and fructose in the presence of sucrose (0.5 g/L)

	Sucrose (0.5 g/L)
Interference	Concn, g/L	A 1	A2	Measured, g/L	Recovery, %
Control	0.5	0.608	0.876	0.499	99.9
Raffinose	33.4	0.609	0.964	0.624	125
	33.4	0.591	0.958	0.618	124
	16.7	0.609	0.920	0.561	112
	16.7	0.590	0.911	0.553	111
	8.34	0.609	0.894	0.525	105
	8.34	0.590	0.894	0.529	106
Mannose	50	0.607	0.547	0.035	7.1
	50	0.611	0.554	0.041	8.2
	25	0.610	0.582	0.082	16.5
	25	0.590	0.576	0.080	15.9
	12.5	0.609	0.647	0.175	35.1
	12.5	0.589	0.638	0.169	33.8
	6.25	0.610	0.886	0.512	102
	6.25	0.590	0.882	0.512	102
Fructose	100	0.610	0.709	0.260	52.0
	100	0.610	0.707	0.259	51.8
	50	0.609	0.787	0.373	74.6
	50	0.590	0.776	0.362	72.4
	25	0.609	0.844	0.454	90.9
	25	0.589	0.836	0.448	89.5
	12.5	0.610	0.874	0.495	99.0
	12.5	0.588	0.868	0.494	98.9

Table 2 shows other substances that have been tested for assay interference. A concentration of 10 g/L of the interfering substance was measured in the presence of 250 mg/L sucrose and 250 mg/L D-glucose (725 mg/L apparent sucrose) and the recovery was determined. For sulphite, a recovery of more than 300% was observed. The interference is already obvious after adding sulphite into reagent 1. This effect was no longer observed at 0.5 g/L sulphite or lower. The other substances did not have an interfering effect in the determination of sucrose and D-glucose.

Table 2.

Interference towards important organic acids, glycerol, sulphite, and other naturally occurring substances in the presence of sucrose (0.25 g/L) and D-glucose (0.25 g/L)

		Apparent sucrose (0.725 g/L)
Interference	Concn, g/L	A 1	A 2	Measured, g/L	Recovery, %
Blank	—a	0.555	0.477	—	—
Blank	—	0.554	0.474	—	—
Reference	0.725	0.554	0.983	0.717	98.9
Reference	0.725	0.553	0.984	0.718	99.0
NaCl	10	0.556	0.975	0.703	97.0
NaCl	10	0.557	0.969	0.692	95.5
Taurine	10	0.558	0.981	0.712	98.1
Taurine	10	0.556	0.976	0.706	97.4
Glycerol	10	0.554	0.979	0.713	98.4
Glycerol	10	0.554	0.974	0.704	97.1
Ascorbic acid	10	0.582	1.008	0.720	99.3
Ascorbic acid	10	0.587	1.010	0.717	98.9
Citric acid	10	0.548	0.977	0.717	98.9
Citric acid	10	0.549	0.974	0.711	98.1
Acetic acid	10	0.548	0.975	0.713	98.4
Acetic acid	10	0.547	0.975	0.712	98.3
Oxalic acid	10	0.545	0.977	0.721	99.5
Oxalic acid	10	0.545	0.973	0.715	98.6
Benzoic acid	10	0.553	0.988	0.724	99.9
Benzoic acid	10	0.554	0.976	0.705	97.3
D/L-malic acid	10	0.544	0.986	0.735	101
D/L- malic acid	10	0.544	0.980	0.725	100
D/L-lactic acid	10	0.554	0.976	0.706	97.4
D/L-lactic acid	10	0.554	0.973	0.702	96.8
D/L-tartaric acid	10	0.542	0.974	0.719	99.1
D/L-tartaric acid	10	0.543	0.972	0.715	98.7
Gluconic acid	10	0.553	0.982	0.716	98.7
Gluconic acid	10	0.554	0.978	0.710	97.9
Galactaric acid	10	0.553	0.986	0.724	99.9
Galactaric acid	10	0.553	0.981	0.717	98.9
Glucosamine	10	0.553	0.999	0.740	102
Glucosamine	10	0.553	0.994	0.733	101
Sulphite	10	2.204	3.553	2.464	340
Sulphite	10	2.191	3.589	2.528	349
Sulphite	5	1.947	3.320	2.426	335
Sulphite	5	1.955	3.369	2.479	342
Sulphite	0.5	0.559	0.991	0.721	99.5
Sulphite	0.5	0.560	0.971	0.684	94.4
Sulphite	0.1	0.554	0.983	0.715	98.6
Sulphite	0.1	0.554	0.985	0.712	98.2

a

— = Not applicable.

Table 3 shows the sweeteners that have been tested for interference with the determination of sucrose and D-glucose. A concentration of 10 g/L of the interfering substance was measured in the presence of 250 mg/L sucrose and 250 mg/L D-glucose (725 mg/L apparent sucrose) and the recovery was determined. None of the substances showed an interfering effect in the determination of sucrose/D-glucose.

Table 3.

Sweeteners tested for interference in the presence of sucrose (0.25 g/L) and D-glucose (0.25 g/L)

		Apparent sucrose (0.725 g/L)
Interference	Concn, g/L	A 1	A 2	Measured, g/L	Recovery, %
Blank	—a	0.556	0.477	—	—
Blank	—	0.558	0.479	—	—
Reference	0.725	0.555	0.998	0.738	101.8
Reference	0.725	0.553	0.979	0.711	98.0
Acesulfame	10	0.554	0.981	0.715	98.7
Acesulfame	10	0.553	0.978	0.710	97.9
Adventame	10	0.557	0.959	0.679	93.7
Adventame	10	0.555	0.958	0.681	93.9
Aspartame	10	0.555	0.978	0.710	98.0
Aspartame	10	0.553	0.974	0.704	97.2
Cyclamate	10	0.554	0.982	0.714	98.5
Cyclamate	10	0.554	0.977	0.710	97.9
Neohesperidin	10	0.697	1.083	0.696	96.0
Neohesperidin	10	0.693	1.080	0.694	95.8
Neotame	10	0.554	0.977	0.707	97.5
Neotame	10	0.556	0.954	0.674	93.0
Saccharin	10	0.554	0.981	0.714	98.5
Saccharin	10	0.554	0.979	0.710	98.0
Sucralose	10	0.555	0.981	0.713	98.4
Sucralose	10	0.555	0.983	0.718	99.0
Thaumatin	10	0.560	0.970	0.692	95.4
Thaumatin	10	0.559	0.959	0.677	93.4
Alitame	10	0.555	0.983	0.715	98.6
Alitame	10	0.554	0.975	0.706	97.4

a

— = Not applicable.

Table 4 summarizes the results for nutritive sweeteners that have been tested for interference with the determination of sucrose and D-glucose. A concentration of 10 g/L of the interfering substance was measured in the presence of 725 mg/L apparent sucrose and the recovery was determined. None of the substances showed an interfering effect on the determination of sucrose/D-glucose with the exception of corn starch syrup powder, oligofructose, and trehalose.

Table 4.

