OMA Consulting Protocol Review: OMACON-2019-2

AOAC INTERNATIONAL Official Methods of Analysis SM Program

Validation Outline for the BIOMILK 300 LAC Method for the Quantitation of Low Levels of Lactose in Select Food Matrices

For First Action Official Methods SM Status April 2019 Version 1

Prepared for: Sandra Salleres Biolan Microbiosensors, S.L. Parque Científico Tecnológico De Bizkaia Edificio 409 Zamudio/Bizkaia/E48170

Prepared by: Patrick M. Bird Technical Consultant AOAC Research Institute 2275 Research Blvd., Suite 300 Rockville, Maryland 20850 USA

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Table of Contents

1.0

Introduction 1.1

BIOMILK 300 LAC Method

1.2

Validation Overview

2.0

Method Developer Studies 2.1 Overview 2.2

Good Laboratory Practices

2.3 2.4 2.5 2.6 2.7 2.8 2.9

Safety

BIOMILK 300 LAC Methodology

Linearity

Interference and Cross Reactivity

Matrix Studies

Robustness

Stability and Lot-to-Lot

3.0

Report Format

Appendices

1. BIOMILK 300 LAC Instructions Manual 2. SMPR 2018.009 3. Report Format

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1.0

Introduction 1.1

BIOMILK 300 LAC Method

The BIOMILK 300 LAC (Appendix 1) is a compact analytical device that combines a biological component with a physicochemical transducer to detect analytes of interest. The BIOMILK 300 LAC combines the high specificity and selectivity of specific enzymes for each target analyte with an amperometric transduction of that biological signal, which is easily detectable and quantifiable. The technology is based on the Biotest, a working electrode that immobilizes the enzyme together with other electroactive components, which cause a change in electric current when analyte is present. There are two parts to the analysis. In the first phase (ADJUSTING REFERENCE), the device records the electricity signal that circulates in the electrochemical cell that is filled with Measuring Solution. In the second phase (MEASURING), a calibration standard or sample is added to the cell and the device records the electrical signal. The addition of sample or standard increases the electricity current in the cell due to the redox reaction sequence that occurs on the Biotest surface. The device uses the differences in intensities recorded before and after the addition of standard or sample to calculate a current intensity variation that correlates with the lactose concentration by means of a prior calibration of the device. The method is intended for the quantitative determination of lactose in low lactose or lactose free dairy products. The method has been developed with three separate analytical ranges. See Table 1 for specifications on the methods measurement ranges.

Table 1: Specifications of the Measurement Ranges 0-200 mg/L

0-2 g/L

0-6 g/L

Error

15%

15%

15%

Quantitative Range

50-200 mg/L

0.2-2 g/L 200 mg/L

0.5-6 g/L

LOQ

50 mg/L

0.5 g/L

Dilution Required

No

1/10

1/10

1.2

Validation Overview

Validation study designs may vary based on several factors, including but not limited to the intended applicability of the method, the analyte being evaluated, or the validation guidelines used to design the study.

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The BIOMILK 300 LAC method will be validated according to AOAC INTERNATIONAL policies and procedures against performance requirements as outlined in the Standard Method Performance Requirements (SMPRs ® ) for Lactose in Low-Lactose or Lactose- Free Milk, Milk Products, and Products Containing Dairy Ingredients, AOAC SMPR 2018.009 (Appendix 2). The scope of SMPR 20018.009 is to measure lactose found in milk, milk products, and products containing dairy ingredients that are low-lactose or lactose-free. SMPRs® are a unique and novel concept for the analytical methods community. SMPRs are international voluntary consensus standards, developed by stakeholders, that prescribe the minimum analytical performance characteristics to be used during the evaluation of a method. AOAC SMPRs are used by AOAC expert review panels in their evaluation of validation study data for methods being considered for Performance Tested Methods SM or AOAC Official Methods of Analysis SM . This validation has two main parts: Single Laboratory Validation (SLV) to obtain Official Method First Action Status and a Multi-Laboratory Validation to achieve Final Action Status. This study outline will provide guidance on the SLV Studies. Testing generated during the SLV studies must meet the performance requirements as outlined in SMPR 2018.009 to achieve Official Method First Action Status.

2.0

SINGLE LABORATIVE VALIDATION (SLV) STUDIES

2.1

Overview

The SLV studies will generate method performance data that meets or exceeds the requirements set forth in SMPR 2018.009. SLV studies will generate data for the applicable range of the method (linearity), interference and cross-reactivity, recovery (matrix studies), bias, LOD/LOQ, precision, robustness and stability/lot-to-lot. Lactose Test Method Claim: Whole UHT milk, Yogurt (normal, flavored, liquid flavored and Greek), cream, chocolate milk shake, spreadable soft cheese, infant formula, protein powder, ice cream. Analyte:

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2.2

Good Laboratory Practices

2.2.1 Reference materials or standards must be obtained directly from the producer or distributor and be accompanied by a certificate of analysis. 2.2.2 Methods will include appropriate blanks and check standards 2.2.3 One or more technicians should prepare samples and a second or more technicians should perform the assays on “blind” coded samples. 2.2.4 All samples must be fully randomized and labeled with a code so that the analyst performing the assays is unaware of the level of analyte being tested. The BIOMILK 300 LAC assay and its components (KITCLAC, KITMLAC1, KITMLAC2) are not considered a hazardous substance or mixture according to regulation (EC) No. 1272/2008. These substances are not classified as dangerous according to directive 67/548/EEC. The following safety risks may be associated with the test components. Inhalation : May be harmful if inhaled. May cause respiratory tract irritation. Ingestion : May be harmful if swallowed. Skin : May be harmful if absorbed through skin. May cause skin irritation. Eyes : May cause eye irritation. Wear appropriate protective equipment which includes but is not limited to protective eyewear, face shield or safety glasses, lab coat, and appropriate gloves. All work should be conducted in properly equipped facilities utilizing the appropriate safety equipment. Individuals should be trained in accordance with applicable regulatory and company/institution requirements before working with chemicals. BIOMILK 300 LAC Methodology (Appendix 1) 2.4.1 Storage: The Biotests are shipped at room temperature, vacuum packed and with silica gel crystals to protect them from atmospheric humidity. Once delivered, they must be kept between 3-8 o C, before expiration date. 2.4.2 To hydrate the Biotest prior to use (this process should be carried out at least 24 hours prior to use), remove the silica gel crystals from inside the Safety

2.3

2.4

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tube and fill it with Measurement Solution until the base of the Biotest is submerged. Once hydrated, the Biotest can be stored vertically at 3-8 o C for a maximum of 15 days.

