Validation of Enzytec™ Liquid D-Lactic Acid for Enzymatic Determination of D-Lactic Acid in Selected Foods and Beverages: Official Method 2024.06 First Action

Abstract

Background

D- and L-lactic acid are produced naturally by lactic acid bacteria and are found in fermented milk products, pickled vegetables, and cured meats. D-lactic acid is formed by some microorganisms only, e.g., Lactobacillus lactis and Leuconostoc cremoris. D-Lactic acid is not formed or only in traces by “higher organisms,” e.g., by animals. Therefore, the presence of D-lactate may serve as an indicator for microbial contamination or spoilage, assuming that fermentation techniques have not been used.

Objective

To validate the performance of the Enzytec™ Liquid D-Lactic acid for the determination of D-lactic acid in food and beverages such as milk and (fermented) milk products, fermented vegetable products, wines, beer, fruit and vegetable juices, and eggs and egg powder.

Methods

The kit contains two ready-to-use components, which makes handling easy and suitable for automation. D-lactic acids react in the presence of NAD and D-lactate dehydrogenase to pyruvate and NADH. The NADH formed is equivalent to the amount of D-lactic acid converted.

Results

Ascorbic acid, 3-hydroxybutyric acid, and sulfite interfere at concentrations higher than 0.2, 0.2, and 0.05 g/L, respectively. Oxaloacetic acid, pyruvic acid, and D-fructose do not interfere at or below concentrations of 0.2, 1, and 10 g/L, respectively. The calculated LOD when using a test volume of 100 µL is 5.4 mg/L, and the LOQ is 15 mg/L. The practical upper measurement range is 600 mg/L. Relative intermediate precision was between 3.5 and 5.7% for pineapple juice, sauerkraut juice, wine, and liquid egg. A reference material (wine) showed recoveries of 108%. For automation, three applications with different test volumes were validated. Linearity is given from 0.75 up to 3125 mg/L.

Conclusions

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

Highlights

The ready-to-use components of the test kit have a shelf life of at least 24 months.

D- and L-lactic acid are found in many foods and beverages. Produced naturally by lactic acid bacteria, both lactic acids are found in many fermented milk products such as yogurt, and also in pickled vegetables, cured meats, and fish. It is commonly supplemented in foods and beverages (E270; racemate of D- and L-lactic acid) as a nonvolatile acidulant agent. In the wine industry, the course of malolactic fermentation—mostly in red wine—is monitored by following the decreasing level of L-malic acid and the increasing level of D- and L-lactic acid. The content of lactate in beer indicates the presence of Lactobacilli in production. The content of lactate in liquid whole egg or in egg powder gives good information about the hygienic situation of the products. Similarly, the quality of milk and fruit and vegetables can be established by measurement of lactic acid content. Lactate in milk powder indicates the use of neutralized sour milk for the production of milk powder.

D-Lactate is very often measured together with L-lactate. D-Lactic acid is formed by some micro-organisms only, e.g., from Lactobacillus lactis, L. bulgaricus, and Leuconostoc cremoris. D-Lactic acid is not formed or only in traces by “higher organisms,” e.g., by animals and humans (1). Therefore, the presence of D-lactate may serve as an indicator for microbial contamination or spoilage (2), presuming that fermentation techniques have not been used in the production of the foodstuff.

The new Enzytec™ Liquid D-Lactic acid was developed as a replacement for the Roche test kit. In parallel, 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. Especially for the wine and juice sector, the automated analysis of lactic acids is necessary.

Experimental

The study design contains the characterization of the manual format using 4 mL cuvettes. The following sections contain all necessary performance characteristics as required by AOAC Appendix F (3) and are based on a former validation study for ethanol in kombucha and other beverages, which led to Official Method 2017.07 Final Action (4, 5).

1. Linearity.—Linearity check over a range from 5 up to 1200 mg/L D-lactic acid 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.

2. Limits of detection (LOD) and limit of quantification (LOQ; estimates).—According to DIN 32645:2008–11 (based on DIN ISO 11843-2:2008-06) with concentrations ranging from 5 up to 50 mg/L D-lactic acid (in water) analyzed using three independent test kit lots (n = 2 replicates per lot).

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

4. Precision profile.—Data were calculated from the linearity data set (aqueous lactic acid solutions). The calculated RSD values are derived from two replicates from three independent test kit lots each.

5. Selectivity.—For the determination of side activities of the measurement system, the following high concentrated organic acid solutions (20 g/L) were measured using the D-lactic reagents in the absence of D-lactic acid: L-lactic acid, pyruvic acid, formic acid, D-/L-malic acid, butyric acid, citric acid, acetic acid, oxalic acid, propionic acid, L-tartaric acid, and oxaloacetic acid. The last was also tested at concentrations of 2 and 0.2 g/L.

6. Interferences.—Different sugars, sugar alcohols, and substances tested for selectivity were tested in presence of D-lactic acid.

7. Trueness.—Measurement of one reference material (wine) with six replicates using three test kit lots by one analyst.

8. Recovery.—Several different matrices (wine, tomato juice, milk, yogurt, cream, milk powder, egg powder, sausage, liquid egg, grape juice, sauerkraut juice, beer) were spiked to half of their endogenous D-lactic acid concentrations or higher; test portions were extracted (six replicates) and analyzed using at least one test kit lot by one analyst. This experiment was also performed for the automated analyzer in a similar way for the matrix wine.

9. Repeatability.—Determined as part of the intermediate precision for pineapple juice, sauerkraut juice, wine, and liquid egg.

10. Inter-lot precision.—Aqueous D-lactic acid solutions with concentrations of 60, 200, and 400 mg/L were analyzed with six replicates on 1 day by one analyst using three different test kit lots.

11. Intermediate precision.—Measurement of pineapple juice, sauerkraut juice, wine, and liquid egg. Each material was used directly for measurement or extracted before and analyzed using one test kit lot by three different analysts on 2 different days with three extracts per day and two replicates per extract. From these values s_r, RSD_r, s_i, and RSD_i were calculated. This design also allowed for an evaluation of the contribution of analyst, day, extraction, and pipetting steps to the overall precision.

12. Robustness.—Incubation temperature was varied between 18 and 37°C. Incubation time before measuring A2 at 340 nm was varied between 5 and 15 min.

13. Dilutability.—A sauerkraut juice and a wine with concentrations above the measurement range were checked for dilutability within the measurement range.

14. Stability.—All three test kit lots were stored at 2 to 8°C for up to 33 months and tested at regular intervals. One test kit lot was used as an assessment of open kit stability in parallel.

AOAC Official Method 2024.06

D-Lactic Acid in Selected Foods and Beverages

Enzymatic Determination by Enzytec™ Liquid D-Lactic Acid: Manual and Automated Applications

First Action 2024

[Applicable for the quantitative measurement of D-lactic acid in milk and (fermented) milk products, fermented vegetable products, wines, beer, fruit and vegetable juices, and eggs and egg powder. Ascorbic acid, 3-hydroxybutyric acid, and sulfite were found to interfere at concentrations higher than 0.2, 0.2, and 0.05 g/L, respectively. Oxaloacetic acid, pyruvic acid, and D-fructose do not interfere at concentrations at or below 0.2, 1, and 10 g/L, respectively. D-lactic acid is not suitable as a spoilage indicator for fermented products.]

Caution: See Material Safety Data Sheet and Instructions for Use available at https://eifu.r-biopharm.com/food/US/food.

A. Principle

D-Lactic acid reacts in the presence of NAD and D-Lactate dehydrogenase (D-Lactate DH) to form pyruvate and NADH (Figure 2024.06). This reaction is quantitative. The amount of NADH formed is equimolar to the D-lactic acid converted and is measured at a wavelength of 340 nm within 20 min.

Figure 2024.06.

Enzymatic reaction scheme for the determination of D-lactic acid.

The test kit contains two ready-to-use components, which forms the basis for a robust and precise simple quantification of D-lactic acid.

B. Chemicals and Reagents

Items (a)–(b) are available as a test kit (Enzytec Liquid D-Lactic acid, Cat. No. E8245; R-Biopharm AG, Darmstadt, Germany). Refer to kit label for expiry at 2 to 8°C and date of production.

1. Reagent 1.—2 × 50 mL containing D-Lactate DH in buffer.

2. Reagent 2.—2 × 12.5 mL containing NAD in buffer.

3. Enzytec Multi acid standard high.—5000 mg/L of D-lactic acid, Cat. No. E8465 (R-Biopharm).

4. Enzytec Multi acid standard low.—250 mg/L of D-lactic acid, Cat. No. E8460 (R-Biopharm).

   Required but not provided with the test kit:

5. Distilled water.

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

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

8. Carrez I solution.—3.60 g K₄[Fe(CN)₆] × 3 H₂O/100 mL of water.

9. Carrez II solution.—7.20 g ZnSO₄ × 7 H₂O/100 mL of water.

10. Concentrated Carrez I solution.—15 g K₄[Fe(CN)₆] × 3 H₂O/100 mL of water.

11. Concentrated Carrez II solution.—30 g ZnSO₄ × 7 H₂O/100 mL of water.

12. 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.