Sugar substitutes (nutritive sweeteners) tested for interference in the presence of sucrose (0.25 g/L) and D-glucose (0.25 g/L)

		Apparent sucrose (0.725 g/L)
Interference	Concn, g/L	A 1	A 2	Measured, g/L	Recovery, %
Water	—a	0.555	0.475	—	—
Water	—	0.553	0.474	—	—
Reference	0.725	0.554	0.983	0.718	99.0
Reference	0.725	0.554	0.984	0.712	98.2
Sorbitol	10	0.553	0.982	0.717	98.9
Sorbitol	10	0.554	0.982	0.716	98.8
Mannitol	10	0.554	0.983	0.719	99.1
Mannitol	10	0.554	0.980	0.707	97.5
Isomalt	10	0.554	0.980	0.714	98.5
Isomalt	10	0.554	0.981	0.715	98.6
Maltitol	10	0.554	0.978	0.710	98.0
Maltitol	10	0.554	0.978	0.704	97.1
Lactitol	10	0.553	0.976	0.708	97.6
Lactitol	10	0.554	0.978	0.710	97.9
Xylitol	10	0.554	0.977	0.709	97.8
Xylitol	10	0.554	0.977	0.703	97.0
Erythritol	10	0.553	0.973	0.706	97.3
Erythritol	10	0.554	0.978	0.710	98.0
Inulin	10	0.554	0.966	0.691	95.3
Inulin	10	0.554	0.994	0.723	99.7
Isomaltulose	10	0.555	0.983	0.717	98.9
Isomaltulose	10	0.554	0.980	0.708	97.6
Corn syrup powder	10	0.553	5.064	6.484	894
Corn syrup powder	10	0.555	4.349	5.471	755
Oligofructose	10	0.555	1.306	1.174	162
Oligofructose	10	0.555	1.302	1.161	160
Trehalose	10	0.554	1.064	0.833	115
Trehalose	10	0.553	1.047	0.809	112

a

— = Not applicable.

The syrup powder is produced from starch by chemical and/or enzymatic hydrolysis. Therefore, maltodextrins, maltotriose, maltose, and D-glucose will be present in high amounts. It was not possible to obtain pure oligofructose. The certificate from the producer states that the sum for sucrose, D-glucose, and D-fructose is 8%, which would explain the overestimation of 160%. Trehalose is a 1-α-glucopyranosyl-1-α-glucopyranoside and may contain traces of free D-glucose that explains recoveries higher than 100%.

Interference data for the glucose system (reagents 3 and 4) have already been submitted to AOAC as Enzytec Liquid D-Glucose for OMA 2024.03. They will be available as a publication in this journal. For D-glucose, mannose and fructose interfere at concentrations higher than 5.1 g/L and 12.5 g/L, respectively. For OMA 985.09 the D-fructose interference was at the same level when analyzing 100 g/L D-fructose in the presence of 0.5 g/L D-glucose. This high excess of D-fructose compared to D-glucose does not occur under practical conditions in food. Nevertheless, the interference is stated in the instructions for use for the test kit. Sulphite exerts interference at levels higher than 1.25 g/L. Measured concentrations for corn syrup powder, trehalose, and oligofructose were comparable in both systems. The overestimation of D-glucose in maltose can be explained by traces of D-glucose in the maltose reference standard, which was certified by the manufacturer Sigma-Aldrich.

Trueness

Three certified reference materials (NIST SRM 3233 Breakfast Cereals, NIST SRM 1869 Infant/Adult Nutritional Formula II, and LGC 7016 Chocolate Confectionary) were used to characterize for trueness.

All three certified reference materials are certified for their sucrose content and one of them for its glucose content (SRM 1869). Therefore, the content of free D-glucose has to be measured in the same test sample extract for exact calculation of the sucrose content from the apparent sucrose content. Besides a remarkably low RSD value at 2% for all three materials, the mean recovery for sucrose was 96% for the breakfast cereals (Table 5), 99% for the chocolate confectionary (Table 6), and 104% for adult/infant formula (Table 7). For glucose, the recovery for SRM 1869 was 105% (Table 7). Using the procedure described in the European Reference Material (ERM) Application Note No. 1 (9), all data sets were checked for significant differences to the certified value—none were found.

Table 5.

Repeated measurement (n = 6 test portions) of the certified reference material NIST SRM 3233 Breakfast Cereals with a sucrose target value of 13.42 g/100 g for characterization of trueness in three test kit lots by one analyst; values for free D-glucose in the sample were determined and subtracted from the apparent sucrose value as described (see G[e])

NIST SRM 3233
Replicate	Apparent sucrose, g/L	D-glucose, g/L	Sucrose, g/L	Sucrose, g/100 g	Sucrose rec., %
	Lot 1
1	7.09	0.44	6.24	12.46	92.9
2	7.16	0.44	6.32	12.52	93.3
3	7.23	0.54	6.20	12.22	91.1
4	7.26	0.45	6.40	12.63	94.1
5	7.29	0.45	6.42	12.64	94.2
6	7.34	0.44	6.50	12.92	96.2
	Lot 2
1	7.37	0.44	6.53	13.03	97.1
2	7.53	0.44	6.68	13.24	98.7
3	7.64	0.54	6.61	13.03	97.1
4	7.37	0.45	6.51	12.86	95.8
5	7.55	0.45	6.69	13.15	98.0
6	7.43	0.44	6.59	13.10	97.6
	Lot 3
1	7.28	0.44	6.44	12.86	95.8
2	7.47	0.44	6.63	13.13	97.8
3	7.63	0.54	6.60	13.00	96.9
4	7.56	0.45	6.70	13.22	98.5
5	7.36	0.45	6.50	12.78	95.3
6	7.42	0.44	6.58	13.08	97.5
			Mean	12.88	96.0
			SD	0.29
			RSD, %	2.23

Table 6.

Repeated measurement (n = 6 test portion) of the certified reference material LGC 7016 Chocolate Confectionary with a sucrose target value of 46.5 g/100 g for characterization of trueness in three lots by one analyst; values for free D-glucose in the sample were determined and subtracted from the apparent sucrose value as described (see G[e])

LGC 7016
Replicate	Apparent sucrose, g/L	D-glucose, g/L	Sucrose, g/L	Sucrose, g/100 g	Sucrose rec., %
	TC1
1	9.27	0.02	9.22	45.64	98.1
2	9.39	0.02	9.35	46.47	99.9
3	9.48	0.01	9.46	46.02	99.0
4	9.34	0.02	9.30	45.63	98.1
5	9.60	0.02	9.55	46.89	100.8
6	9.61	0.02	9.57	46.64	100.3
	TC 2
1	9.28	0.02	9.24	45.70	98.3
2	9.37	0.02	9.33	46.36	99.7
3	8.87	0.01	8.84	43.02	92.5
4	9.34	0.02	9.30	45.65	98.2
5	9.57	0.02	9.53	46.75	100.5
6	9.59	0.02	9.54	46.52	100.0
	TC 3
1	9.25	0.02	9.21	45.55	98.0
2	9.38	0.02	9.34	46.43	99.8
3	9.50	0.01	9.48	46.10	99.1
4	9.40	0.02	9.36	45.93	98.8
5	9.62	0.02	9.57	46.97	101.0
6	9.61	0.02	9.57	46.65	100.3
			Mean	46.05	99.0
			SD	0.89
			RSD, %	1.93

Table 7.