2.4.3 Reagent Preparation:

2.4.3.1 LAC Measurement Solution (MS): Both LAC1 Measurement Kit and LAC2 Measurement Kit contain 8 tubes (MLAC1 or MLAC2). To prepare the Measurement Solution, which is needed to prepare

Reagents and Calibration Standards and to carry out the measurements, dilute the contents of one tube (MLAC1 or MLAC2) according to Table 2.

Table 2: Measurement Solution (MS) Preparation LAC1 Measurement Tube (MLAC1)

LAC2 Measurement Tube (MLAC2)

Volume of Distilled Water

250 mL

1 L

2.4.3.2 LAC Calibration Standards and Reagents: The LAC Calibration Kit contains 6 tubes of powdered CLAC1 Calibration Reagent, 4 tubes of powdered CLAC2 Calibration Reagent, along with 2 empty tubes to prepare the LAC1 Calibration Standard, 2 empty tubes to prepare the LAC2 Calibration Standard and 3 empty tubes to prepare the Measurement Standard. 2.4.3.2.1 CLAC1 and CLAC2 are prepared using measurement solution as outlined in Table 3. To prevent lose of reagent, introduce the pipette tip through the perforated stopper when adding the measurement solution. Store at 3-8 o C for up to 7 days. 2.4.3.2.2 The LAC Calibration Standards (LAC1, LAC2 and

Measurement Standard) are prepared daily by diluting the CLAC1 and CLAC2 Calibration Reagents. These standards are used to calibrate the equipment. For milk products, dilute the standards according to the specifications in Table 3.

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Table 3: Preparing the Calibration Reagents and Standard 0-200 mg/L 0-2 g/L 0-6 g/L

LAC1 Calibration Reagent (CLAC1) LAC1 Calibration Standard LAC2 Calibration Reagent (CLAC2) LAC2 Calibration Standard

10 mL MS (*)

10 mL MS (*)

10 mL MS (*)

4 mL MS + 1 mL CLAC1

4.9 mL MS + 0.1 mL CLAC1

4.9 mL MS + 0.1 mL CLAC1

5 mL MS (*)

5 mL MS (*)

5 mL MS (*)

9.75 mL MS + 0.25 mL CLAC2

9.75 mL MS + 0.25 mL CLAC2

9.75 mL MS + 0.25 mL CLAC2

7.75 mL MS + 2mL CLAC1 + 0.25 mL CLAC2

9.55 mL MS + 0.2 mL CLAC1 + 0.25 mL CLAC2

9.05 mL MS + 0.2 mL CLAC1 + 0.75 mL CLAC2

Measurement Standard

2.4.3.2.3 The storage conditions of each of the prepared reagents are specified in Table 4.

Table 4: Reagent Storage Conditions Biotest CLAC1/ CLAC2

Measurement Reagent

Standards

Sample

Dry

3-8 o C/ e.d 3-8 o C/ 15 days

r.t./e.d/*

-

r.t./e.d/*

-

Hydrated

3-8 o C/ 7 days

Daily

r.t./15 days

2 hours

e.d.: until expiration date r.t.: room temperature *: keep in a dark and dry place

2.4.3.2.4 If other milk products are to be analyzed, with a glucose content different from ~23 g/l (hydrolysis of the total content of lactose of milk), the LAC1

Calibration Standard and the Measurement Standard must be changed in order to reflect the different glucose content. Use the following formula:

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2.4.3.2.5 Where C is the desired glucose content in (g/l) and V is the calculated volume of CLAC1 in ml. 2.4.4 Assembling the Biosensor: Connect both the Biotest (placing it in the Biotest carrier and screwing it into place) and the reference electrode to their respective connectors. For safety reasons, it is recommended to never disconnect the reference electrode. When the equipment is disassembled after analyses are completed, the reference electrode remains protected with the Storage Solution supplied (4M KCl).

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2.4.4.1 Fill the measurement cuvette with 10 mL of Measurement Solution and put it in the cuvette holder making sure that all the components of the electrochemical cell are submerged. 2.4.4.2 Connect the device to a 100-240 VAC socket and press the “On” switch that is beside the screen for 2 seconds. 2.4.4.3 In the BIOTEST option: select the parameter to be analyzed. Press », enter the Biotest code (this code is on the Biotest ID label), press » and finally select the Measurement Range to be used and press Save.

2.4.4.4 The ANALYSIS option takes you to the main analysis menu: Activate, Calibrate, Standard and Sample.

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2.4.4.5 Once a day, the Biotest must be activated and calibrated before starting to perform the analysis. These two operations should also be carried out after the device has been inactive for long periods (30 minutes). 2.4.4.6 Once the Biosensor is set up, make sure that the measurement cuvette contains 10 mL of Measurement Solution and that all the components of the electrochemical cell are submerged. 2.4.5 Activation: Press activate, check that the cuvette is correctly fitted, and press CONTINUE to begin the activation process. 2.4.5.1 Wait until the device records the measurement target and it issues a sound signal. Inject the Measurement Standard into the measurement cuvette, using a micropipette, and wait until the process ends (see Table 5). 2.4.5.2 The message “Biotest activated. Ready for calibration” will appear on the screen 2.4.5.3 Change the content of the cuvette with 10 mL of new Measurement Solution.