13. Perchloric acid, 1 M in water.

C. Apparatus and Supplies

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

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

2. pH meter.—FiveEasy™ pH/mV bench meter (Mettler-Toledo, Giessen, Germany).

3. Beakers.

4. Fluted paper filters.—X100 Grade 3 hw 150 mm filter paper, circles, Ahlstrom-Munksjo Munktell, Helsinki, Finland.

5. Plastic tubes.—50 mL, recloseable, conical tube, item number 339652, Nunc A/S, Roskilde, Denmark.

6. Magnetic stirrer.—Cimarec™ Poly 15 (Fisher Scientific, Schwerte, Germany).

7. Graduated flasks.—50, 100, 200 mL.

8. Reaction tubes.—2 mL; recloseable.

9. Syringe filters.—Minisart^® High Flow syringe filter 16 532 K (Sartorius).

10. Disposable plastic cuvettes.—1 cm light path.

11. Micropipettes.—20–200 and 100–1000 μL.

12. Multipette.—To dispense 2 mL aliquots of reagent 1 and 500 µL of reagent 2 for manual pipetting.

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

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

15. Vortex mixer.

16. Ultrasonic bath.—Sonorex Super RK 31 (Bandelin, Berlin, Germany).

17. Microcentrifuge.—Mikrozentrifuge 5427 R (Eppendorf SE, Hamburg, Germany).

D. General Preparations

1. Store the kit at 2–8°C. Let all kit components come to room temperature 20–25°C before use. Do not freeze any of the kit components.

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

3. Pipet the test or control solution with a variable micropipet and the reagent 1 and 2 solution with a multi-stepper pipet to ensure good mixing. Use a single tip for each of these components.

4. Components and procedures of the test kit have been standardized for use in this procedure. Do not interchange components between kits of different batches (lot numbers).

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

6. Keep in mind that solid matrixes can be inhomogeneous; therefore, grind a representative part of the samples very well and homogenize before weighing.

E. Test Solution Preparation

1. Use clear test samples directly, or after dilution with distilled water. For a D-lactic acid concentration between 15 and 600 mg/L, use 100 µL test volume. For concentrations between 1.5 and 60 mg/L, use 1000 µL. For test solutions with concentrations close to the LOQ, it is recommended to increase the test volume [see G(g)], check the pH value of the test solution and neutralize the pH value in case of any doubt; test solutions must be clear.

2. Red wines need to be diluted only (typically 1:5 with water); use 100 µL test volume.

3. For turbid test samples (e.g., sauerkraut juice, pineapple juice): Filter by using fluted paper filter or syringe filter or centrifuge the test solution in a reaction tube (recommended 3000 g for at least 5 min) until a clear filtrate or supernatant is obtained. Use 100 µL (or more if necessary) of fruit juices undiluted; fermented vegetable juice needs to be diluted before measurement; use 100 µL test volume.

4. Degas matrixes containing carbon dioxide (e.g., beer) by aid of a short ultrasound burst (10 s), filter if not clear, and use 100 µL of the test solution.

5. Adjust pH value of strongly acidic matrixes (e.g., white wine and fruit juices if used undiluted) by adding 1 M KOH until pH value is between 6.5 and 7.5; bring to a known volume with distilled water.

6. Use PVPP when testing juices/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 (paper filter or syringe filter) or centrifuge at 3000 g for at least 5 min in a reaction tube until a clear supernatant is obtained.

7. Sausages.—Weigh 5 g homogenized test sample in a 50 mL Falcon tube and add 20 mL of 1 M perchloric acid, vortex to suspend and rotate for 10 min, transfer with approx. 40 mL distilled water in a beaker; add 5 M KOH until stirring until pH value is approx. 7, and transfer in a 100 mL graduated flask; dilute to the mark with distilled water and store for 20 min at 2 to 8°C in a refrigerator; filter through a fluted paper filter and use 100 µL of the extract.

8. Yogurt and cream cheese.—Weigh 2 g of test sample in a 100 mL graduated flask, add distilled water and shake to suspend, and dilute to the mark with distilled water; in the case of cream cheese, add 10 mL distilled water to 2 g of test sample, suspend, add another 10 mL, and repeat this procedure until the material is fully suspended, then dilute to 100 mL with distilled water; filter through a fluted paper filter or centrifuge at 3000 g for at least 5 min in a reaction tube until a clear supernatant is obtained. Use 1000 µL for analysis.

9. Milk and tomato juice.—Pipet 1 mL of milk or 5 mL of tomato juice in a 50 mL graduated flask, add 10 mL of distilled water, add 2.5 mL Carrez I solution, shake, and add 2.5 mL Carrez II solution; shake and add 5 mL 0.1 M NaOH; shake and dilute with distilled water to the mark; filter through a fluted paper filter or centrifuge at 3000 g for at least 5 min in a reaction tube until a clear supernatant is obtained; use 1000 µL for analysis.

10. Cream and whole milk powder.—Weigh 2 g test sample in a 100 mL graduated flask, add 10 mL of distilled water, add 5 mL Carrez I solution, shake, and add 5 mL Carrez II solution; shake and add 10 mL 0.1 M NaOH; shake and dilute with distilled water to the mark; filter through a fluted paper filter or centrifuge at 3000 g for at least 5 min in a reaction tube until a clear supernatant is obtained; use 1000 µL for analysis.

11. Whole egg powder.—Weigh 2 g test sample in a 50 mL Falcon tube, then add 10 mL of distilled water and 1 drop of 1-octanol; shake and incubate for 15 min in a boiling water bath; cool the tube to room temperature and transfer in a 50 mL graduated flask; add 2 mL concentrated Carrez I solution, shake, add 2 mL concentrated Carrez II solution, shake, and add 1 M NaOH until pH value is approx. 8 (normally approx. 200 µL of 1 M NaOH are needed); dilute with distilled water to the mark, shake and filter through a fluted paper filter, and use 1000 µL for analysis.

12. Liquid egg.—Weigh 5 g test sample in a 50 mL Falcon tube, then add 10 mL of distilled water and 1 drop of 1-octanol; shake and incubate for 15 min in boiling water bath; cool the tube to room temperature and transfer in a 25 mL graduated flask; add 1 mL concentrated Carrez I solution, shake, add 1 mL concentrated Carrez II solution, shake, and dilute to the mark with 0.1 M NaOH; shake and filter through a fluted paper filter and use 1000 µL for analysis.

13. In the case of higher test volumes (up to 1000 µL), check the pH value of the test solution and neutralize the pH value in case of any doubt; test solutions must be clear.

F. Determination

For a test volume of 100 µL in the manual application, the linear range is between 15 and 600 mg/L. The LOD is 5.4 mg/L. For the automated applications, the measurement ranges are from 5.0 to 625 mg/L (Basic), 30 to 3125 mg/L (High range), and 0.75 to 62.5 mg/L (Sensitive).

1. Manual assay in 4 mL cuvettes

   1. It is recommended to use control solutions like references or standard solutions such as Multi Acid standard described in B(c) and (d).

   2. Pipet the test or control solution with a variable micropipet and the reagent 1 and 2 solution with a multi-stepper pipet to ensure good mixing.

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

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

   5. Pipet 2 mL of reagent 1 (R1) in each cuvette.

   6. Add 100 µL of distilled water (blank), test or control solutions; mix carefully, e.g., using disposable plastic spatulas.

   7. Incubate for 3 min between 20 and 37°C.

   8. Read and document absorbance (A1) in a spectrophotometer set at 340 nm for each cuvette.

   9. Add 500 µL of reagent 2 (R2) in each cuvette and mix well.

   10. Incubate for 15 min between 20 and 37°C.

   11. Read and document absorbance (A2) in a spectrophotometer set at 340 nm for each cuvette.

   12. It is possible to increase the test volume up to 1000 µL to achieve an increase in assay sensitivity. The volumes for R1 and R2 remain unchanged; however, the calculation must be changed as described in Section G.

   13. If a creep reaction occurs, the reaction is not finished after 15 min and typically shows a constant ΔA increase. To calculate the analyte-specific ΔA value, plot the absorbance values against time and calculate a linear regression to obtain the increase in ΔA per minute that is related to the creep reaction. Extrapolate the absorbance to the time of addition of reagent 2 (see AOAC OMA 2024.07 for the explanatory Figure 2024.07B).

2. Automation on a Pictus 500 device

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

   2. High range application: Two-point linear calibration with 0 mg/L (water) and 5000 mg/L (use Enzytec Multi acid standard high).

   3. Basic range application: Two-point linear calibration with 0 mg/L (water) and 1000 mg/L (use Enzytec Multi acid standard high and dilute 1 + 4 with water).