Repeated measurement (n = 6 test portions) of the certified reference material NIST SRM 1869 Infant/Adult Nutritional Formula II; milk/whey/soy-based with a sucrose target value of 27.88 ± 0.60 g/100 g (k = 2) and a glucose target of 2.00 ± 0.45 g/100 g (k = 2) for characterization of trueness in one lot by one analyst; values for free D-glucose in the sample were determined and subtracted from the apparent sucrose value as described (see G[e])

Replicate	Apparent sucrose, g/L	D-glucose, g/L	D-glucose, g/100 g	D-glucose rec., %	Sucrose, g/L	Sucrose, g/100 g	Sucrose rec., %
1	29.28	1.861	2.040	102	25.75	28.21	101
2	32.17	2.031	2.131	107	28.31	29.70	107
3	28.63	1.869	2.138	107	25.07	28.68	103
4	27.87	1.777	2.090	104	24.49	28.80	103
5	33.09	2.093	2.121	106	29.11	29.50	106
6	31.46	1.971	2.079	104	27.72	29.25	105
Mean			2.10	105		29.02	104
SD			0.04			0.56
RSD, %			1.78			1.93

Additionally, trueness for the D-glucose system was checked with NIST SRM 3282 Cranberry Juice with a certified D-glucose value of 8.50 ± 0.6 g/L (k = 2). The D-glucose value was measured with six replicates using three test kit lots. The mean concentration from all measurements was 8.71 ± 0.16 g/L, which is a mean recovery of 102%. The standard wine (blue label) with a certified value of 29.57 g/L was measured using all three test kit lots and found to have a mean concentration of 29.82 g/L (see OMA 2024.03).

Recovery Using Spiked Matrix Samples

This performance characteristic was determined during validation for blueberry ice cream, sweetened condensed milk, orange juice, carrot juice, and shandy because no (certified) reference materials were available for these matrixes. The spiking concentration for each matrix was dependent on the naturally occurring values for D-glucose and sucrose. It was the intention to spike between half the natural amount and the natural amount of each sugar (Tables 8–12). In the case of carrot juice, less than half the natural concentration was spiked.

Table 8.

Recovery experiment: Naturally incurred blueberry ice cream samples were spiked with 10 g/100 g sucrose and 5 g/100 g D-glucose to give a concentration of 19.5 g/100 g apparent sucrose; naturally incurred and spiked samples were extracted and analyzed in n = 6 test portions using the Enzytec Liquid Combi Sucrose/D-Glucose kit; mean concentrations from naturally incurred samples were subtracted from spiked values; sucrose values were calculated as described in G(e)

	Blueberry ice cream
	Apparent sucrose		D-glucose		Sucrose
	Measured, g/100 g	Difference, g/100 g	Measured, g/100 g	Difference, g/100 g	Measured, g/100 g	Difference, g/100 g
Non-spiked	16.21		2.97		10.57
	16.15		3.00		10.46
	16.17		2.98		10.51
	16.20		2.98		10.53
	15.93		2.91		10.40
	16.19		2.92		10.65
Mean (non-spiked)	16.14		2.96		10.52
Spiked 5 g/100 g D-glucose, 10 g/100 g sucrose, 19.5 g/100 g apparent sucrose	35.61	19.47	8.05	5.09	20.31	9.79
	35.79	19.65	8.09	5.14	20.42	9.89
	35.31	19.17	8.05	5.09	20.03	9.51
	35.34	19.19	8.02	5.06	20.09	9.57
	35.85	19.71	8.07	5.11	20.52	10.00
	35.83	19.69	8.05	5.09	20.53	10.01
Mean		19.48		5.10		9.80
SD		0.25		0.02		0.22
RSD, %		1.27		0.47		2.20
Recovery, %		100		102		98

Table 9.

Recovery experiment: Naturally incurred sweetened condensed milk samples spiked with sucrose to give a concentration of 25 g/100 g sucrose; naturally incurred and spiked extracts were extracted and analyzed in n = 6 test portions using the Enzytec Liquid Combi Sucrose/D-Glucose kit; mean concentrations from naturally incurred samples were subtracted from spiked values; sucrose values were calculated as described in G(e)

	Sweetened condensed milk
	Apparent sucrose		D-glucose		Sucrose
	Measured, g/100 g	Difference, g/100 g	Measured, g/100 g	Difference, g/100 g	Measured, g/100 g	Difference, g/100 g
Non-spiked	44.23		0.43		43.73
	44.46		0.31		44.19
	43.96		0.21		43.88
	44.74		0.29		44.51
	43.79		0.43		43.29
	43.96		0.20		43.91
Mean (non-spiked)	44.19		0.31		43.92
Spiked 25 g/L sucrose	69.51	25.32	0.47	0.15	67.35	23.43
	70.97	26.77	0.41	0.10	68.88	24.96
	70.99	26.80	0.52	0.21	68.70	24.78
	70.91	26.71	0.39	0.08	68.85	24.94
	71.04	26.85	0.42	0.11	68.94	25.02
	71.01	26.81	0.46	0.14	68.84	24.92
Mean		26.54		0.13		24.67
SD		0.60		0.05		0.62
RSD, %		2.27		34.8		2.49
Recovery, %						99

Table 10.

Recovery experiment: Naturally incurred orange juice was spiked with 20 g/L sucrose and 10 g/L D-glucose (39 g/L apparent sucrose); naturally incurred and spiked extracts were analyzed in n = 6 test portions using the Enzytec Liquid Combi Sucrose/D-Glucose kit; mean concentrations from naturally incurred samples were subtracted from spiked values; sucrose values were calculated as described in G(e)

	Orange juice
	Apparent sucrose		D-Glucose		Sucrose
	Measured, g/L	Difference, g/L	Measured, g/L	Difference, g/L	Measured, g/L	Difference, g/L
Non-spiked	80.86		22.38		38.33
	80.79		22.49		38.07
	79.23		22.42		36.63
	80.75		22.60		37.82
	80.10		22.40		37.54
	79.21		22.13		37.17
Mean (non-spiked)	80.16		22.40		37.59
Spiked 10 g/L D-glucose, 20 g/L sucrose, 39 g/L apparent sucrose	119.9	39.77	32.09	9.69	58.95	21.36
	120.3	40.14	32.51	10.11	58.53	20.93
	119.8	39.68	32.38	9.98	58.32	20.72
	121.2	41.03	32.56	10.15	59.34	21.74
	120.2	40.02	32.45	10.05	58.52	20.93
	119.7	39.56	32.48	10.08	58.01	20.41
Mean		40.03		10.01		21.02
SD		0.53		0.17		0.47
RSD, %		1.33		1.67		2.24
Recovery, %		103		100		105

Table 11.

Recovery experiment: Naturally incurred carrot juice was spiked with 15 g/L sucrose and 5 g/L D-glucose (24.5 g/L apparent sucrose); naturally incurred and spiked extracts were analyzed in n = 6 test portions using the Enzytec Liquid Combi Sucrose/D-Glucose kit; mean concentrations from naturally incurred samples were subtracted from spiked values; sucrose values were calculated as described in G(e)

	Carrot juice
	Apparent sucrose		D-glucose		Sucrose
	Measured, g/L	Difference, g/L	Measured, g/L	Difference, g/L	Measured, g/L	Difference, g/L
Non-spiked	58.91		10.13		39.66
	58.90		10.37		39.19
	59.54		10.24		40.08
	59.37		10.12		40.13
	57.31		10.34		37.65
	59.60		10.16		40.30
Mean (non-spiked)	58.94		10.23		39.50
Spiked 5 g/L D-glucose, 15 g/L sucrose, 24.5 g/L apparent sucrose	84.35	25.41	15.24	5.01	55.40	15.90
	83.56	24.63	15.35	5.12	54.40	14.90
	83.74	24.81	15.25	5.03	54.76	15.26
	82.56	23.62	15.44	5.21	53.22	13.72
	84.09	25.15	15.15	4.92	55.30	15.80
	83.52	24.58	15.21	4.98	54.63	15.12
Mean		24.70		5.05		15.11
SD		0.62		0.10		0.79
RSD, %		2.51		2.06		5.21
Recovery, %		101		101		101

Table 12.