Table 5: Addition volumes for activation, calibration and analysis 0-200 mg/L 0-2 g/L

0-6 g/L 650 µL 650 µL 650 µL 650 µL

LAC1 Calibration Standard LAC2 Calibration Standard Measurement Standard

2 mL 2 mL 2 mL 2 mL

2 mL 2 mL 2 mL 2 mL

Sample

2.4.6 Calibration: The calibration consists of 3 points and it is performed by means of adding the LAC1 Calibration Standard once and the LAC2

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Calibration Standard twice to the measurement cuvette at three different times. 2.4.6.1 To trace the calibration line, the device will take into account the changes in intensity caused by the different additions and the relevant concentrations of each Calibration Standard. 2.4.6.2 Press Calibrate, fill the cuvette with 10 mL of new Measurement Solution and press CONTINUE. 2.4.6.3 Wait until the device issues a sound signal. Press CONTINUE and inject, in this order, the LAC1 Calibration Standard into the measurement cuvette (first addition (1/3)), using a micropipette (see Table 5). Note: It is important to add the Calibration Standard immediately after pressing the CONTINUE button to record the current intensity correctly at the time of adding it. 2.4.6.4 Wait until the device records the change in intensity and issues a sound signal. Press CONTINUE again and inject the LAC2 Calibration Standard into the measurement cuvette (second addition (2/3)) using a micropipette (see Table 5). 2.4.6.5 Wait until the device records the change in intensity and issues a sound signal. Press CONTINUE again and inject the LAC2 Calibration Standard again (third addition, (3/3)) using a micropipette (see Table 5). 2.4.6.6 As a result of the calibration, the parameters of the linear regression obtained in the interpolation of the three calibration points will appear on the screen.

2.4.6.7 R and m values equal to or over 0.9990 and 6.000, respectively, will be considered valid to proceed with the analysis. Typical values for b do not exceed 100.

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2.4.6.8 If the regression is not valid, change the measurement cuvette, put it on Stand by for 30 seconds and calibrate again. If the regression is valid (“calibration OK” appears), continue to Standard Measurement. 2.4.7 Measurement Standard Analysis: The calibration status should be tested using the Standard option after a series of measurements (10-15) or if the device has not been running for a minimum of 30 minutes. 2.4.7.1 Press Standard in the analysis option, fit the cuvette correctly and fill with 10 mL of Measurement Solution and press CONTINUE. 2.4.7.2 Wait until the device issues a sound signal. Press CONTINUE and inject Measurement Standard into the cuvette (See Table 5). 2.4.7.3 Wait until the “Biosensor calibrated” or “Biosensor recalibrate” message appears. 2.4.7.4 Replace the contents of the cuvette with 10 mL of new LAC Measurement Solution. If “Biosensor calibrated” message appeared, proceed to Sample Preparation. If “Biosensor recalibrate” message appeared, return to step 2.4.7. or check the proper preparation of the Calibration Standards. 2.4.8 Sample Pretreatment, Dilution and Analysis: A preliminary sample pretreatment or dilution step is needed to perform the analysis of the sample. 2.4.9 Activated charcoal (0-200 mg/L range): Liquid samples need to be pretreated before being analyzed in the 0-200 mg/L range. 2.4.9.1 Add a capsule of charcoal activated in 50 mL of sample and divide

the sample in half to speed up the mixing process. Shake until the active charcoal is homogeneously mixed with the milk sample and let it react for at least 30 minutes before analyzing. Shake it occasionally.

2.4.9.2 Test kits may also contain a pretreatment tube instead of a capsule. In that case, inject 5 mL of milk sample inside the pretreatment tube, shake until the mixture is homogeneous and analyze the sample as usual. 2.4.10 Dilution (0-2 g/L range and 0-6 g/L range): Samples (milk and other dairy products) in the 0-2 g/L and 0 -6 g/L ranges need to be diluted. 2.4.10.1 To do so, mix 1 mL of sample with 9 mL of Measurement

Solution and shake it to homogenize. Once the samples are prepared, they must be analyzed within a maximum of 2 hours.

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During that time the samples can be kept at ambient temperature. 2.4.11 General sample pretreatment: High viscous samples (Greek yogurt, high protein yogurt, crème caramel or others) must be liquified before analyzing in order to be able to pipette them inside the measurement cuvette. Make use of an Ultra-turrax or a mincer. 2.4.11.1 Occasionally, an extra step of heating is needed. For

example, for spreadable or soft cheese, heat in a microwave at low power for a few seconds in order to melt it before analyzing. In case of high viscous samples, the electrodes must be

2.4.11.2

cleaned with distilled water between analysis.

2.4.11.3

Butter cannot be analyzed by BIOMILK 300 LAC as it is not

miscible in aqueous solution.

2.4.11.4

The presence of added Vitamin C (ascorbic acid and derivatives, E-300, E301 and E-302) may interfere in the BIOMILK 300 LAC result. Please contact Biolan for support.

2.4.12 Sample analysis 2.4.12.1

Press “Sample”, fit the cuvette correctly with 10 mL of

Measurement Solution and press CONTINUE.

2.4.12.2

Wait until the device issues a sound signal. Press CONTINUE and inject the sample into the cuvette (See Table 5). The result will be shown on the screen as mg/L or g/L of

2.4.12.3

lactose, depending on the range used.

2.4.12.4

To save the result of an analysis, press SAVE and enter a

code.

2.4.12.5

Press the RECORDS option of the main screen and select LACTOSE to see the recorded data. Additionally, there is the option of downloading the recorded data from the device to a computer, using the specific software developed by Biolan.

2.4.12.6

To delete the data saved, go to RECORDS and press

DELETE ALL.

2.4.12.7

Replace the contents of the cuvette with 10 mL of new LAC

Measurement Solution before analyzing a new sample.