   4. Sensitive range application: Four-point calibration with 0, 11.1, 33.3, and 100 mg/L prepared by diluting Enzytec Multi Acid standard [Section B(d)].

   5. To ensure that these calibrations are valid over a longer period of, e.g., 1 week, aqueous control solutions [Section B(c) and (d)] should be analyzed with every run. If these control solutions are not within specifications [see H(b)], a recalibration must be done.

   6. Determination

      1. Add 200 µL reagent 1.

      2. Add test solutions, control solution or calibrator/water; volumes depend on the application:

         1. 2 µL for High range application.

         2. 10 µL for Basic range application.

         3. 100 µL for Sensitive application.

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

      4. Incubate for 2 min at 37°C.

      5. Read A1 at 340 nm.

      6. Add 50 µL reagent 2.

      7. Incubate for 10 min at 37°C.

      8. Read A2 at 340 nm.

G. Calculations

1. Calculate ΔA for every test or control solution:

   $$\Delta A\mathrm{ }=\mathrm{ }(A2\mathrm{ }-\mathrm{ }df\mathrm{ }\times \mathrm{ }A1)\mathit{test}\mathrm{ }or\mathrm{ }\mathit{control}\mathrm{ }–\mathrm{ }(A2\mathrm{ }-\mathrm{ }df\mathrm{ }\times \mathrm{ }A1)\mathit{reagent}\mathrm{ }\mathit{blank}$$
   where df is a dilution factor depending on the test volume:

   $$100\mathrm{ }\mathrm{\mu }L,\mathrm{ }df\mathrm{ }=\mathrm{ }(\mathit{test}\mathrm{ }\mathit{volume}\mathrm{ }+\mathrm{ }R1)/(\mathit{test}\mathrm{ }\mathit{volume}\mathrm{ }+\mathrm{ }R1\mathrm{ }+\mathrm{ }R2)\mathrm{ }=\mathrm{ }0.808$$

    

   $$1000\mathrm{ }\mathrm{\mu }L,\mathrm{ }df\mathrm{ }=\mathrm{ }(\mathit{test}\mathrm{ }\mathit{volume}\mathrm{ }+\mathrm{ }R1)/(\mathit{test}\mathrm{ }\mathit{volume}\mathrm{ }+\mathrm{ }R1\mathrm{ }+\mathrm{ }R2)\mathrm{ }=\mathrm{ }0.857$$
2. Calculate concentrations for every test or control solution (100 µL test volume):

   $$c\mathrm{ }=\mathrm{ }(V\mathrm{ }\times \mathrm{ }MW\mathrm{ }\times \mathrm{ }\Delta A)/(\epsilon \mathrm{ }\times \mathrm{ }d\mathrm{ }\times \mathrm{ }v)$$
   where V = final volume; MW = molecular weight of D-lactic acid; ε = absorption coefficient of NADH at 340 nm; d = light path within cuvette; v = test volume.

   $$D-\mathit{Lactic}\mathrm{ }\mathit{acid},\mathrm{ }mg/L\mathrm{ }=$$

    

   $$(2.600\mathrm{ }mL\mathrm{ }\times \mathrm{ }90.1\mathrm{ }g\mathrm{ }\times \mathrm{ }\mathit{mole}-1\mathrm{ }\times \mathrm{ }\Delta A)/$$

    

   $$(6.3\mathrm{ }L\mathrm{ }\times \mathrm{ }\mathit{mmole}-1\mathrm{ }\times \mathrm{ }cm-1\mathrm{ }\times \mathrm{ }1\mathrm{ }cm\mathrm{ }\times \mathrm{ }0.1\mathrm{ }mL)$$
   or

   $$D-\mathit{Lactic}\mathrm{ }\mathit{acid},\mathrm{ }mg/L\mathrm{ }=\mathrm{ }371.8\mathrm{ }\times \mathrm{ }\Delta A$$

3. If a test solution was diluted before measurement, this result must be multiplied with the dilution factor.

4. When using a test volume of 1000 µL, the calculation will change as follows:

   $$D-\mathit{Lactic}\mathrm{ }\mathit{acid},\mathrm{ }mg/L\mathrm{ }=$$

    

   $$(3.500\mathrm{ }mL\mathrm{ }\times \mathrm{ }90.1\mathrm{ }g\mathrm{ }\times \mathrm{ }\mathit{mole}-1\mathrm{ }\times \mathrm{ }\Delta A)/$$

    

   $$(6.3\mathrm{ }L\mathrm{ }\times \mathrm{ }\mathit{mmole}-1\mathrm{ }\times \mathrm{ }cm-1\mathrm{ }\times \mathrm{ }1\mathrm{ }cm\mathrm{ }\times \mathrm{ }1.0\mathrm{ }mL)$$
   or

   $$D-\mathit{Lactic}\mathrm{ }\mathit{acid},\mathrm{ }mg/L\mathrm{ }=\mathrm{ }50.06\mathrm{ }\times \mathrm{ }\Delta A$$
5. Calculation in solid matrixes:

   $$D-\mathit{Lactic}\mathrm{ }\mathit{acid},\mathrm{ }mg/100\mathrm{ }g\mathrm{ }=$$

    

   $$D-\mathit{Lactic}\mathrm{ }\mathit{acid}\mathrm{ }\mathit{test}\mathrm{ }\mathit{solution},\mathrm{ }mg/L/$$

    

   $$(\mathit{test}\mathrm{ }\mathit{sample}\mathrm{ }\mathit{weight}\mathrm{ }in\mathrm{ }\mathit{test}\mathrm{ }\mathit{solution},\mathrm{ }mg/L)\times 100$$

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

7. Results close to LOQ.—If the result is between 15 and 30 mg/L for a test volume of 100 µL, repeat measurement with 200 µL; if lower than 15 mg/L, repeat measurement with 500 or 1000 µL test volume for better accuracy (trueness, precision).

   Measurement range for 200 µL test volume: 7.5 to 300 mg/L.

   Measurement range for 1000 µL test volume: 1.5 to 60 mg/L.

H. System Suitability Tests/Analytical Quality Control

1. For each analytical run, an aqueous control solution (e.g., Enzytec Multi acid standard low, 250 mg/L of D-lactic acid) should be analyzed. If available, use certified reference materials.

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

3. 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 pipets for accuracy, and (4) check photometer for correct wavelength.

Results and Discussion

General Remarks

In addition to manual testing, 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 of the format in which the test kit is used, each single performance characteristic was not repeated for both manual and automated applications.

Linearity/Measurement Range

Twenty-six test solutions with concentrations between 5 and 1200 mg/L were measured in triplicate using each of the three different lots. The mean concentration from all three lots was plotted against the target concentration.

As can be seen in Figure 1, the upper limit of the linear measurement range is under ideal conditions (aqueous solution and fresh test kits) around 500 mg/L. All RSD values are below 5% for concentrations between 40 and 500 mg/L. Below concentrations of 40 mg/L RSD values were at or below 10%. The measurement range covers a broad range and is even given at concentrations as low as 15 mg/L (Figure 2).

Figure 1.

Measured D-lactic acid concentrations for aqueous solutions with concentrations between 0.005 and 1.200 g/L with standard deviation for three independent measurements for each lot (closed circles); the relative standard deviation in % is also given (open triangles); open circles depict values out of the linear measurement range.

Figure 2.

Measured concentration values for aqueous solutions with concentrations between 5 and 50 mg/L with standard deviation for three independent measurements for each lot (closed circles); the relative standard deviation in % is also given (open triangles).

From the plot shown in Figure 1, it is clear that the upper limit of linearity is 500 mg/L for a new test kit lot, whereas the lower limit of linearity is the LOQ (15 mg/L; see Estimation of LOQ) by definition. Figure 3 shows that even long-term storage (24 months at 2–8°C) does not lead to a reduced range of linearity; the graphs in Figures 1 and 3 are nearly identical in the upper part. There is a tendency toward higher variability for concentrations below 20 mg/L, which could be attributed to analyst variability.

Figure 3.

Linearity of the system between 5 and 1200 mg/L out of data derived from two lots with three replicate (closed circles) and a sample volume of 100 µL after storage for 24 months at 2–8°C; relative standard deviation in % is also given (open triangles); open circles depict values out of the linear measurement range.

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

Estimation of LOD

The LOD was determined by measuring 10 test solutions in the very low measurement range (5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg/L) with a test volume of 100 µL. Each test solution was measured in triplicate with each reagent lot. For the evaluation, the estimation procedure according to DIN 32645 (comparable to DIN ISO 11843–2) was used. A detailed description of the calculation can be found in AOAC OMA 2017.05 (4). Estimated LOD values varied between 3.2 and 5.4 mg/L.