Results of naturally incurred and spiked matrix samples; a naturally incurred shandy sample was spiked with 10 g/L sucrose and 10 g/L D-glucose (29 g/L apparent sucrose); naturally incurred and spiked extracts were analyzed in n = 6 test portions using the Enzytec Liquid Combi Sucrose/D-Glucose kit; mean concentrations from naturally incurred samples were subtracted from spiked values; sucrose values were calculated as described in G(e)

	Shandy
	Apparent sucrose		D-Glucose		Sucrose
	Measured, g/L	Difference, g/L	Measured, g/L	Difference, g/L	Measured, g/L	Difference, g/L
Non-spiked	42.47		17.68		8.87
	42.16		17.98		8.00
	43.81		17.86		9.87
	43.36		17.76		9.61
	41.91		17.91		7.88
	42.68		17.81		8.85
Mean (non-spiked)	42.73		17.83		8.85
Spiked 10 g/L D-glucose, 10 g/L sucrose, 29 g/L apparent sucrose	72.85	30.12	28.24	10.41	19.20	10.35
	72.59	29.86	28.30	10.47	18.82	9.97
	73.23	30.50	28.32	10.49	19.42	10.57
	72.85	30.12	28.11	10.28	19.44	10.60
	73.29	30.56	28.25	10.41	19.62	10.78
	73.06	30.33	27.94	10.11	19.97	11.12
Mean		30.25		10.36		10.57
SD		0.27		0.14		0.39
RSD, %		0.88		1.38		3.66
Recovery, %		104		104		106

Each spiked and un-spiked matrix was extracted/diluted and analyzed with six test portions. With the exception of one set of results for shandy, all mean recoveries were between 98 and 105% for blueberry ice cream (Table 8), sweetened condensed milk (Table 9), orange juice (Table 10), carrot juice (Table 11), and shandy (Table 12).

Additionally, recovery data for the D-glucose system are available for red wine, white wine, wheat beer, tomato juice, and orange juice. The mean recovery of the spiked matrixes was between 92.7 and 101% (see AOAC OMA 2024.03).

Precision of Repeatability

As observed in other enzymatic assays, pipetting of one extract into different cuvettes seems to be one main contributor to imprecision. To characterize the sucrose/D-glucose system, one sample extract from ice cream and orange juice was pipetted as six replicates into cuvettes and measured. The experiment was repeated on 2 more days by one analyst. As can be seen in Table 13, RSD values were between 0.5 and 1.7% and comparable to the ones shown in Tables 8–12.

Table 13.

Characterization of repeatability; one extract of each spiked test solution was analyzed in n = 6 cuvettes on 3 consecutive days; ice cream with sucrose and D-glucose concentrations of 10 and 2.5 g/100 g, respectively, was spiked with 10 g/100 g of sucrose and 5 g/100 g of D-glucose; orange juice with sucrose and D-glucose concentrations of 38 and 22 g/L, respectively, was spiked with 20 g/L of sucrose and 10 g/L of D-glucose

	Apparent sucrose			D-glucose			Sucrose
	Day 1	Day 2	Day 3	Day 1	Day 2	Day 3	Day 1	Day 2	Day 3
Orange juice, g/L	116.4	118.4	119.0	31.96	31.83	32.24	55.32	57.89	57.42
	116.4	119.5	119.1	31.97	31.88	32.11	55.32	59.02	57.54
	119.3	119.0	119.3	32.07	31.67	32.52	58.23	58.49	57.73
	118.2	119.3	120.4	32.16	32.22	33.09	57.16	58.73	58.79
	118.6	119.2	118.7	32.30	31.74	32.28	57.57	58.66	57.13
	118.9	116.1	118.9	32.29	31.76	32.26	57.87	55.62	57.32
Mean	117.9	118.6	119.2	32.13	31.85	32.42	56.91	58.07	57.65
SD	1.28	1.26	0.59	0.15	0.19	0.36	1.28	1.26	0.59
RSD, %	1.09	1.06	0.50	0.47	0.61	1.10	2.26	2.16	1.03
Blueberry ice cream, g/100 g	34.00	33.04	33.30	7.34	7.24	7.40	19.92	19.19	19.27
	32.26	33.10	32.14	7.50	7.38	7.43	18.17	19.25	18.11
	33.45	33.35	33.14	7.55	7.34	7.38	19.36	19.50	19.12
	33.37	33.35	32.88	7.42	7.33	7.33	19.29	19.50	18.85
	33.21	32.68	32.97	7.33	7.29	7.34	19.12	18.83	18.95
	33.41	32.98	33.40	7.36	7.17	7.41	19.32	19.13	19.38
Mean	33.28	33.09	32.97	7.41	7.29	7.38	19.20	19.23	18.95
SD	0.57	0.25	0.45	0.09	0.08	0.04	0.57	0.25	0.45
RSD, %	1.72	0.76	1.37	1.22	1.04	0.54	2.98	1.31	2.38

The repeatability of the D-glucose system was characterized with spiked tomato juice, white wine, orange juice, wheat beer, and red wine. RSD values were between 0.25 and 1.98% (see AOAC OMA 2024.03).

Inter-Lot Precision

To characterize for differences between test kit lots for the sucrose/D-glucose system, an inter-lot precision experiment was set up by analyzing three different aqueous solutions and two certified reference materials with six replicates analyzed on 1 day by one analyst using all three kit lots. This was necessary to show that all lots were produced under routine and comparable conditions.

The results are shown in Table 14 and prove that all three lots were comparable over the measurement range. All RSD values were at or below 2% for the test sample with the lowest concentrations of sucrose. At higher concentrations, the RSD values were not higher than 1%. As expected from results for the characterization of trueness, the precision for the extracts of the two reference materials also showed a very good precision. For the D-glucose system, comparable RSD values at 2% for a D-glucose concentration of 0.0238 g/L and below 1% for higher concentrations were observed (see AOAC OMA 2024.03).

Table 14.

Results of three aqueous solutions and two reference materials measured by one person with n = 6 replicates in all three test kit lots using the sucrose/D-glucose system on 1 day

	NIST SRM 3233	LGC 7016	0.050 g/L sucrose	0.75 g/L sucrose	1.50 g/L sucrose
	Conc., g/L	Conc., g/L	Conc., g/L	Conc., g/L	Conc., g/L
Lot 1	12.46	45.64	0.050	0.74	1.49
	12.52	46.47	0.049	0.75	1.50
	12.22	46.02	0.050	0.75	1.50
	12.63	45.63	0.048	0.73	1.48
	12.64	46.89	0.048	0.74	1.50
	12.92	46.64	0.048	0.75	1.50
RSD, %	1.83	1.15	2.14	0.78	0.58
Lot 2	13.03	45.70	0.049	0.74	1.47
	13.24	46.36	0.049	0.74	1.49
	13.03	43.02	0.050	0.74	1.49
	12.86	45.65	0.048	0.74	1.47
	13.15	46.75	0.049	0.74	1.49
	13.10	46.52	0.049	0.74	1.49
RSD, %	1.01	3.00	1.13	0.43	0.75
Lot 3	12.86	45.55	0.049	0.75	1.51
	13.13	46.43	0.049	0.75	1.51
	13.00	46.10	0.048	0.76	1.52
	13.22	45.93	0.050	0.74	1.49
	12.78	46.97	0.048	0.75	1.51
	13.08	46.65	0.048	0.76	1.50
RSD, %	1.28	1.11	1.37	0.63	0.63

Intermediate Precision

To get an idea about intermediate or laboratory-internal reproducibility, three test kit lots were tested on three different days using three different photometers by three lab technicians in a laboratory (Table 15).

Table 15.