2.5

Method Range (Linearity) Study

The method range (linearity) study determines the applicable range of the method extending beyond the set of standards or calibrators supplied with the test kit. SMPR

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2018.009 identifies three separate analytical ranges, dependent on the type of product evaluated: 5-100 mg/100g for infant formula and adult nutritionals; 10-100 mg/100g for lactose free milk, milk products and products containing dairy ingredients; >100-2,000 mg/100g for low-lactose milk, milk products and products containing dairy ingredients. 2.5.1 A stock solution of 4g/L of D-Lactose monohydrate, CAS Number: 64044- 51-5, ≥99%) in LAC Measurement Solution containing 23 g/L of glucose will be prepared to make the specified lactose standards. (0.2 g D-Lactose monohydrate and 1.15 g D-Glucose anhydrous in 50 mL LAC Measurement Solution) 2.5.1.1 For the 0-200 mg/L calibration range the following concentrations will be prepared: 0, 25, 50, 75, 100, 125, 150, 175 and 200 mg/L. 2.5.1.2 For the 0-2 g/L calibration range the following concentrations will be prepared: 0, 10, 25, 50, 75, 1,00, 125, 150, 175 and 200 mg/L. 2.5.1.3 For the 0-6 g/L calibration range the following concentrations will be prepared: 0, 25, 75, 1,50, 225, 3,00, 375, 450, 424 and 600 mg/L. Note* For the 0 - 2 g/L and 0 - 6 g/L range, the sample is diluted 1/10 prior to analysis so a calibration range of 0-200 mg/L and 0 – 600 mg/L is used. 2.5.2 Test a minimum of 2 replicates at each concentration and randomize and blind code the samples. 2.5.3 Perform the assay according to the candidate method instructions for use (Section 2.4 or Appendix 1) on the samples. Testing will be performed following instructions for milk. 2.5.4 If a sample is above the measurement range, follow method instructions for re-analysis. 2.5.5 Plot results vs. concentration and analyse for linearity. 2.5.6 Plot residuals vs. concentration and look for non-random patterns. 2.5.6.1 If a non-random pattern is discovered, find a model to reduce or eliminate it. 2.5.6.2 Repeat the linearity and residual analyses with the new model.

Interference and Cross Reactivity

2.4

Interference studies will be conducted in lactose free milk to evaluate both positive and negative interferences of the method. Compounds will include different monosaccharides and disaccharides (in order to test cross reactivity of

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the enzyme), alcohol sugars, ascorbic acid and sorbic acid as examples of antioxidant molecules that could interfere with the detection system, and OMEGA3, oleic oil and some vitamins normally present in commercial enriched milk. 2.4.1 Prepare samples by diluting potential cross-reactive compounds to levels typically found in milk. Compounds include:

2.4.1.1 Glucose – 40 g/L 2.4.1.2 Fructose – 40 g/L 2.4.1.3 Galactose – 40 g/L 2.4.1.4 Sacarose – 40 g/L 2.4.1.5 Allolactose – 1 g/L 2.4.1.6 Lactulose – 1 g/L 2.4.1.7 Epilactose - 1 g/L 2.4.1.8 Ascorbic Acid - 0.25 g/L 2.4.1.9 Sorbic Acid – 0.25 g/L 2.4.1.10 OMEGA3 – 1.5 g/L 2.4.1.11 Oleic Oil – 12.5 g/L 2.4.1.12 Vitamin B12 - 0.003 g/L 2.4.1.13 Vitamin B6 – 0.012 g/L

2.4.1.14 Glycerol – 50 g/L 2.4.1.15 Sorbitol – 50 g/L 2.4.1.16 Mannitol – 50 g/L 2.4.1.17 Lactitol – 50 g/L 2.4.2 Stock solutions of each analyte will be prepared at levels typically found in dairy products. 2.4.3 Sample analysis will be conducted using the 0-200 mg/L calibration range 2.4.4 Testing will be conducted in milk containing ~40 mg/L lactose. 2.4.5 Randomize and blind-code samples. 2.4.6 Test according to the candidate method instructions for use. 2.4.7 Decode results and report any positive or negative interference.

2.5

Matrix Studies

The matrix study will determine the bias, recovery, repeatability precision, LOD, and LOQ. The following matrices will be evaluated: Whole UHT milk, Yogurt (normal, flavored, liquid flavored and Greek), cream, chocolate milk shake, spreadable soft cheese, infant formula, protein powder, ice cream.

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2.5.1 Prepare and homogenize each matrix such that there are a minimum 6 known concentration levels of lactose, including zero (if possible), to cover the analytical range of the method. 2.5.2 Aliquots of 50 mL of each matrix will be spiked with the appropriate volume of a stock solution of D-lactose of 4 g/L or 60 g/L in LAC Measurement Solution, in order to achieve the desired concentration. 2.5.3 For liquid samples analyzed at the 0-200 mg/L range, the pretreatment with activated charcoal will be performed before the addition of lactose. 2.5.4 For semi solid samples analyzed at 0-2 g/L or 0-6 g/L range, the addition will be performed before the 1/10 dilution required for analysis. 2.5.5 Each of the three analytical ranges will be evaluated for each matrix. 2.5.5.1 For the 0-200 mg/L calibration range a minimum of 6 contamination levels between 0 and 200 mg/L should be used. 2.5.5.2 For the 0-2 g/L calibration range a minimum of 6 contamination levels between 0 and 2 g/L should be used. 2.5.5.3 For the 0-6 g/L calibration range a minimum of 6 contamination levels between 0 and 6 g/L should be used. 2.5.6 Randomize and blind code 2 sample replicates per concentration per matrix. 2.5.7 From each blinded sample, analyze according to the candidate method (2.4 and Appendix 1). 2.5.8 Decode and tabulate the replicate data and calculate the mean at each concentration. 2.5.9.1 Graph replicate candidate method results vs. known concentration and perform regression analysis. 2.5.9.2 Determine recovery and bias at each concentration from the comparison of the candidate method means to the known histamine concentration as follows: 2.5.9.2.1 Recovery = [mean cand /known spike] x 100 2.5.9.2.2 Bias = mean cand – known spike 2.5.9.3 Determine accuracy and precision for the candidate method at each concentration for each matrix as follows: 2.5.9 Bias, Recovery and Repeatability Precision Determination

n

∑

2

(

)

1 X X n − − i

2.5.9.3.1 s r =

1

i

=

16

2.5.9.3.2 RSD r = [s r /mean cand ] x 100

2.5.10 LOD and LOQ Determination

2.5.10.1 2.5.10.2

Graph s r vs. mean for the candidate method results.