Estimation of LOQ

The LOQ was evaluated from the same measurement data as above (see Limit of Detection). The calculated LOQ is between 5.7 and 9.8 mg/L. A low concentration of 10 mg/L (Figure 2) still resulted in an RSD at about 15% (n = 9; three test kit lots). At 15 mg/L, the resulting RSD is approximately 10%, which is acceptable for an LOQ. Greater test volumes were tested for the automated application (see Automation on a Pictus 500 Spectrophotometric Analyzer). After 24 months of storage, the RSD value at 15 mg/L is 18%, which might be attributed to analyst variability (results not shown).

Selectivity Study: Side Activities

None of the tested organic acids showed side activities at these concentrations with the exception of 100 g/L L-lactic acid, which exerts a negligible activity of 0.04% (Table 1).

Table 1.

Substances tested for side reactivity in the absence of D-lactic acid

Substance	Target				Measured
	g/L	A 1	A 2	ΔA	g/L
L-Lactic acid	100	0.065	0.751	0.112	0.042
	10	0.066	0.465	0.014	0.005
Pyruvic acid	3	0.066	0.434	0.006	0.002
	1	0.058	0.421	0.000	0.000
	0.5	0.065	0.429	0.002	0.001
Formic acid	20	0.1	0.509	0.003	0.001
D/L-Malic acid	40	0.066	0.481	0.004	0.001
Butyric acid	20	0.063	0.474	−0.002	−0.001
Citric acid	20	0.063	0.472	−0.003	−0.001
Acetic acid	20	0.068	0.475	−0.005	−0.002
Oxalic acid	20	0.07	0.476	−0.005	−0.002
Propionic acid	20	0.063	0.47	−0.006	−0.002
L-Tartaric acid	20	0.063	0.473	−0.003	−0.001
Oxaloacetic acid	20	0.187	0.618	0.05	0.019
	2	0.134	0.56	0.035	0.013
	0.2	0.128	0.551	0.031	0.012
Ascorbic acid	20	0.234	1.153	0.537	0.2
	2	0.109	0.681	0.167	0.062
3-Hydroxy-butyric acid	20	0.065	2.524	2.05	0.762
	2	0.064	0.759	0.286	0.106
SO2	20	0.063	4.136	3.659	1.361
	2	0.063	1.251	0.778	0.289
	0.2	0.062	0.551	0.074	0.028

In contrast, ascorbic acid, 3-hydroxybutyric acid, and sulfite exerted side activity (Table 1) and were analyzed in more detail (Table 2). Using the LOQ of 15 mg/L as a limit, ascorbic acid, 3-hydroxy butyric acid, and sulfite were found to have a low activity at concentrations higher than 0.2, 0.2, and 0.1 g/L, respectively.

Table 2.

Detailed analysis of ascorbic acid, 3-hydroxybutyric acid, and SO₂ for side reactivity in the absence of D-lactic acid; LOQ of the system is 10 mg/L; mean of two replicates is shown

Substance	Target				Measured
	g/L	A 1	A 2	ΔA	g/L
Ascorbic acid	1	0.119	0.636	0.121	0.045
	0.5	0.113	0.586	0.076	0.028
	0.2	0.110	0.547	0.039	0.015
	0.1	0.108	0.529	0.023	0.008
	0.02	0.107	0.507	0.002	0.001
3-Hydroxy-butyric acid	0.2	0.107	0.514	0.008	0.003
	0.15	0.106	0.510	0.005	0.002
	0.1	0.106	0.511	0.006	0.002
	0.05	0.104	0.505	0.009	0.003
	0.02	0.107	0.503	0.004	0.002
SO2	0.2	0.104	0.576	0.080	0.030
	0.15	0.104	0.558	0.061	0.023
	0.1	0.105	0.532	0.035	0.013
	0.05	0.106	0.511	0.014	0.005
	0.02	0.107	0.511	0.012	0.004

Selectivity Study: Interference

During validation, the above-mentioned substances (formic acid, D-/L-malic acid, butyric acid, citric acid, acetic acid, oxalic acid, propionic acid, L-tartaric acid, sorbic acid) were also tested in the presence of 0.2 g/L D-lactic acid. No interference of the measurement of D-lactic acid was detected when these substances were present at a concentration of 20 g/L (Table 3).

Table 3.

Substance tested for interference in the presence of 0.2 g/L D-Lactic acid

Substance	Interferant	Target				Measured	Recovery
	g/L	g/L	A 1	A 2	ΔA	g/L	%
Pyruvic acid	3	0.1	0.058	0.651	0.229	0.085	85.1
	1	0.1	0.065	0.680	0.253	0.094	94.0
	0.5	0.1	0.065	0.685	0.258	0.096	95.8
Formic acid	20	0.2	0.129	1.107	0.534	0.199	99.3
DL-Malic acid	20	0.2	0.130	1.107	0.533	0.198	99.1
Butyric acid	20	0.2	0.130	1.105	0.534	0.199	99.3
Citric acid	20	0.2	0.129	1.108	0.534	0.199	99.3
Acetic acid	20	0.2	0.130	1.108	0.538	0.200	100
Oxalic acid	20	0.2	0.129	1.108	0.534	0.199	99.4
Propionic acid	20	0.2	0.131	1.098	0.527	0.196	98.0
L-Tartaric acid	20	0.2	0.129	1.105	0.531	0.198	98.8
Sorbic acid	20	0.2	0.124	1.075	0.533	0.198	99.2
Oxaloacetic acid	20	0.2	0.281	1.197	0.501	0.186	93.2
	2	0.2	0.132	1.105	0.529	0.197	98.4
Ethanol	100	0.1	0.065	0.703	0.276	0.103	103
	30	0.1	0.065	0.695	0.268	0.100	99.8
	10	0.1	0.066	0.692	0.265	0.098	98.4

The recovery of D-lactic acid was always between 98 and 100%. Pyruvic acid and oxaloacetic acid did not show interference up to 1 and 2 g/L, respectively, but lower recoveries can be expected when the amounts are increased (Table 3). When performing this experiment with ascorbic acid, 3-hydroxy butyric acid, or sulfite, recoveries of 200% and higher were observed (results not shown). Therefore, a more detailed experiment was conducted to characterize these interferences in a better way. As can be seen in Table 4, 3-hydroxybutyric acid, ascorbic acid, and SO₂ interfere at or higher than 0.2, 0.2, and 0.05 g/L, respectively.

Table 4.

Detailed analysis of interference of 3-hydroxy-butyric acid, ascorbic acid, and SO₂ at different concentrations in the presence of 0.1 g/L D-lactic acid

Substance	Interferant	Target				Measured	Recovery
	g/L	g/L	A 1	A 2	ΔA	g/L	%
3-Hydroxy-butyric acid	2	0.1	0.109	0.866	0.369	0.137	137
	1	0.1	0.109	0.812	0.316	0.117	117
	0.5	0.1	0.107	0.789	0.293	0.109	109
	0.2	0.1	0.108	0.776	0.280	0.104	104
	0.1	0.1	0.107	0.773	0.277	0.103	103
Ascorbic acid	2	0.1	0.129	0.948	0.436	0.162	162
	1	0.1	0.117	0.858	0.356	0.132	132
	0.5	0.1	0.110	0.807	0.311	0.116	116
	0.2	0.1	0.105	0.766	0.274	0.102	102
	0.1	0.1	0.103	0.752	0.262	0.097	97
SO2	1	0.1	0.103	1.067	0.582	0.216	216
	0.5	0.1	0.103	0.848	0.363	0.135	135
	0.2	0.1	0.104	0.830	0.345	0.128	128
	0.1	0.1	0.103	0.792	0.308	0.114	114
	0.05	0.1	0.108	0.770	0.281	0.104	104
	0.025	0.1	0.106	0.765	0.277	0.103	103

Glucose, sucrose, lactose, and galactose do not interfere at concentrations of 100 g/L (Table 5). Sugar alcohols (glycerol and sorbitol) do not interfere at concentrations of 10 g/L. In contrast, fructose does interfere above 10 g/L.

Table 5.