Characterization of laboratory-internal reproducibility (intermediate precision) by three analyst (one lot each) analyzing each material on 3 different days with five test portions per day

			Aqueous sucrose solutions
			0.050 g/L		0.750 g/L		1.500 g/L
Analyst	Day	Lot	sucrose, g/L	Rec., %	sucrose, g/L	Rec., %	sucrose, g/L	Rec., %
1	1	1	0.050	99.5	0.754	100.5	1.511	100.7
			0.048	96.2	0.759	101.2	1.514	100.9
			0.048	96.9	0.750	100.1	1.506	100.4
			0.050	100.3	0.749	99.8	1.504	100.2
			0.048	97.0	0.751	100.2	1.511	100.8
			0.052	103.2	0.755	100.7	1.502	100.1
2	1	2	0.049	98.8	0.740	98.6	1.479	98.6
			0.050	100.5	0.749	99.9	1.516	101.1
			0.050	99.8	0.753	100.4	1.510	100.7
			0.048	95.7	0.737	98.2	1.478	98.6
			0.049	97.3	0.743	99.1	1.489	99.3
			0.047	94.9	0.743	99.1	1.497	99.8
3	1	3	0.052	104.6	0.762	101.6	1.522	101.4
			0.051	102.6	0.762	101.6	1.546	103.1
			0.049	98.5	0.761	101.4	1.530	102.0
			0.048	96.7	0.757	100.9	1.512	100.8
			0.050	99.6	0.760	101.4	1.516	101.1
			0.052	104.5	0.752	100.3	1.515	101.0
1	2	3	0.049	97.9	0.741	98.9	1.488	99.2
			0.049	97.8	0.745	99.4	1.503	100.2
			0.049	98.9	0.741	98.8	1.496	99.8
			0.050	99.6	0.746	99.5	1.514	100.9
			0.049	97.5	0.749	99.9	1.498	99.9
			0.050	100.5	0.742	98.9	1.504	100.3
2	2	1	0.047	95.0	0.741	98.8	1.487	99.1
			0.050	99.4	0.744	99.2	1.496	99.8
			0.048	95.7	0.746	99.5	1.497	99.8
			0.051	102.3	0.737	98.3	1.476	98.4
			0.048	95.4	0.747	99.6	1.500	100.0
			0.048	96.4	0.745	99.3	1.495	99.7
3	2	2	0.049	98.8	0.754	100.5	1.517	101.1
			0.050	100.0	0.757	100.9	1.444	96.3
			0.051	102.5	0.756	100.8	1.377	91.8
			0.051	101.6	0.751	100.2	1.507	100.5
			0.049	98.7	0.758	101.1	1.527	101.8
			0.050	100.3	0.757	100.9	1.523	101.5
1	3	2	0.050	99.2	0.751	100.1	1.496	99.7
			0.050	100.1	0.747	99.6	1.499	100.0
			0.050	100.2	0.748	99.7	1.496	99.7
			0.049	99.0	0.743	99.1	1.491	99.4
			0.050	99.2	0.744	99.2	1.483	98.9
			0.050	99.9	0.736	98.2	1.489	99.3
2	3	3	0.049	98.0	0.738	98.4	1.483	98.8
			0.049	97.0	0.749	99.9	1.500	100.0
			0.050	99.0	0.748	99.7	1.506	100.4
			0.048	95.6	0.739	98.5	1.488	99.2
			0.048	96.2	0.745	99.3	1.492	99.5
			0.044	88.8	0.749	99.9	1.501	100.1
3	3	1	0.051	102.3	0.749	99.9	1.509	100.6
			0.052	103.9	0.678	90.4	1.515	101.0
			0.049	98.3	0.762	101.6	1.508	100.5
			0.047	94.1	0.747	99.6	1.492	99.4
			0.048	96.8	0.752	100.3	1.517	101.2
			0.046	92.8	0.753	100.4	1.516	101.0
Mean			0.049	98.6	0.748	99.7	1.500	100.0
SD			0.0015		0.012		0.023
RSD (%)			3.00		1.59		1.56

The measurement was made using three aqueous solutions with different concentrations of sucrose using the sucrose/D-glucose system. Each analyst performed the experiment on different days.

As can be seen in Table 15, the precision clearly depends on the concentration. The lowest concentration resulted in the highest RSD of 3%, whereas higher concentrations of aqueous solutions showed quite low RSD values at or below 1.6%. All recoveries were acceptable.

For the D-glucose system a slightly different experiment was used using three test kit lots each tested by a different analyst on two different days with three replicates on each day. At a D-glucose concentration of 23.8 mg/L, the RSD value was at 6%, while higher concentrations had values at or below 2% (see AOAC OMA 2024.03).

Ruggedness Study

These experiments were undertaken to show the influence of variable parameters on test kit results. These parameters are known to be subject to variation during use of the test kit. The parameters tested for their ruggedness were incubation temperature (18, 25, and 37°C; corresponding to 64, 77, and 99°F) and incubation times (5, 10, and 15 min) for the first incubation step (sucrose cleavage) and the second step (D-glucose measurement). Table 16 shows the results of these experiments. Besides occasional higher variation of the control sample with the lowest sucrose concentration, there were no important differences.

Table 16.

Ruggedness study at incubation temperatures of 18, 25, and 37°C (64–99°F) with different incubation times for measurement of A1 or A2 (5, 10, and 15 min) and a fixed time of 15 min for A1 or A2. Test solutions were aqueous solutions (Solution A, B, and C) with known sucrose values

A2 15 min							A1 15 min
Sucrose	18°C		25°C		37°C		18°C		25°C		37°C
Target, g/L	Measured, g/L	Rec., %	Measured, g/L	Rec., %	Measured, g/L	Rec., %	Measured, g/L	Rec., %	Measured, g/L	Rec., %	Measured, g/L	Rec., %
	A1 5 min						A2 5 min
0.05	0.048	95.5	0.049	98.8	0.051	101	0.046	91.2	0.052	103.8	0.055	110
0.05	0.054	107.4	0.050	100	0.051	102	0.048	96.1	0.053	105.4	0.048	96.7
0.75	0.720	96.1	0.739	98.5	0.756	101	0.739	98.5	0.740	98.7	0.726	96.8
0.75	0.731	97.4	0.743	99.0	0.745	99.4	0.740	98.6	0.747	99.6	0.744	99.1
1.5	1.444	96.3	1.471	98.0	1.487	99.1	1.473	98.2	1.481	98.7	1.450	96.6
1.5	1.457	97.1	1.491	99.4	1.484	98.9	1.476	98.4	1.510	100.6	1.458	97.2
	A1 10 min						A2 10 min
0.05	0.050	99.6	0.047	94.7	0.051	102	0.047	93.8	0.052	103.0	0.059	118
0.05	0.050	99.3	0.050	100	0.048	96.2	0.049	97.9	0.052	104.2	0.048	95.4
0.75	0.732	97.6	0.740	98.7	0.731	97.4	0.742	99.0	0.740	98.7	0.726	96.8
0.75	0.737	98.3	0.746	99.5	0.734	97.9	0.744	99.2	0.749	99.9	0.743	99.0
1.5	1.467	97.8	1.482	98.8	1.459	97.3	1.480	98.7	1.482	98.8	1.448	96.5
1.5	1.477	98.5	1.494	99.6	1.485	99.0	1.485	99.0	1.511	100.7	1.459	97.3
	A1 15 min						A2 15 min
0.05	0.046	91.7	0.051	103	0.063	125	0.046	91.7	0.051	102.8	0.063	125
0.05	0.049	98.7	0.052	103	0.048	96.3	0.049	98.7	0.052	103.1	0.048	96.3
0.75	0.744	99.2	0.740	98.6	0.727	96.9	0.744	99.2	0.740	98.6	0.727	96.9
0.75	0.748	99.7	0.746	99.5	0.742	99.0	0.748	99.7	0.746	99.5	0.742	99.0
1.5	1.485	99.0	1.482	98.8	1.447	96.5	1.485	99.0	1.482	98.8	1.447	96.5
1.5	1.488	99.2	1.511	101	1.456	97.1	1.488	99.2	1.511	100.7	1.456	97.1

In principle, the enzymatic conversion of sucrose to D-glucose and D-fructose is finished within 5 min. Especially at 18°C, the rate of conversion is slower (curves not shown) so it can be assumed that a stored test kit at the end of its shelf life could not reach 100% conversion within 5 min. Therefore, in order to achieve good recoveries, the following incubation times are recommended: A1 after 15 min and A2 after 15 min for incubation temperatures between 20 and 37°C (68–99°F).