Calculate the LOD as

( ) b s m

0 3.3 1 1.65 + −

X

LOD =

where is the mean analytical value of the known negative matrix; is the intercept of the plot; and is the slope of the plot. 0 X b s m

2.5.10.3

Estimate the LOQ as

LOQ = 3 x LOD

2.5.10.4

Validate the LOQ by spiking each matrix at or near the estimated LOQ and testing 10 replicates to demonstrate acceptable precision.

2.5.11 Acceptance Criteria 2.5.11.1

For each matrix evaluated, the following performance requirements as outlined in SMPR 2018.009 (Figure 1) should be met.

Figure 1: SMPR 2018.009 Performance Requirements

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2.6

Robustness Evaluation

The idea for the robustness testing is to deliberately introduce minor, reasonable variations in a test kit method and observe the effects. These minor variations should be of a magnitude that might well be expected to occur when the test kit is used by an end user. If the results are altered, test kit users should be warned not to depart by more than some stated amount from the specified conditions. Presumably most of these variations will show negligible effects, but manufacturers and users benefit if just one sensitive condition is determined, and users are appropriately warned. The kit manufacturer is expected to make a good faith effort to determine which parameters are most likely to affect the analytical performance and determine the range of variations that can occur without adversely affecting analytical results. These recommended factors for robustness testing are based on the technical materials provided by Biolan Microbiosensors. AOAC INTERNATIONAL recommends testing the following operational parameters:

Table 6. Robustness Parameters Parameter Designation A. Sample Pre -Treatment Time

Value 1 20 min 1.8 mL 1.8 mL

Value 2 30 min 2.0 mL 2.0 mL

Value 3 40 min 2.2 mL 2.2 mL

B. Sample Volume

C. LAC1 volume during calibration

Table 7. Factorial Design for Robustness Testing Treatment Combination A

Parameter Value

B

C

1 2 3 4 5 6 7

20 min 20 min 20 min 20 min 40 min 40 min 40 min

1.8 mL 1.8 mL 2.2 mL 2.2 mL 1.8 mL 1.8 mL 2.2 mL

1.8 mL 2.2 mL 1.8 mL 2.2 mL 1.8 mL 2.2 mL 1.8 mL

18

8 9

40 min 30 min

2.2 mL 2.0 mL

2.2 mL 2.0 mL

Analysis 2.6.1 Sample analysis will be conducted using the 0-200 mg/L calibration range 2.6.2 Testing will be conducted in milk containing ~100 mg/L lactose. 2.6.3 Test according to the candidate method instructions for use. 2.6.4 Randomize the treatment combinations. 2.6.5 Test 2 replicates for each treatment combination. 2.6.6 Analyze data by ANOVA, multifactor regression or generalized linear model software to determine whether any parameter change affects the results. For the lot-to-lot consistency study and stability study, two separate evaluations will be performed. The first study will evaluate the stability of the Biotest under dry conditions and the second will evaluate the Biotest after being rehydrated. At least 3 lots will be evaluated. The Biotest kit, stored at 3-8°C with each electrode vacuum packed and packaged with silica gel crystals to reduce humidity, has a shelf life of 6 months before hydration. After hydration, the Biotest has a shelf life of 15 days. Lot-to-Lot Consistency and Stability Study Rehydration Stability - The study plan is to test a minimum of three lots of Biotest kit (from the beginning, middle and end of dry shelf life) at day 0, 7 and 15 after rehydration. Analysis 2.7.1 Sample analysis will be conducted using the 0-200 mg/L calibration range 2.7.2 Testing will be conducted in milk containing ~100 mg/L lactose. 2.7.3 Test according to the candidate method instructions for use. 2.7.4 Test 2 replicates for each time point or each lot of test kits. 2.7.5 Calculate the mean and s r for each time point or lot. Dry Stability - The study plan is to test a minimum of three lots of Biotest kit at 7 days, 45 days, 8 months and 14 months.

2.7

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2.7.6 Use a generalized linear model to determine whether time points and/or lots differ.

3.0

REPORT FORMAT

A validation study report in the format shown in Appendix 4 is to be submitted by the method developer at the completion of data collection. This report is to incorporate all the results generated through SLV studies. A call for experts will be made and an AOAC Expert Review Panel (ERP) will be conveyed to review the method. Once reviewed and any revisions are made, the ERP will determine if the test kit is acceptable for First Action Official Method of Analysis SM status. If acceptable, the method developer must submit the report for publication in the Journal of the AOAC INTERNATIONAL .