Sugars and sugar alcohols tested for interference in the presence of 0.1 g/L D-lactic acid

Substance	Interferant	Target				Measured	Recovery
	g/L	g/L	A 1	A 2	ΔA	g/L	%
Glucose	100	0.1	0.103	0.761	0.275	0.102	102
Sucrose	100	0.1	0.109	0.769	0.280	0.104	104
Lactose	100	0.1	0.105	0.762	0.275	0.102	102
Galactose	100	0.1	0.104	0.763	0.277	0.103	103
Glycerol	10	0.1	0.102	0.758	0.273	0.102	102
Sorbitol	10	0.1	0.102	0.755	0.270	0.101	101
Fructose	100	0.1	0.105	0.888	0.401	0.149	149
	75	0.1	0.106	0.862	0.374	0.139	139
	50	0.1	0.105	0.823	0.336	0.125	125
	25	0.1	0.103	0.778	0.292	0.109	109
	20	0.1	0.102	0.780	0.298	0.111	111
	15	0.1	0.102	0.772	0.290	0.108	108
	12.5	0.1	0.103	0.775	0.292	0.109	109
	10	0.1	0.105	0.778	0.278	0.103	103
	5	0.1	0.105	0.769	0.269	0.100	100

Trueness

For characterization of trueness, a standard wine of the German Wine Analysts (Label “hellblau” lot 1050807 with a D-lactic acid concentration at 0.722 ± 0.0373 g/L) was analyzed with n = 6 test portions per each the three test kit lots by one analyst. As can be seen in Table 6, the RSD values are below 1.4% and the recoveries are between 107 and 109%. These slightly elevated recoveries may be the result of the target concentration of this wine, which was derived from a Proficiency Testing Round where several methods were used. Comparing the measured mean concentration and the certified value resulted in no statistical difference (6). Trueness of the enzymatic test system was also checked during the characterization for Intermediate precision by using another standard wine (Label “orange” lot 1081608 with a D-lactic acid concentration at 1.07 ± 0.076 g/L). The mean recovery of this material was 103%.

Table 6.

Characterization for trueness using one Standard Wine

	Reference Wine
	target: 0.722 g/L
	Lot 1	Lot 2	Lot 3
Replicate	g/L	g/L	g/L
1	0.802	0.769	0.792
2	0.784	0.773	0.803
3	0.791	0.773	0.781
4	0.773	0.776	0.786
5	0.773	0.783	0.793
6	0.781	0.767	0.777
Mean	0.784	0.774	0.789
SD	0.011	0.006	0.009
RSD, %	1.42	0.73	1.19
Recovery, %	108.6	107.1	109.2

Recovery Using Spiked Matrix Samples

For characterization of recovery, an aqueous control solution, a spiked grape juice, and a spiked wine were used and analyzed with n = 6 test portions in each of the three lots. For spiking, an equimolar mixture of D- and L-lactic acid were used. As can be seen in Table 7, the RSD values are below 2% and the recovery is between 98 and 104%.

Table 7.

Recovery of D-lactic acid from an aqueous control solution, a spiked wine, and a spiked grape juice

Target, g/L	Control solution	Wine, spiked	Grape juice, spiked
	0.135	0.200	0.200
Lot 1	0.142	0.195	0.202
	0.143	0.195	0.205
	0.142	0.194	0.203
	0.140	0.197	0.201
	0.139	0.196	0.200
	0.141	0.196	0.204
Lot 2	0.140	0.196	0.202
	0.140	0.192	0.202
	0.137	0.193	0.201
	0.137	0.191	0.202
	0.136	0.195	0.200
	0.139	0.197	0.202
Lot 3	0.139	0.199	0.202
	0.143	0.200	0.201
	0.144	0.198	0.202
	0.142	0.198	0.202
	0.145	0.202	0.205
	0.141	0.199	0.200
Mean, g/L	0.141	0.196	0.202
SD, g/L	0.0025	0.0028	0.0015
RSD, %	1.78	1.42	0.72
Recovery, %	104	98	101

Results for analysis of spiked sauerkraut juice and beer are shown in Table 8. Mean recoveries were between 98 and 101%. RSD values for spiked and non-spiked matrixes were remarkably low with values between 0.23 and 0.52%.

Table 8.

Recovery of D-lactic acid in sauerkraut juice and beer spiked at two levels and analyzed in one test kit lot

	Sauerkraut juice
	Non-spiked	Spiked, 1.5 g/L	Difference	Recovery
	g/L	g/L	g/L	%
	4.547	6.029	1.461	97.4
	4.577	6.068	1.500	100.0
	4.571	6.032	1.464	97.6
	4.541	6.029	1.461	97.4
	4.564	6.024	1.456	97.1
	4.607	6.053	1.485	99.0
Mean	4.568	6.039	1.471	98.1
SD	0.024	0.017	0.017
RSD, %	0.52	0.29	1.19

	Beer
	Non-spiked	Spiked, 0.1 g/L	Difference	Recovery
	g/L	g/L	g/L	%
	0.146	0.246	0.100	100.2
	0.145	0.245	0.100	99.6
	0.145	0.246	0.100	100.2
	0.146	0.246	0.101	101.1
	0.145	0.246	0.100	100.4
	0.146	0.247	0.101	101.4
Mean	0.145	0.25	0.100	100.5
SD	0.000	0.001	0.001
RSD, %	0.23	0.26	0.64

Table 9 summarizes results for non-spiked and spiked whole egg. In this case, two different low levels of D-lactic acid were spiked since this organic acid is a spoilage parameter. Mean recoveries varied between 95 and 97%. Again, RSD values for the six-fold extraction/measurement of spiked test samples were excellent. In the case of the non-spiked whole egg, the measured mean value of 5.4 mg/kg showed a higher RSD value of 22%, which can be explained by the closeness to the LOQ of the system (15 mg/L if 100 µL is applied). For extraction of egg, the test sample was clarified with Carrez and 500 µL was applied for measurement.

Table 9.

Recovery of D-lactic acid in whole egg spiked at two different levels and analyzed in one test kit lot

	Liquid whole egg
	Non-spiked	Spiked, 50 mg/kg	Difference	Recovery
	mg/kg	mg/kg	mg/kg	%
	3.4	53.8	48.4	96.8
	5.0	53.4	48.0	96.1
	5.4	52.0	46.5	93.1
	7.0	53.3	47.9	95.8
	5.8	54.1	48.7	97.3
	5.8	52.1	46.7	93.4
Mean	5.4	53.1	47.7	95.4
SD	1.19	0.88	0.88
RSD, %	22.04	1.66	1.8

	Non-spiked	Spiked, 100 mg/kg	Difference	Recovery
	mg/kg	mg/kg	mg/kg	%
	3.4	104.4	99.0	99.0
	5.0	100.3	94.9	94.9
	5.4	103.1	97.7	97.7
	7.0	102.7	97.3	97.3
	5.8	100.1	94.7	94.7
	5.8	101.4	96.0	96.0
Mean	5.4	102.0	96.6	96.6
SD	1.19	1.69	1.69
RSD, %	22.04	1.66	1.8

Table 10 summarizes results for a broad range of food matrixes spiked at one level with six test portions and analyzed in one lot. For spiking, a matrix-specific mixture of D- and L-lactic acid was used. Mean recoveries for milk and (fermented) products (milk, yogurt, cream, whole milk powder) varied between 90 and 99%. RSD values for the six-fold extraction/measurement of un-spiked test samples containing D-lactic acid were between 4.7 (yogurt) and 6.1% (milk powder), which is excellent for levels of approximately 4 mg/100 g. For spiked milk and milk products, RSD values varied from a very low 0.5 for yogurt and 9% for milk. Red wine often contains high natural contents of D-lactic acid due to the malo-lactic fermentation. Recovery and RSD value were 99% and below 1%, respectively. Tomato juice contains low concentrations of D-lactic acid of approximately 30 mg/L and an acceptable RSD value of 2.2%. After spiking an additional 15 mg/L to tomato juice, the calculated recovery was 105% with an RSD value of 2.6%. Despite a mean D-lactic acid content of less than 3 mg/100 g, the RSD value for the un-spiked egg powder is 7.6%. Spiking additional 5 mg/100 g led to a mean recovery of 101% and an RSD value of 5.6%. The matrix with the most complicated extraction is sausage. The mean endogenous content of D-lactic acid is approximately 60 mg/100 g with a RSD value of 3.9%. Spiking of 60 mg/100 g resulted in a mean recovery of 95%. All matrixes were analyzed using a test volume of 1000 µL with the exception of red wine and sausage, where 100 µl were applied.

Table 10.