For the glucose system, the recommended incubation times and temperatures were A1 = 3 min and A2 = 15 min for incubation temperatures between 20°C (68°F) and 37°C (99°F). If needed, incubation times can be shortened to 2 min (A1) and 10 min (A2) at a temperature of 37°C.

Dilutability

Dilutability is characterized to check whether a high-concentration test solution can be measured correctly when diluted with deionized water within the measurement range. For the determination in the sucrose/D-glucose system, two certified reference materials were used and extracted. Dilution was made with deionized water to give concentrations within the measurement range. Each diluted extract was analyzed with two replicates per run. As can be seen in Figures 5 and 6, a test solution diluted to a measured value of 2000 mg/L is already in the linear range of the system, which perfectly matches the characterization of linearity.

Figure 5.

Results for dilutability of NIST SRM 3233 (sucrose: 13.42 g/100 g); the regression equation was calculated from all data points; n = 2 per dilution were analyzed in two independent runs.

Figure 6.

Results for dilutability of LGC 7016 (sucrose: 46.5 g/100 g); the regression equation for was calculated from all data points; n = 2 per dilution were analyzed in two independent runs.

Dilutability for the D-glucose system was shown for white wine, orange juice, and tomato juice. The upper linear limit was between 1500 and 2000 mg/L D-glucose (see AOAC OMA 2024.03).

Real-Time Stability Study

To characterize the shelf life of the (unopened) test kit, a stability study using all three test kit lots (pilot scale lots) was performed over a period of 30 months (Table 17). Neither differences between lots nor trends towards higher or lower concentrations were observed. Therefore, the real-time stability of unopened test kits was set to 29 months. The components of the D-glucose system were also checked in the same way and revealed a stability of 29 months.

Table 17.

Characterization of real-time stability of the enzymatic system over a maximum period of 30 months using three pilot scale lots (lot 1 to 3); three different control samples were analyzed in duplicate (Rep. 1 and Rep. 2) by different analysts; at zero months the kits were only tested by using one control sample

		Sucrose solution		Multi-sugar standard		Standard wine
		Target: 1.5 g/L sucrose		Target: 1.45 g/L apparent sucrose		Target: 56.18 g/L apparent sucrose
Lot	Month	Rep. 1	Rep. 2	Rep. 1	Rep. 2	Rep. 1	Rep. 2
1	0	—a	—	—	—	57.47	57.24
	6	1.501	1.495	1.480	1.472	59.76	59.71
	9	1.508	1.502	1.483	1.477	55.70	56.04
	12	1.511	1.505	1.472	1.484	53.93	53.96
	16	1.513	1.506	1.483	1.481	56.43	55.93
	20	1.482	1.487	1.461	1.475	56.40	57.32
	24	1.485	1.501	1.462	1.468	54.15	54.34
	30	1.506	1.514	1.454	1.462	56.58	56.34
2	0	1.484	1.51	1.434	1.436	58.56	59.02
	0	1.496	1.493	1.486	1.478	54.81	55.30
	0	1.498	1.496	1.480	1.479	55.07	55.32
	6	1.485	1.489	1.436	1.464	56.19	55.72
	9	1.487	1.490	1.504	1.516	58.07	57.97
	12	1.502	1.501	1.468	1.473	54.68	54.59
	16	1.495	1.499	1.468	1.463	55.35	54.94
	20	1.486	1.494	1.462	1.465	53.31	53.20
	24	1.495	1.504	1.444	1.446	51.50	53.80
	30	1.515	1.507	1.456	1.447	57.34	57.54
3	0	1.467	1.464	1.436	1.458	54.34	56.47
	0	1.499	1.499	1.468	1.469	57.98	57.59
	0	1.485	1.493	1.459	1.463	56.84	57.30
	6	1.504	1.510	1.481	1.477	58.24	57.97
	9	1.487	1.494	1.463	1.462	54.38	55.35
	12	1.488	1.497	1.461	1.471	54.44	54.94
	16	1.453	1.511	1.451	1.475	55.05	55.95
	20	1.496	1.501	1.437	1.444	53.66	53.49
	24	1.501	1.52	1.471	1.449	56.71	57.32
	30	1.510	1.509	1.455	1.447	57.56	57.28
	Mean, g/L	1.496		1.466		55.902
	SD, g/L	0.013		0.017		1.792
	RSD, %	0.85		1.14		3.21
	Rec., %	99.8		101.1		99.5

a

— = Not applicable.

Stability Study on Transportation

To investigate the influence of harsh transport conditions, a simulated transport stability was performed. The conditions that were simulated included shaking and temperature changes. All components of one lot were placed on a horizontal shaker at room temperature and agitated for 6 h; 400 revolutions per minute (rpm) were used at the beginning and changed later on to 150 rpm. Afterwards the components were refrigerated for 18 h at 2–8°C (36–46°F) followed by 7 h at room temperature on a horizontal shaker (150 rpm). Components were incubated at 37°C (99°F) for 18 h and after cooling down to room temperature measured, on the same day, with aqueous sucrose standards. Afterwards, the stressed components were stored at 2–8°C (36–46°F) and were re-checked after 9, 15, and 27 months in the QC department. As can be seen in Table 18, the simulation of transportation shows no effects on functionality and quality of the components (ANOVA was not significant; P > 0.50).

Table 18.

Results after transport stability for one lot using three different control samples

				After simulated transport
		Reference		0 Months		9 Months		15 Months		27 Months
	Apparent sucrose target, g/L	Measured, g/L	Rec., %	Measured, g/L	Rec., %	Measured, g/L	Rec., %	Measured, g/L	Rec., %	Measured, g/L	Rec., %
Control A (aqueous)	0.05	0.0517	103	0.0474	94.9	0.0487	97.3	0.0464	92.8	0.0486	97.2
	0.05	0.0504	101	0.0487	97.5	0.0471	94.1	0.0476	95.1	0.0476	95.3
Control B (aqueous)	0.75	0.748	99.7	0.716	95.5	0.730	97.4	0.717	95.6	0.737	98.3
	0.75	0.735	98.0	0.719	95.9	0.744	99.2	0.705	93.9	0.740	98.7
Control C (aqueous)	1.50	1.489	99.3	1.461	97.4	1.501	100	1.459	97.3	1.461	97.4
	1.50	1.470	98.0	1.464	97.6	1.495	99.7	1.465	97.7	1.469	98.0

The D-glucose system was checked 24 months after the simulated transport and also showed no significant tendencies towards higher or lower values.