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AOAC SMPR 2018.009

Milk and milk products.— Milk is defined as the normal mammary secretion of a milk animal, intended for consumption as liquid milk or for further processing Milk product is defined as a product obtained by any processing of milk. Although a milk product shall be made from milk, the definition does not hinder the milk from being subjected to various processing steps before it becomes an end product. Composite milk product is a product of a milk product and other food(s) where the milk constituents are an essential part in terms of quantity of the final product. [Bulletin of IDF 397 (2005) The Codex General Standard for the Use of Dairy Terms, Its Nature, Intent and Implications ] Recovery. —Fraction or percentage of analyte that is measured when the test sample is analyzed using the entire method. Repeatability. — Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator (in the same laboratory) and repeating during a short time period. Expressed as the repeatability standard deviation (SD r ); or % repeatability relative standard deviation (%RSD r ). Reproducibility .—Variation arising when identical test materials are analyzed in different laboratory by different operators on different instruments. The standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility standard deviation (SD R ); or % reproducibility relative standard deviation (%RSD R ). 5 Method Performance Requirements See Table 2. 6 System Suitability Tests and/or Analytical Quality Control Suitable methods will include blanks, and appropriate check standards. 7 Validation Guidance Recommended level of validation: AOAC Official Methods of Analysis S M . Method data packages must include relevant data regarding interferences and instabilities, such as listed in Table 2. Not all interferences are likely to occur in all matrices. Method developers are responsible for assessing interferences with the method. 8 Maximum Time-to-Results None 9 Reference and Harmonization Materials See Tables 3 and 4 and Figure 2. Refer to Annex F: Development and Use of In-House Reference Materials in Appendix F: Guidelines for Standard Method Performance Requirements , 21st Ed. of the Official Methods of Analysis of AOAC INTERNATIONAL (2019). Available at http:// www.eoma.aoac.org/app_f.pdf Approved by the AOAC Stakeholder Panel on Strategic Food Analytical Methods (SPSFAM). Final Version Date: August 2018.

Standard Method Performance Requirements (SMPRs ® ) for Lactose in Low-Lactose or Lactose- Free Milk, Milk Products, and Products Containing Dairy Ingredients Intended Use: Method for Confirming Compliance with Regulatory Standards and Dispute Resolution 1 Purpose AOAC SMPRs describe the minimum recommended performance characteristics to be used during the evaluation of a method. The evaluation may be an on-site verification, a single- laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC stakeholder panels composed of representatives from the industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC expert review panels in their evaluation of validation study data for method being considered for Performance Tested Methods SM or AOAC Official Methods of Analysis SM , and can be used as acceptance criteria for verification at user laboratories. 2 Applicability Measure lactose found in milk, milk products, and products containing dairy ingredients that are low-lactose or lactose-free. The analytical method must account for potential interferences ( see Table 1) in these matrices. This scope includes “lactose-free” infant formulas and adult nutritionals. 3 Analytical Technique Any analytical technique(s) that measures the analyte(s) of interest and meets the following method performance requirements is/are acceptable. 4 Definitions Infant formula .—Breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infant during the first months of life up to the introduction of appropriate complementary feeding (Codex Standard 72-1981) Infant Formula and Formulas for Special Medical Purposes – 0–12 month of age; Follow-Up Formula – from 6–12 months and for young children; Young Children – 12–36 months of age; Foods for Special Medical Purposes Nutritionally complete specially formulated food for adults, consumed in liquid form, which may constitute the sole source of nourishment (AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals; 2010). Made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch and amino acids, with and without intact protein. Lactose .—β-D-galactopyranosyl-(1→4)-D-glucose. CAS No. 63-42-3 ( see Figure 1). Limit of detection (LOD) .— The lowest concentration or mass of analyte in a test sample that can be distinguished from a true blank sample at a specified probability level. Limit of quantitation (LOQ) .— The lowest level of analyte in a test sample that can be quantified at a specified level of precision.

© 2018 AOAC INTERNATIONAL

Table 1. Potential interferants Other nontarget mono-, di-, and trisaccharides (especially lactulose and allo- lactose) Enzymatic activity (beta-galactosidase) Organic acid activity Glucose with higher degrees of polymerization Sugar alcohols to include:

Table 2. Method performance requirements

Milk, milk products, and products containing dairy ingredients

Infant formula

Analytical range, mg/100 g 5–100

10–100 85–115

>100–2000

Recovery, %

85–115

90–110

RSD r RSD

, %

≤10 ≤15

≤10 ≤15

≤7

R ≤10 Note : Requirements are for foods and beverages as received. For infant formula and adult nutritional products, concentrations apply to “ready-to-feed” liquids, reconstituted powders (for infant formula products, 25 g into 200 g water). , %

 Glycerol  Erythritol  Xylitol  Sorbitol  Mannitol

 Maltitol  Lactitol  Isomalt Hydroxylated compounds (nontargeted carbohydrates, sugar alcohols, sugar acids, sucralose, etc.) Salts, such as sodium chloride Amine-containing compounds (glucosamine HCl, amino acids, peptides, glycoproteins, etc.)

Table 3. Reference materials No.

SRM name Analyte Value U Units Value type Baby Food Composite Lactose 0.5 0.1 % Reference

NIST SRM 2383a

Table 4. Harmonization materials National Milk Laboratories (UK)  http://www.qclscientific.com/pdfs/Dairy%20Calibration%20  Standards%20rev.4.pdf MUVA (Germany)  https://www.muva.de/muva/web.nsf/id/pa_reference_materials_e.html

Figure 1. Lactose.

Figure 2. Reference and harmonization materials.

© 2018 AOAC INTERNATIONAL

LACTOSE Instructions Manual

For the determination of lactose in free and low lactose dairy products

CONTENTS

1. BIOMILK 300 Technology and Application…………………...4

2. Equipment Needed for Analysis...........................................6

3. Tailored Preparation…………………………………………….7

a) Biotest Storage and Use ................................................ ..7

b) Reagent Preparation........................................................7

4. Setting Up the Biosensor.....................................................10

a) Assembly of the Biosensor...............................................10

b) Activation and Calibration of the Biotest.

Measurement Standard Analysis.........................................12

5. Sample Pretreatement, Dilution and Sample Analysis.........15

6. Recommendations for Use...................................................17

7. Annex………………………………………………………….…18

8. Safety Section.…..……………………………………………...19

Version: V.LAC.12.18.I.B

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1. BIOMILK 300 Technology and Application

A biosensor is a compact analytical device that combines a biological component with a physicochemical transducer to detect analytes of interest.