Recovery of D-lactic acid in red wine, tomato juice, milk, yogurt, cream, whole milk powder, whole egg spiked, and sausage spiked at one level in six test portions and analyzed in one test kit lot; indicated spike levels are given as the D-lactic acid level

	Red wine	Tomato juice	Milk	Yogurt	Cream	Whole milk powder	Whole egg powder	Sausage
	g/L	mg/L	mg/L	mg/100 g	mg/100 g	mg/100 g	mg/100 g	mg/100 g
	Non-spiked
	534	29.08	−2.94	4.22	−0.40	3.60	2.91	58.96
	541	28.75	−2.17	3.78	−0.08	3.33	2.59	65.44
	533	29.73	−2.44	4.04	−0.24	3.30	2.99	60.99
	543	28.38	−0.27	4.02	−0.20	2.97	2.55	60.38
	534	28.55	−2.38	4.35	−0.55	3.40	2.96	61.11
	543	29.98	−1.60	4.13	−0.20	3.33	2.60	58.88
Mean	538	29.08	−1.97	4.09	−0.28	3.32	2.77	60.96
SD	4.70	0.650	0.94	0.19	0.17	0.20	0.21	2.40
RSD, %	0.87	2.23	—	4.7	—	6.1	7.6	3.9
Spike level	250 mg/L	15 mg/L	20 mg/L	100 mg/100 g	100 mg/100 g	10 mg/100 g	5 mg/100 g	60 mg/100 g
	794	42.982	59.69*	93.7	96.7	13.6	7.7	120.94
	783	44.848	19.51	94.0	101.9	12.7	7.5	111.91
	785	44.568	15.84	93.0	99.4	13.2	8.1	114.25
	785	46.511	17.98	94.4	98.7	13.2	7.9	116.97
	780	44.858	15.90	94.1	100.2	13.5	7.2	119.92
	784	45.338	17.98	93.8	97.1	13.3	8.5	124.33
Mean	785	44.851	17.44	93.8	99.0	13.3	7.8	118.1
SD	4.71	1.146	1.57	0.48	1.94	0.32	0.44	4.57
RSD, %	0.60	2.55	8.99	0.51	2.0	2.4	5.6	3.9
Recovery, %	98.9	105	97.0	89.8	99.3	99.3	100.8	95.2

Inter-lot Precision

To characterize differences between test kit lots, an inter-lot precision experiment was set up by analyzing three different aqueous solution with n = 6 test portions on 1 day by one analyst in all three kit lots. This is necessary to show that all lots were produced under routine and comparable conditions. The results are shown in Table 11 and prove that all three lots were comparable over the whole measurement range. All RSD values were at or below 1.3% with the exception for the lowest concentration, where RSD values were 5.4% and lower.

Table 11.

Characterization of inter-lot precision using three different aqueous control solutions with concentrations of 60, 200, and 400 mg/L analyzed on one day in all three lots by one analyst with six replicates

Target, g/L	Lot 1	Lot 2	Lot 3
	g/L	g/L	g/L
0.060	0.061	0.056	0.060
0.060	0.062	0.055	0.060
0.060	0.059	0.056	0.057
0.060	0.063	0.063	0.062
0.060	0.059	0.057	0.060
0.060	0.061	0.056	0.062
Mean, g/L	0.061	0.057	0.060
RSD, %	2.57	5.44	3.14
0.200	0.193	0.195	0.198
0.200	0.190	0.196	0.195
0.200	0.190	0.196	0.197
0.200	0.196	0.197	0.196
0.200	0.195	0.196	0.197
0.200	0.195	0.194	0.196
Mean, g/L	0.193	0.196	0.197
RSD, %	1.29	0.52	0.47
0.400	0.391	0.392	0.393
0.400	0.386	0.386	0.398
0.400	0.387	0.394	0.395
0.400	0.390	0.388	0.393
0.400	0.390	0.387	0.393
0.400	0.389	0.388	0.386
Mean, g/L	0.389	0.389	0.393
RSD, %	0.48	0.81	0.96

Intermediate Precision

For the evaluation of intermediate precision, three different control solutions distributed over the measuring range were measured. Each solution was measured in n = 6 replicates using each reagent lot by three analysts on 3 different days. In total, 18 measurements were received for every solution. As shown in Table 12, the RSD values are not higher than 1.3% and even lower for higher concentrations.

Table 12.

Characterization of laboratory-internal reproducibility (intermediate precision) in three aqueous control solutions analyzed by three analysts in three lots on different days with six replicates; mean, standard deviation (SD), and relative standard deviation (RSD) are calculated

			Aqueous control solutions
Person/day	Lot	Replicate	0.060 g/L	0.200 g/L	0.400 g/L
1	3	1	0.061	0.197	0.393
1	3	2	0.058	0.197	0.396
1	3	3	0.060	0.196	0.390
1	3	4	0.060	0.199	0.391
1	3	5	0.060	0.200	0.388
1	3	6	0.061	0.197	0.392
2	2	1	0.059	0.193	0.391
2	2	2	0.059	0.195	0.389
2	2	3	0.059	0.195	0.390
2	2	4	0.059	0.196	0.391
2	2	5	0.060	0.196	0.391
2	2	6	0.060	0.196	0.390
3	1	1	0.059	0.197	0.393
3	1	2	0.060	0.197	0.392
3	1	3	0.059	0.197	0.392
3	1	4	0.060	0.197	0.396
3	1	5	0.061	0.197	0.394
3	1	6	0.060	0.197	0.394
Mean, g/L			0.060	0.197	0.392
SD, g/L			0.0008	0.0016	0.0022
RSD, %			1.30	0.79	0.56

To get an idea about intermediate precision in matrix, one test kit lot was tested on 2 different days using three different photometers by three analysts in a laboratory (Table 13). The use of three different test kit lots was omitted because it was shown several times before that the parameter “lot” does not contribute very much to the overall variation of results.

Table 13.

Characterization of laboratory-internal reproducibility (intermediate precision) by three analysts extracting or diluting the materials and analyzing them on two different days with three extracts per day and analyzing each extract in two cuvettes

				Pineapple	Sauerkraut	Wine	Egg
Analyst	Day	Extract	Cuvette	g/L	g/L	g/L	g/kg
1	1	1	1	0.103	5.047	1.249	0.811
			2	0.104	4.851	1.140	0.803
		2	1	0.104	4.885	1.144	0.798
			2	0.104	4.849	1.144	0.801
		3	1	0.102	4.837	1.165	0.795
			2	0.104	4.867	1.152	0.796
	2	1	1	0.109	4.786	1.109	0.800
			2	0.107	4.790	1.120	0.805
		2	1	0.109	4.770	1.097	0.791
			2	0.108	4.727	1.135	0.795
		3	1	0.109	4.733	1.131	0.798
			2	0.109	4.772	1.101	0.789
2	1	1	1	0.114	5.146	1.149	0.747
			2	0.113	5.049	1.233	0.741
		2	1	0.111	4.960	1.132	0.751
			2	0.113	4.926	1.080	0.766
		3	1	0.112	4.772	1.125	0.719
			2	0.107	4.869	1.121	0.713
	2	1	1	0.103	4.592	1.091	0.733
			2	0.108	4.546	1.142	0.731
		2	1	0.107	4.228	1.090	0.722
			2	0.112	4.240	1.098	0.757
		3	1	0.111	4.331	1.118	0.740
			2	0.108	4.400	1.120	0.745
3	1	1	1	0.107	4.305	1.017	0.776
			2	0.101	4.381	1.021	0.769
		2	1	0.103	4.379	0.997	0.789
			2	0.110	4.450	1.041	0.771
		3	1	0.109	4.209	1.008	0.768
			2	0.108	4.427	1.043	0.774
	2	1	1	0.105	4.542	1.077	0.752
			2	0.101	4.495	1.095	0.758
		2	1	0.107	4.370	1.069	0.684
			2	0.103	4.284	1.093	0.709
		3	1	0.103	4.427	1.070	0.717
			2	0.102	4.543	1.068	0.716
			Mean	0.107	4.633	1.105	0.762
			SD	0.004	0.266	0.055	0.034
			RSD, %	3.47	5.73	4.94	4.44

The measurement was made with one standard wine, sauerkraut juice, pineapple juice, and liquid whole egg. All test samples were naturally contaminated. They were extracted by each analyst with n = 3 on each of the 2 days and analyzed in two cuvettes per extract. Each analyst conducted the experiment on different days within a period of 2 weeks.

As can be seen in Table 13, the analysis of all matrixes (pineapple juice, sauerkraut juice, wine, whole egg) resulted in an overall RSD of less than 5.7%. The concentration tested ranged between 0.1 and 4.6 g/L of D-lactic acid.

This experiment was especially designed to calculate repeatability, intermediate precision, and the contribution of each type of precision (analyst, day, extraction, and cuvette) by a nested ANOVA design. Table 14 shows the results for repeatability s(r) and intermediate precision s(i) together with their relative measures given in percentage (RSD). All RSD_i values are at or below 6%. Since RSD_i is at least twice as much as RSD_r, it can be concluded that test solution preparation such as extraction or centrifugation are not the main drivers of total precision but analyst and day (see  Table 15).

Table 14.

Characterization of intermediate and repeatability precision from the nested analysis of variance

Performance characteristic	Pineapple	Sauerkraut	Wine	Egg
Mean, g/L	0.107	4.633	1.105	0.762
s(r), g/L	0.0024	0.113	0.030	0.017
RSD(r), %	2.29	2.44	2.76	2.21
s(i), g/L	0.004	0.290	0.061	0.038
RSD(i), %	3.74	6.26	5.48	5.00

Table 15.