Stability Study on Freezing

To simulate an unintended freezing of the test kit components, the whole test kit of one lot was frozen for 24 h at −20°C (−4°F). Afterwards, the components were allowed to warm up to room temperature and were frozen again at −20°C (−4°F). After 24 h, the components were thawed and, after warming up to room temperature, finally measured with aqueous sucrose and D-glucose standards against unstressed components. It was observed visually that components in reagent 2 and reagent 4 precipitated after freezing. A check for functionality revealed a complete loss of enzymatic activity. A user will be informed by reading the test kit insert not to freeze the components of this test kit.

Stability Study of Already Opened and Reused Components

The shelf life of an unopened test kit is not the only stability parameter that needs to be characterized. In-use stability is as important for a test kit user because normally the test kit will be used over a period of weeks or months after opening and removing reagents from the bottles for the first time. Therefore, an experiment was set up over a period of 27 months using one test kit lot. As can be seen in Table 19, there were no tendencies towards higher or lower values for the control samples after 27 months. The glucose system showed the same in-use stability of at least 27 months.

Table 19.

Characterization of in-use stability of the enzymatic system over a maximum period of 27 months using one pilot scale lot; three different control samples were analyzed in duplicate by different analysts.

	Apparent sucrose	Day 0	6 Months	12 Months	18 Months	27 Months
	target, g/L	Measured, g/L	Measured, g/L	Measured, g/L	Measured, g/L	Measured, g/L
Control A (aqueous)	0.05	0.0517	0.0458	0.0462	0.0485	0.0492
	0.05	0.0526	0.0511	0.0474	0.0478	0.0482
Control B (aqueous)	0.75	0.762	0.718	0.719	0.700	0.725
	0.75	0.765	0.714	0.712	0.697	0.729
Control C (aqueous)	1.50	1.507	1.468	1.451	1.437	1.435
	1.50	1.530	1.472	1.457	1.440	1.438
Blank A1 (OD340 nm)b	—a	0.4281	0.4058	0.4037	0.4122	0.3968

a

— = Not applicable.

b

OD = Optical density.

Automation on a Pictus 500 Spectrophotometric Analyzer

Comments on validation parameters independent of automation.—Side-reactivity to other related sugars/sugar alcohols and interfering substances will not be characterized on the automated analyzer here because there is no known effect that an automated process will have to change the reactivities towards these substances.

The pipetting environment within a closed automated process is much more regulated than the normal laboratory environment (including the analyst). For measurement, all reagents are cooled to 8 ± 2°C (42–50°F), while the reaction zone where the analysis takes place is set to 37°C (99°F). This ensures a quick enzymatic reaction and highly reproducible results. Therefore, the characterization of incubation times and temperatures was not repeated. Incubation at 37°C (99°F) and the necessary incubation times are described for the 4 mL cuvette manual application (Table 16). There is also no practical reason to analyze test kit components that were tested for their stabilities against transport and short-term storage at 37°C (99°F).

As already mentioned, the Pictus 500 can automatically change between the three applications in case the concentration is below or above the basic measurement range. Therefore, it was decided not to characterize each application for an LOD but to characterize a proper LOQ.

Data for the automated use of the D-glucose system are given in more detail in the AOAC OMA 2024.03. Only summarized data are presented here for the glucose system.

• LOQ.—The lower end of the measurement range is the LOQ where acceptable recovery and precision are met. Internal requirements are a recovery between 95 and 105%, and an RSD value equal or lower than 10%. For each of the three applications, aqueous solutions with different concentrations of sucrose were analyzed at least five times. The concentration was calculated from the calibration of the system.

  Figure 7 shows the results for the sucrose/D-glucose system for the basic application with a test volume of 10 µL, which is, despite a factor of 10 in volume, the identical ratio of test volume to reagents as the manual format with a test volume of 100 µL with 2000 µL reagent 1 and 500 µL reagent 2. The automated analyzer has an LOQ of 15 mg/L sucrose using the criteria described before. The manual format had a calculated LOQ of about 17 mg/L sucrose where the precision was still sufficient. The D-glucose system had an LOQ of 18 mg/L D-glucose.

  Since sucrose and D-glucose are often present at very high concentrations in food such as jams and chocolate, the automated high-range application with a low test volume of 2 µL was introduced to analyze these matrixes without dilution prior to measurement. As can be seen in Figure 8, for the sucrose/D-glucose system, the LOQ for this application is 75 mg/L sucrose. This application was not investigated in the manual format because this would require test volumes of 20 µL that are challenging for untrained analysts. The Pictus 500 showed RSD values at or quite below 5% for a test volume of 2 µL. The LOQ for the D-glucose system was 75 mg/L D-glucose. Below this value, recovery will be above 105%, which is not acceptable (see H).

  In case trace analysis of sucrose is necessary, the sensitive-range application with a test volume of 100 µL was investigated for its LOQ (Figure 9). An LOQ of 3.8 mg/L sucrose can be claimed for this application where recovery and precision requirements were met. Lower than this value, recoveries were clearly below 95%. The D-glucose system had an LOQ of 2.4 mg/L using a 100 µL test volume.

• Linearity.—The most important parameter for an automated application is the linear range because in the case of enzymatic analysis the analyte is often present in the matrix and its proper quantification only depends on the proper choice of test volume and calibration. For each of the three applications using the sucrose/D-glucose system the optimal linear measurement range was characterized. Figure 10 shows that the upper measurement value is (at least) 1900 mg/L sucrose for a test volume of 10 µL. The D-glucose system is linear up to 1900 mg/L D-glucose in the basic application.

  For the high-range application with a test volume of 2 µL, the upper measurement value is 9500 mg/L (Figure 11). This is a factor of 5 higher compared to the basic application and perfectly fits to the increased test volume of 10 µL. The D-glucose system had an upper measurement value of 10000 mg/L D-glucose when applying 2 µL test solution in the system.

  For the sensitive application with a test volume of 100 µL, the upper measurement value is 190 mg/L (Figure 12). It is always recommended to include control samples at the upper level to check for linearity. The D-glucose system is linear up to 190 mg/L when 100 µL test solution is applied.

• On-board and calibration stability.—Data on these important characteristics will be provided for the Final Action decision because these experiments are ongoing. Analysts use the assay in many different ways over a longer period, e.g., some will leave the reagents in the analyzer until they are empty. Others will store them after each working day in the fridge to prevent deterioration. The setup of such an experiment is, therefore, challenging.

• Precision and recovery.—The precision of the automated pipetting was characterized for all three application using the sucrose/D-glucose system. In all cases, two aqueous solutions were used because characterization of different matrixes was already done during the validation of the manual application. To check for trueness, one standard wine was applied diluted for all three applications.

  The aqueous solution with a target value of 1500 mg/L apparent sucrose contains sucrose at a level of 1500 mg/L. The second aqueous solution with a target of 1450 mg/L apparent sucrose is the Enzytec Multi-sugar standard low with D-glucose and sucrose at a level of 500 mg/L. According to G(e), the target concentration is 1450 mg/L apparent sucrose.

Figure 10.

Characterization of linearity for the basic application with 10 µL test volume; RSD values are given as open triangles and measured values as closed circles.

Figure 11.

Characterization of linearity for the high-range application with 2 µL test volume; RSD values are given as open triangles and measured values as closed circles.

Figure 12.

Characterization of linearity for the sensitive application with 100 µL test volume; RSD values are given as open triangles and measured values as closed circles.

Figure 2024.05A.

Enzymatic reaction scheme for the determination of sucrose and D-glucose.

Figure 7.

Confirmation of LOQ for the basic application with 10 µL test volume; RSD values are given as open triangles and recoveries as closed circles.