In the case of BIOMILK 300, BIOLAN combines the high specificity and selectivity of specific enzymes for each analyte with an amperometric transduction of that biological signal, which is easily detectable and quantifiable. The technology is based on the development of the Biotest, a working electrode that immobilises the enzyme together with other electroactive components, which causes a change in the electric current when it comes into contact with the analyte to be detected

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How is the analysis performed?

Any of the processes performed in the BIOMILK 300 (activation, calibration, analysis, etc.) comprise two different parts.

The first phase is when the device records the electricity signal that circulates in the electrochemical cell (establishing the target) as soon as the process begin (ADJUSTING REFERENCE), when the measurement cuvette is only filled with Measurement Solution. The second phase (MEASURING) begins with the addition of the Calibration Standard or of the sample to the measurement cuvette, once the device has issued a sound signal, indicating that the target of each measurement has been established correctly.

This addition increases the electricity current in the cell due to the redox reaction sequence that occurs on the Biotest surface.

The device uses the difference in intensities recorded before and after the addition to calculate a current intensity variation that correlates with the lactose concentration by means of a prior calibration of the device. BIOMILK 300 LAC is a BIOLAN application to stablish quantitatively the lactose level in low lactose or lactose free dairy products by means of three different calibration ranges:

Table 1: Specifications of the Measurement Ranges. 0-200 mg/l 0-2 g/l

0-6 g/l

15%

15%

15%

Error

Quantitative Range

50-200 mg/l

0,2-2 g/l

0,5-6 g/l

50 mg/l

200 mg/l

0,5 g/l

LOQ

NO

1/10

1/10

Dilution

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2. Equipment Needed for the Analysis Equipment needed for the analysis Biosensor: device, measurement cuvette, reference electrode and feeder (comes in the pack). LAC Biotest.

LAC Pretreatment. LAC Calibration Kit. LAC Measurement Kit. 100-1000 µl and 0.5-5 ml pipette. Distilled water.

BIOLAN references BM300-01: Biosensor.

BTLAC10 : LAC Biotest for 10 analysis. BTLAC20 : LAC Biotest for 20 analysis. BTLAC50 : LAC Biotest for 50 analysis. KITCLAC : LAC Calibration Kit (6 LAC1 Calibration Reagent Tubes (CLAC1) and 4 LAC2 Calibration Reagent Tubes (CLAC2), for 25 working days). KITMLAC1 : LAC1 Measurement Kit (8 LAC1 Measurement tubes (MLAC1), for 160 analysis). KITMLAC2 : LAC2 Measurement Kit (8 LAC2 Measurement tubes (MLAC2), for 640 analysis). ELEREFN : Reference electrode with storage solution (SOLALMER). Recommended Equipment 1 litre bottle, to prepare the Measurement Solution. Homogenizer. Dispenser that can be clipped on to the 1-litre bottle, to quickly dispense the Measurement Solution.

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3. Tailored Preparation

a) Biotest storage and use The Biotests are dispatched at room temperature, vacuum packed and with silica gel crystals to protect them from atmospheric humidity. Once delivered, they must be kept in the same way, between 3-8ºC, before expiration date. To hydrate the Biotest prior to use (this process should be carried out at least 24 hours prior to use), remove the silica gel crystals from inside the tube and fill it with Measurement Solution until the base of the Biotest is submerged. Once hydrated, the Biotest can be stored vertically at a temperature between 3-8ºC for a maximum of 15 days.

b) Reagent preparation

• LAC Measurement Solution (MS):

Both LAC1 Measurement Kit and LAC2 Measurement Kit contain 8 tubes (MLAC1 or MLAC2) to prepare the Measurement Solution, which is needed to prepare Reagents and Calibration Standards and to carry out the measurements. See Table 2 for the Measurement Solution preparation.

Table 2: Measurement Solution (MS) preparation.

LAC1 Measurement tube (MLAC1)

LAC2 Measurement tube (MLAC2)

250 ml

1 litre

Volume of distilled water

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• LAC Calibration Standards and Reagents:

The LAC Calibration Kit contains 6 tubes (CLAC1) of LAC1 powdered Calibration Reagent, 4 tubes (CLAC2) of LAC2 powdered Calibration Reagent, together with 2 empty tubes to prepare the LAC1 Calibration Standard, 2 empty tubes to prepare the LAC2 Calibration Standard and 3 empty tubes to prepare the Measurement Standard. The LAC1 and LAC2 Calibration Standards are prepared by diluting the LAC1 and LAC2 Calibration Reagents, respectively, and these are the reagents to be used to calibrate the equipment. The Measurement Standard is used to test the calibration status (see Table 3).

Table 3: Preparing the Calibration Reagents and Standards.

0-200 mg/l

0-2 g/l

0-6 g/l

LAC1 Calibration Reagent (CLAC1) LAC1 Calibration Standard LAC2 Calibration Reagent (CLAC2)

10 ml MS (*)

10 ml MS (*)

10 ml MS (*)

4 ml MS + 1ml CLAC1

4,9 ml MS + 0,1ml CLAC1

4,9 ml MS + 0,1ml CLAC1

5 ml MS (*)

5 ml MS (*)

5 ml MS (*)

9,75 ml MS + 0,25 ml CLAC2

9,75 ml MS + 0,25 ml CLAC2

9,25 ml MS + 0,75 ml CLAC2

LAC2 Calibration Standard

7,75 ml MS + 2 ml CLAC1 + 0,25 ml CLAC2

9,55 ml MS + 0,2 ml CLAC1 + 0,25 ml CLAC2

9,05 ml MS + 0,2 ml CLAC1 + 0,75 ml CLAC2

Measurement Standard

(*) Prepare it 24 hours before being used.