Characterization of contribution of each variance component (analyst, day, extract, and cuvette) to total precision within one laboratory

Contributor to total precision	Pineapple %	Sauerkraut %	Wine %	Egg %
Mean, g/L	0.107	4.633	1.105	0.762
Analyst	19.9	21.3	48.7	55.1
Day	41.83	63.5	26.1	25.4
Extract	0.00	10.16	1.78	14.1
Residual (Cuvette)	38.28	5.0	23.5	5.38

Ruggedness Study

These experiments were undertaken during validation to show the influence of parameters on test kit results. These parameters are known to be subject to variation during use of the test kit. The parameter tested for its ruggedness was the incubation time before measuring A2 at 18, 25, and 37°C. Incubation times before reading A1 were 1 min at 37°C or 3 min between 18 to 25°C. Table 16 shows the results of this set of experiments using three aqueous control solutions. Even for the shortest incubation time and lowest temperature, recoveries for the control solutions were between 98 and 99%.

Table 16.

Variation of incubation temperature (18, 25, and 37°C) and incubation time (5, 10, and 15 min) using three different aqueous control solutions analyzed with two replicates; incubation times before reading A 1 were 1 minute at 37°C or 3 minutes between 18 and 25°C

	A 2 5 min		A 2 10 min		A 2 15 min
	18°C
Target	Measured	Rec.	Measured	Rec.	Measured	Rec.
g/L	g/L	%	g/L	%	g/L	%
0.06	0.059	98.9	0.059	98.8	0.059	98.6
0.06	0.059	98.0	0.059	98.2	0.059	98.7
0.20	0.195	97.7	0.195	97.7	0.195	97.6
0.20	0.197	98.7	0.198	98.9	0.198	99.0
0.40	0.394	98.4	0.395	98.8	0.395	98.7
0.40	0.391	97.8	0.393	98.3	0.395	98.7

	25°C
	Measured	Rec.	Measured	Rec.	Measured	Rec.
Target, g/L	g/L	%	g/L	%	g/L	%
0.06	0.061	102	0.061	102	0.061	102
0.06	0.059	98.9	0.059	98.5	0.059	98.5
0.20	0.195	97.4	0.195	97.4	0.194	97.2
0.20	0.195	97.3	0.194	97.2	0.194	97.0
0.40	0.393	98.3	0.394	98.5	0.394	98.4
0.40	0.391	97.7	0.391	97.7	0.391	97.8

	37°C
	Measured	Rec.	Measured	Rec.	Measured	Rec.
Target, g/L	g/L	%	g/L	%	g/L	%
0.06	0.059	98.1	0.058	97.3	0.058	96.7
0.06	0.059	97.9	0.058	97.0	0.058	96.6
0.20	0.193	96.7	0.193	96.4	0.192	96.0
0.20	0.194	97.1	0.194	97.0	0.194	97.0
0.40	0.384	96.0	0.385	96.3	0.388	96.9
0.40	0.386	96.4	0.386	96.5	0.386	96.6

Dilutability

For measurement of dilutability, two different matrixes with high lactic acid contents were used. These test samples were diluted over the whole measuring range and measured in duplicate in one lot. As shown in Figures 4 and 5, dilutability for red wine and sauerkraut juice is really good within the measurement range up to about 0.5 g/L. At the lower end of concentrations, it was interesting to see that the theoretical LOQ characterization was robust because dilutability was as low as 15 to 50 mg/L (in matrix). One should bear in mind that the LOQ characterization was performed using aqueous solution with no matrix.

Figure 4.

Results for dilutability of a red wine (lactic acid concentration was around 0.711 g/L before dilution); equation for regression was calculated without the data marked with open circles; n = 2 per dilution were analyzed in two independent runs.

Figure 5.

Results for dilutability of sauerkraut juice (D-lactic acid concentration was around 4.381 g/L before dilution); equation for regression was calculated without the data marked with open circles; n = 2 per dilution were analyzed in two independent runs.

Real-Time Stability Study

To characterize the shelf life of the (unopened) test kit, a stability study with five different test kit lots was performed over a period of a maximum of 33 months (Table 17). Neither differences between lots nor trends toward higher or lower concentrations were observed.

Table 17.

Real-time stability study up to 33 months of three different pilot scale lots analyst by a total of four analysts using two control solutions and the standard wine

			Aqueous solution				Standard wine
			Target: 0.450 g/L		Target: 0.150 g/L		Target: 0.722 g/L
Lot	Month	Analyst	Rep. 1	Rep. 2	Rep. 1	Rep. 2	Rep. 1	Rep. 2
TC 1	6 mo	1	0.463	0.460	0.151	0.149	0.781	0.772
	9 mo	2	0.462	0.461	0.150	0.151	0.755	0.745
	12 mo	3			0.148	0.150	0.748	0.759
	15 mo	4	0.466	0.463	0.151	0.151	0.777	0.755
	18 mo	3	0.457	0.460	0.149	0.151	0.739	0.740
	24 mo	3	0.452	0.454	0.149	0.149	0.712	0.728
	27 mo	3	0.451	0.453	0.148	0.150	0.726	0.740
	30 mo	3	0.445	0.446	0.147	0.147	0.748	0.750
	33 mo	3	0.441	0.443	0.147	0.146	0.730	0.734
TC 2	6 mo	1	0.460	0.459	0.151	0.150	0.785	0.757
	9 mo	2	0.465	0.461	0.152	0.150	0.774	0.728
	12 mo	3			0.147	0.149	0.740	0.75
	15 mo	4	0.463	0.462	0.151	0.151	0.748	0.765
	18 mo	3	0.457	0.459	0.150	0.150	0.731	0.730
	24 mo	3	0.445	0.446	0.149	0.148	0.730	0.719
	27 mo	3	0.437	0.441	0.145	0.147	0.708	0.729
	30 mo	3	0.418	0.417	0.141	0.141	0.717	0.724
	30 mo	3	0.422	0.425	0.142	0.143	0.735	0.746
	33 mo	3	0.404	0.404	0.137	0.138	0.687	0.685
TC 3	6 mo	1	0.457	0.458	0.149	0.148	0.774	0.753
	9 mo	2	0.462	0.461	0.152	0.15	0.769	0.832
	12 mo	3			0.148	0.153	0.745	0.752
	15 mo	4	0.467	0.461	0.155	0.151	0.769	0.754
	18 mo	3	0.456	0.459	0.150	0.151	0.731	0.725
	24 mo	3	0.449	0.451	0.148	0.151	0.716	0.725
	27 mo	3	0.442	0.445	0.147	0.147	0.718	0.732
	30 mo	3	0.431	0.429	0.145	0.143	0.748	0.741
	33 mo	3	0.420	0.419	0.139	0.141	0.707	0.702
Mean, g/L			0.448		0.148		0.742
SD, g/L			0.016		0.004		0.024
RSD, %			3.56		2.46		3.26
Rec., %			99.7		98.7		103

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 were placed on a horizontal shaker at room temperature and agitated for 6 h. A total of 400 rpm was used at the beginning and later adjusted to 150 rpm. Afterwards, the components were refrigerated for 18 h at 4°C, followed by 7 h at room temperature on a horizontal shaker (150 rpm). Components were incubated at 37°C for 18 h and finally measured as usual. When comparing the stressed conditions with a test kit stored at 4°C, there was no difference between both data sets directly after the simulated transport was finished. Afterwards, test kits were tested every 6 months to show that there is no effect on the long-term stability of the kit. Until month 27, there is no trend toward lower or higher values (Table 18).

Table 18.

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

			After simulated transportation
		Reference	0 mo	6 mo	12 mo	18 mo	24 mo	27 mo
	Target	Measured	Measured	Measured	Measured	Measured	Measured	Measured
	g/L	g/L	g/L	g/L	g/L	g/L	g/L	g/L
Control solution A	0.06	0.058	0.061	0.061	0.060	0.057	0.061	0.061
	0.06	0.058	0.061	0.058	0.063	0.059	0.061	0.061
Control solution B	0.20	0.199	0.197	0.196	0.200	0.201	0.199	0.195
	0.20	0.198	0.196	0.197	0.193	0.200	0.198	0.196
Control solution C	0.40	0.389	0.393	0.391	0.390	0.397	0.394	0.388
	0.40	0.389	0.394	0.388	0.388	0.399	0.395	0.389

Stability Study on Freezing

To simulate an unintended freezing of the test kit components, the whole test kit was frozen for 24 h at –20°C. Afterwards, the components were allowed to warm up to room temperature and were frozen again at –20°C. After 24 h, the components were thawed and used for measurement. The two freeze-thaw cycles have no influence on the correct recovery of the measured control solutions (Table 19).

Table 19.