Figure 8.

Confirmation of LOQ for the high-range application with 2 µL test volume; RSD values are given as open triangles and recoveries as closed circles.

Figure 9.

Confirmation of LOQ for the sensitive-range application with 100 µL test volume; RSD values are given as open triangles and recoveries as closed circles.

The standard wine was already used to characterize real-time stability. The target level is 561.8 mg/L apparent sucrose and is the result of a D-glucose level of 29.57 g/L, which is equivalent to 56.18 g/L apparent sucrose. The sample has to be diluted 1:100 before measurement depending on the application.

Table 20 shows the results for the basic-range application with a test volume of 10 µL. As expected for automated pipetting, RSD values below 0.5% were obtained for concentrations of 1500 mg/L. The validity of the two-point calibration (0 and 1500 mg/L) was also checked with these three solutions. Recoveries ranged from 99 to 104% and were thus clearly within specifications.

Table 20.

Characterization of precision for the basic-range (10 µL test volume) application using two aqueous control solutions and a standard wine; an asterisk marks a single outlier according to Grubbs (P < 0.01); results are shown as apparent sucrose

	Aqueous solution		Standard wine
Replicate	Target: 1500 mg/L	Target: 1450 mg/L	Target: 561.8 mg/L
1	1534	1497	556.2
2	1542	1504	560.8
3	1549	1487	558.9
4	1541	1499	558.1
5	1539	1506	556.2
6	1541	1504	556.2
7	1539	1502	557.3
8	1541	1502	559.5
9	1537	1501	444.4*
10	1535	1505	558.8
Mean, mg/L	1540	1501	558
SD, mg/L	4.32	5.33	1.64
RSD, %	0.28	0.36	0.29
Recovery, %	102.7	103.5	99.3

For the D-glucose system, RSD values were clearly below 1% for aqueous solutions and a red wine. Mean recoveries were between 96 and 100%.

Table 21 shows the results for the high-range application with a test volume of 2 µL. As expected for automated pipetting and the small volume, RSD values of 0.5% were obtained for the aqueous control solutions. The standard wine had an RSD value of 1.2%. The validity of the two-point calibration (0 and 7500 mg/L) was also checked with these three solutions. Recoveries ranged between 98 and 103% and were thus clearly within specifications. The standard wine comes with a certificate so that the trueness of the system was established.

Table 21.

Characterization of precision for the high-range (2 µL test volume) application using two aqueous solutions and a standard wine; results are given as apparent sucrose

	Aqueous solution		Standard wine
Replicate	Target: 1500 mg/L	Target: 1450 mg/L	Target: 561.8 mg/L
1	1523	1498	555.5
2	1505	1489	547.8
3	1506	1491	548.0
4	1514	1507	544.8
5	1503	1510	546.1
6	1508	1500	567.6
7	1509	1502	553.5
8	1525	1489	557.4
9	1519	1510	554.1
10	1503	1490	552.2
Mean, mg/L	1511	1499	553
SD, mg/L	8.33	8.60	6.71
RSD, %	0.55	0.57	1.21
Recovery, %	100.8	103.4	98.4

For the D-glucose system, RSD values were clearly below 2.5% for aqueous solutions and 3.9% for a red wine. Mean recoveries were between 97 and 101%.

Table 22 shows the results for the sensitive-range application with a test volume of 100 µL. For this application, a four-point calibration (0, 15, 45, and 150 mg/L) was established and validated. RSD values were at or less than 1%. The validity of the four-point calibration was also checked with these three solutions. Recoveries ranged between 99 and 104% and were thus within specifications.

Table 22.

Characterization of precision for the sensitive range application (100 µL test volume) using two aqueous control solutions and a standard wine (dilution 1:1000); results are given as apparent sucrose

	Aqueous solution		Standard wine
Replicate	Target: 150 mg/L	Target: 145 mg/L	Target: 56.18 mg/L
1	152.2	149.5	55.25
2	152.8	153.6	56.32
3	153.1	151.9	55.82
4	151.9	150.0	55.67
5	153.1	148.6	55.60
6	152.9	152.0	55.17
7	151.4	150.7	56.09
8	151.1	149.3	55.72
9	152.6	149.7	54.98
10	153.2	149.4	55.15
Mean, mg/L	152.4	150.5	56
SD, mg/L	0.75	1.55	0.44
RSD, %	0.49	1.03	0.79
Recovery, %	101.6	103.8	98.9

For the D-glucose system, RSD values were clearly below 1.4% for aqueous solutions and a red wine. Mean recoveries were between 101 and 103%.

To characterize one of the applications for trueness, NIST SRM 1869 was analyzed for apparent sucrose and D-glucose. The content of sucrose was calculated by subtracting the D-glucose content form the apparent sucrose content. As can be seen in Table 23, mean recovery for D-glucose was 99%, while recovery for sucrose was 94.1%. RSD values were in both cases below 3%.

Table 23.

Repeated measurement (n = 6 test portions) of the certified reference material NIST SRM 1869 Infant/Adult Nutritional Formula II; milk/whey/soy-based with a sucrose target value of 27.88 ± 0.60 g/100 g (k = 2) and a D-glucose target of 2.00 ± 0.45 g/100 g (k = 2) for characterization of trueness in the high-range application mode of the spectrophotometric analyzer using a test volume of 10 µL; values for D-glucose in the sample were subtracted from the apparent sucrose value automatically by the Pictus 500 as described [see G(e)]

Replicate	Weight, g	Volume, mL	Dilution	Apparent sucrose, mg/L	D-glucose, mg/L	D-glucose, g/100 g	D-glucose rec., %	Sucrose, mg/L	Sucrose, g/100 g	Sucrose rec., %
1	1.013	11.1	5	5265	360.1	1.97	98.6	4580	25.10	90.0
2	1.077	11.3	5	5945	395.1	2.07	103.6	5194	27.25	97.7
3	0.988	11.3	5	5168	336.0	1.92	96.1	4530	25.90	92.9
4	0.961	11.3	5	5135	339.5	2.00	99.8	4490	26.40	94.7
5	1.115	11.3	5	5906	385.3	1.95	97.6	5174	26.22	94.0
6	1.071	11.3	5	5730	372.4	1.96	98.2	5022	26.50	95.0
Mean						1.98	99.0		26.23	94.1
SD						0.05			0.71
RSD. %						2.61			2.71

Conclusions

In summary, the data from the in-house validation study proved that the performance claims for food and beverages such as juices, chocolate, breakfast cereals, ice cream, sweetened condensed milk, wine, beer, and soft drinks are fulfilled. The method is robust and accurate for both applications (manual and automated). The Enzytec Liquid Combi Sucrose/D-Glucose kit fulfils the range, accuracy, and precision requirements outlined in SMPR 2018.001 for the applicable matrixes included in the validation study. The test kit was accepted as an AOAC Official Method of Analysis℠ for quantification of sucrose and D-glucose in the claimed matrixes.

Acknowledgments

We would like to thank Patricia Meinhardt (R-Biophamr Inc., Washington, MO, USA), Rebecca Ziegler (R-Biopharm AG, Darmstadt, Germany), Ute Maelzer-Funk (R-Biopharm AG, Darmstadt, Germany), Alexander Muhl (R-Biopharm AG, Darmstadt, Germany), and Tina Dubois (R-Biopharm AG, Darmstadt, Germany) for their practical contributions. We would like to thank the entire AOAC Expert Review Panel and especially Ruth Ivory (Neogen, Ayr, Scotland) for their excellent contributions.

Conflict of Interest

Both authors were employed by the company manufacturing the test kit while conducting the study.