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The Calibration Reagent tubes have a pre-perforated stopper into which the tip of the pipette is introduced to prevent losses of reagent. The storage conditions of each of the prepared reagents are specified in Table 4.

Table 4. Reagent storage conditions.

CLAC1/ CLAC2 r.t. /e.d/ *

Measurement Reagent

Biotest

Standards

Sample

3-8ºC / e.d.

-

r.t. /e.d/ *

-

Dry

3-8ºC/ 15 days 3-8ºC /7 days daily

r.t./ 15 days 2 hours

Hydrated

e.d.: until expiration date r.t.: room temperature *: keep in a dark and dry place

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4. Setting Up the Biosensor

a) Assembly of the Biosensor To assembly the Biosensor correctly:

• Connect both the Biotest (placing it in the Biotest carrier and screwing it into place) and the reference electrode to their respective connectors.

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• Fill the measurement cuvette with 10 mL of Measurement Solution and put it in the cuvette holder making sure that all the components of the electrochemical cell are submerged.

• Connect the device to a 100-240 VAC socket and press the On switch that is beside the screen for 2 seconds. In the BIOTEST option: select the parameter to be analysed, press », enter the Biotest code (this code is on the Biotest ID label), press » and finally select the Measurement Range to be used and press SAVE.

The ANALYSIS option takes you to the main analysis menu: Activate, Calibrate, Standard and Sample.

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b) Activation and Calibration of the Biotest. Measurement Standard Analysis. Once a day, the Biotest has to be activated and calibrated before starting to perform the analysis. These two operations should also be carried out after the device has been inactive for long periods (over 2 hours). Once the Biosensor is set up (see Point 4.1.), make sure that the measurement cuvette contains 10 ml of Measurement Solution and that all the components of the electrochemical cell are submerged. 1.1. Press Activate, check that the cuvette is correctly fitted and press CONTINUE to begin the activation process. 1.2. Wait until the device records the measurement target and it issues a sound signal. Inject the Measurement Standard into the measurement cuvette, using a micropipette, and wait until the process ends (see Table 5). 1.3. Change the content of the cuvette with 10 ml of new Measurement Solution. The calibration consists on 3 points and it is performed by means of adding the LAC1 Calibration Standard once and the LAC2 Calibration Standard twice to the measurement cuvette at three different times. To trace the calibration line, the device will take into account the changes in intensity caused by the different additions and the relevant concentrations of each Calibration Standard. 2. Calibration: 1.Activation:

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2.1. Press Calibrate, fill the cuvette with new Measurement Solution and press CONTINUE. 2.2. Wait until the device issues a sound signal. Press CONTINUE and inject, in this order, the LAC1 Calibration Standard into the measurement cuvette (first addition (1/3)), using a micropipette (see Table 5). 2.3. Wait until the device records the change in intensity and issues a sound signal. Press CONTINUE again and inject the LAC2 Calibration Standard into the measurement cuvette (second addition (2/3)) using a micropipette (see Table 5). 2.4. Wait until the device records the change in intensity and issues a sound signal. Press CONTINUE again and inject the LAC2 Calibration Standard again (third addition, (3/3)) using a micropipette (see Table 5). As a result of the calibration, the parameters of the linear regression obtained in the interpolation of the three calibration points will appear on the screen. It is important to add the Calibration Standards immediately after pressing the CONTINUE button to record the current intensity correctly at the time of adding it.

R and m values equal to or over 0.9990 and 6.000, respectively, will be considered valid to proceed with the analysis. Typical values for b do not exceed 100.

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Table 5: Addition volumes for activation, calibration and analysis.

0-200 mg/l

0-2 g/l

0-6 g/l

2 ml 2ml 2ml 2ml

2 ml 2 ml 2 ml 2 ml

650 µl 650 µl 650 µl 650 µl

LAC1 Calibration Standard

LAC2 Calibration Standard

Measurement Standard

Sample

The general procedure of calibration is intended for the analysis of milk samples. If other dairy products are to be tested, please see annex for more details.

3. Measurement Standard analysis:

The calibration status should be tested using the Standard option after a series of measurements or if the device has not been running for a long period. 3.1. Press Standard, fit the cuvette correctly with 10 ml of Measurement Solution and press CONTINUE. 3.2. Wait until the device issues a sound signal. Press CONTINUE and inject Measurement Standard into the cuvette (See Table 5). 3.3. A "Standard OK" or "Calibrate again" message will appear on the screen.

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5.Sample Pretreatment, Dilution and Analysis A preliminary sample pretreatment or dilution step is needed to perform the analysis of the sample. Liquid samples need to be pretreated before being analysed in the 0-200 mg/l range. To do so, add a capsule of charcoal activated in 50 ml of sample and cut it in half to speed up the mixing process. Shake until the active charcoal is homogeneously mixed with the milk sample and let it react for at least 30 minutes before analysing. Shake it occasionally. Eventually you might receive a pretreatment tube instead of a capsule. In that case, inject 5ml of milk sample inside the pretreatment tube, shake until the mixture is homogeneous and analyse the sample as usual. Activated charcoal (0-200 mg/l range)

Dilution (0-2 g/l range and 0-6 g/l range)

Samples (milk and other dairy products) in the 0-2 g/l and 0 -6 g/l ranges need to be diluted. To do so, mix 1 ml of sample with 9 ml of Measurement Solution and shake it to homogenize. Once the samples are prepared, they have to be analysed in a maximum of 2 hours and can be kept at ambient temperature.

General sample pretreatment

High viscous samples (greek yogurt, high protein yogurt, crème caramel or others) must be liquefy before analysing in order to be able to pipette them inside the measurement cuvette. Make use of an Ultra-turrax or a mincer. Occasionally, an extra step of heating is needed. For example, for spreadable or soft cheese, heat in a microwave at low power for a few seconds in order to melt it before analysing.

In case of high viscous samples, the electrodes must be cleaned with distilled water between analysis.

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