Influence of the test kit performance after freezing at −20°C in comparison with a kit stored at 2–8°C

			−20°C	4°C
Kit lot		Target	Measured	Measured
		g/L	g/L	g/L
TC1	Control solution A	0.06	0.060	0.060
		0.06	0.059	0.059
	Control solution B	0.20	0.198	0.199
		0.20	0.197	0.195
	Control solution C	0.40	0.394	0.390
		0.40	0.389	0.390
TC2	Control solution A	0.06	0.060	0.060
		0.06	0.061	0.058
	Control solution B	0.20	0.196	0.196
		0.20	0.202	0.196
	Control solution C	0.40	0.398	0.396
		0.40	0.393	0.391
TC3	Control solution A	0.06	0.062	0.061
		0.06	0.061	0.059
	Control solution B	0.20	0.196	0.194
		0.20	0.196	0.194
	Control solution C	0.40	0.393	0.394
		0.40	0.391	0.391

In-Use Stability Study

Test kit components from three different lots were opened every 3 months up to 27 months. At each time point, control solutions were analyzed. According to these data, the in-use stability is at least 27 months, and no indication of deterioration was observed (Table 20). Nevertheless, it should be noted that achieving the long shelf life specified is dependent on the cleanliness of the equipment used to pipet out of the component vials.

Table 20.

Characterization of the in-use stability over a period of 27 months using three aqueous control solutions in one test kit lot

		0 mo	6 mo	12 mo	18 mo	24 mo	27 mo
	Target	Measured	Measured	Measured	Measured	Measured	Measured
	g/L	g/L	g/L	g/L	g/L	g/L	g/L
Control solution A	0.06	0.060	0.062	0.059	0.064	0.062	0.060
	0.06	0.059	0.063	0.058	0.061	0.059	0.064
Control solution B	0.20	0.199	0.200	0.198	0.204	0.200	0.202
	0.20	0.195	0.202	0.200	0.203	0.202	0.205
Control solution C	0.40	0.390	0.399	0.398	0.400	0.399	0.400
	0.40	0.390	0.395	0.395	0.404	0.400	0.404

Automation on a Pictus 500 Spectrophotometric Analyzer

Comments on validation parameters independent on automation.—Side-reactivity to other related organic acids or other substances and interfering substances were not characterized on the automated analyzer because there is no known effect that an automated process will change the reactivity toward these substances.

The pipetting environment within a closed automate is much more regulated than the normal laboratory environment (including the analyst). For measurement, all reagents are cooled at 8°C ± 2°C, while the reaction zone where the analysis takes place is tempered to 37°C. 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 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, freezing, and short-term storage at 37°C. 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 a LOD.

LOQ

The lower end of the measurement range is the limit of quantification where acceptable recovery and precision are met. Our internal requirements are a recovery higher than 95% and an RSD equal or lower than 10%. For each of the three applications, aqueous solutions with different concentrations of D-Lactic acid were analyzed at least five times. The concentration was calculated from the calibration of the system.

Figure 6 shows the results for the Basic range application with a test volume of 10 µL, which is—despite a factor of 10 in volumes—the identical ratio of test volume to reagents as the manual format with a test volume of 100 µL, 2000 µL reagent 1 and 500 µL reagent 2. The automated analyzer has an LOQ of 5 mg/L because for this concentration the RSD value is clearly below 10% and the recovery below 105% (Figure 6). The manual format exerted a calculated LOQ of about 10 mg/L, but the precision at this point is still not sufficient. Data from a precision plot (Figure 2) gave an LOQ of 15 mg/L for the manual format.

Figure 6.

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

Since D-lactic acid is often present at quite high concentrations in food such as fermented milk products, wine, and fermented vegetable (juices), 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 7, the LOQ for this application is 30 mg/L. This application was not investigated for the manual format because this would require test volumes of 20 µL, which is challenging for untrained analyst. The Pictus 500 shows RSD values at or below 6% for a test volume of 2 µL.

Figure 7.

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.

In case analysis of trace levels of D-lactic acid is necessary, the Sensitive range application with a test volume of 100 µL was investigated for its LOQ (Figure 8). A LOQ of 0.75 mg/L can be claimed for this application where recovery and precision requirements were met.

Figure 8.

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.

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 test sample and its proper quantification only depends on the proper choice of test volume and calibration. For each of the three applications, the optimal linear measurement range was characterized. Figure 9 shows that the upper measurement range is 625 mg/L for a test volume of 10 µL.

Figure 9.

Characterization of linearity for the Basic range application with 10 µL test volume; the open circle was not included in linear regression; RSD values are given as open triangles and measured concentrations as closed circles.

For the High range application with a test volume of 2 µL, the upper measurement range is 3125 mg/L (Figure 10). This is a factor of five compared to the Basic application and perfectly fits to the increased test volume of 10 µL.

Figure 10.

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

For the Sensitive range application with a test volume of 100 µL, the upper measurement range is 62.5 mg/L (Figure 11). It is always recommended to include control samples at the upper range to check for linearity.

Figure 11.

Characterization of linearity for the Sensitive range application with 100 µL test volume; the open circle was not included in linear regression; RSD values are given as open triangles and measured concentrations as closed circles.

On-Board and Calibration Stability

Data on these important characteristics will be provided for the Final Action decision because these experiments are ongoing. Customers use the assay in many different ways over a longer period. For example, some will leave the reagents in the automate until they are empty. Others will store them after each working day in the refrigerator 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 applications. Two aqueous solutions were used because the characterization of different matrixes was already done during the validation of the manual application. To check for trueness, one standard wine was applied for all applications.

Table 21 shows the results for the Basic range application with a test volume of 10 µL. As expected for automated pipetting, RSD values at or below 1% were obtained for concentrations between 107 and 450 mg/L. The validity of the calibration was also checked with these three solutions. Recoveries ranged between 95 and 100% and were thus clearly within specifications.

Table 21.

Characterization of precision for the Basic range (10 µL test volume) application using two aqueous solutions and a reference wine

	Aqueous solution		Reference wine
Target	450	150	107
Replicate	mg/L	mg/L	mg/L
1	448.5	149.3	100.8
2	450.2	147.8	101.0
3	449.2	149.1	101.1
4	454.4	150.9	102.0
5	452.2	150.2	102.2
6	444.6	148.1	99.1
7	447.0	148.7	100.6
8	448.3	149.0	101.5
9	451.4	150.3	102.3
10	452.3	150.3	102.1
Mean	449.8	149.4	101.3
SD	2.89	1.02	0.98
RSD, %	0.64	0.69	0.97
Recovery, %	100.0	99.6	94.7

Table 22 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 at or below 2% were obtained for concentrations between 107 and 450 mg/L. Recoveries ranged between 100 and 102% and were thus clearly within specifications. The standard wine comes with a certificate so that trueness of the system was established for the application with the smallest volume of 2 µL.

Table 22.

Characterization of precision for the High range (2 µL test volume) application using two aqueous solutions and a standard wine sample

	Aqueous solution		Reference wine
Target	450	150	107
Replicate	mg/L	mg/L	mg/L
1	455.6	152.2	109.5
2	449.0	152.5	110.8
3	457.2	155.4	111.3
4	457.3	156.4	106.4
5	447.4	150.6	107.0
6	445.5	151.9	106.8
7	452.7	150.4	105.0
8	457.0	154.8	109.3
9	456.5	151.2	104.3
10	440.1	146.5	104.8
Mean	451.8	152.2	107.5
SD	6.04	2.87	2.54
RSD, %	1.34	1.88	2.37
Recovery, %	100.4	101.5	100.5

Table 23 shows the results for the Sensitive range application with a test volume of 100 µL. RSD values were at 1% or lower for concentrations between 10.7 and 45 mg/L. The validity of the four-point calibration was also checked with the three solutions. Recoveries ranged between 99 and 101% for aqueous solutions. The reference wine had to be diluted before measurement by 1:100, which could be the reason for the lowered recovery of 88%.

Table 23.

Characterization of precision for the Sensitive range application (100 µL test volume) using two aqueous solutions and a reference wine

	Aqueous solution		Reference wine
Target	45	15	10.7
Replicate	mg/L	mg/L	mg/L
1	44.61	15.22	9.55
2	44.54	15.45	9.48
3	44.00	15.02	9.32
4	44.20	14.98	9.32
5	44.20	15.21	9.28
6	44.46	15.11	9.47
7	44.61	15.20	9.42
8	43.95	14.96	9.28
9	44.23	14.97	9.32
10	44.33	15.16	9.28
Mean	44.31	15.13	9.37
SD	0.24	0.15	0.10
RSD, %	0.54	1.02	1.06
Recovery, %	98.5	100.9	87.6

Conclusion

In summary, the data obtained during the in-house validation study has shown that the performance claims for food and beverages such as milk and (fermented) milk products, fermented vegetable products, wines, beer, fruit and vegetable juices, and eggs and egg powder are fulfilled. The method is robust and accurate for both applications (manual and automated). The test kit Enzytec Liquid D-Lactic acid was accepted as an AOAC Official Method of Analysis℠ for quantification of D-lactic acid in the claimed matrixes.

CRediT Author Statement

Markus Lacorn (Conceptualization [Equal], Data curation [Lead], Methodology [Equal], Resources [Equal], Validation [Lead], Writing—original draft [Lead]) and Thomas Hektor (Conceptualization [Equal], Project administration [Lead], Resources [Equal], Supervision [Lead], Writing—review & editing [Lead])

Conflict of Interest

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