PROTOCOL DOCUMENT NO.
Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
1.0 OBJECTIVE
1.1. To carry out validation of the analytical method for testing of aflatoxins so as to
obtain documented evidence that the method consistently produces accurate
results within NMSQCL.
2.0 SCOPE
2.1. This analytical method validation protocol shall be limited to the assay test of
aflatoxins (B1, B2, G1, G2) within the NMSQCL.
2.2. The parameters to be assessed shall include accuracy, precision (repeatability
and intermediate precision), specificity, linearity and range.
3.0 DEFINITIONS & ABBREVIATIONS
3.1. NMSQCL: National Medical Stores Quality Control Laboratory
3.2. CQCO: Chief Quality Control Officer
3.3. PQCO: Principal Quality Control Officer
3.4. SQA: Senior Quality Analyst
3.5. RSD: Relative Standard Deviation
3.6. NMT: Not more than
3.7. NLT: Not less than
3.8. HPLC: High Performance Liquid Chromatogram
3.9. FLD: Fluorescence detector
3.10. SDS: Safety Data Sheet
3.11. LC: Liquid Chromatography
3.12. ml: milliliter
3.13. mg: milligram
3.14. µL: Microliters
3.15. µg: Micrograms
3.16. g/L: Gram per liter
3.17. v/v: volume by volume
3.18. ppb: parts per billion
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PROTOCOL DOCUMENT NO.
Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
4.0 SAFETY PRECAUTIONS
4.1. Always wear gloves, a laboratory coat while carrying out all preparations.
4.2. Always wear a respirator when working with hazardous solvents.
4.3. Always use the available face masks and splash guards when necessary.
4.4. Always read the chemical safety information provided in the SDS.
4.5. Adequately protect from daylight the laboratory where the analyses are carried
out. Protect solutions containing aflatoxin from light as far as possible.
4.6. Aflatoxins are highly dangerous, and extreme care should be exercised in
handling aflatoxin materials.
5.0 MATERIALS AND EQUIPMENT
5.1. Apparatus/equipment
5.1.1. Laboratory glassware
5.1.2. HPLC with FLD
5.1.3. Ultrasonic bath
5.1.4. Micropipettes 5 µL – 100 µL
5.1.5. Analytical balance
5.1.6. Aflatoxin immunoaffinity columns
5.1.7. Whatman filter paper no.1 or equivalent
5.1.8. Photochemical post column derivatization system
5.1.9. Vacuum manifold
5.1.10. Membrane filter (0.45 µm)
5.1.11. High speed blender
5.1.12. Glass syringe
5.1.13. 10ml syringes
5.2. Chemicals and Reagents
5.2.1. Methanol HPLC grade
5.2.2. Potassium bromide
5.2.3. Nitric acid
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PROTOCOL DOCUMENT NO.
Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
5.2.4. Sodium chloride
5.2.5. Purified water
5.2.6. Aflatoxin mixture (2µg/ml B1 & G1, 0.5µg/ml B2 & G2)
6.0 TEST PROCEDURE
6.1. Appearance of the product
6.1.1. Describe the product carefully giving details of the following; brand name (if
any), generic name, dosage form, strength, marketing authorization holder,
manufacturer, batch number, expiry date, color, shape, trademarks, packaging
and any other noticeable parameters.
6.2. Assay Method
6.2.1. Preparation of aflatoxin mixture stock solution.
Draw 125µL of aflatoxin mixture using the glass syringe into an umber colored
25ml volumetric flask that is at ambient temperature. Top up to the 25ml mark
with a solution of methanol: purified water (50:50). The resultant solution
should contain about 10µg/l of aflatoxin B1& G1 and about 2.5µg/l of aflatoxin
B2& G2.
6.2.2. Preparation of sample solution.
Weigh 25g of sample and 2g of sodium chloride into a high-speed blender
jar. Add 125ml of methanol: purified water (60:40, v: v) into the jar, cover
and blend for 1 minute at high speed. Dilute the extract with 125ml of
purified water. Mix well by swirling followed by filtering approximately 40-50
ml of sample extract through Whatman No. 1 filter paper immediately.
Transfer 10ml of the filter (equivalent to 1g of sample) into the 10 ml
syringe barrel for passage through the prepared immunoaffinity column at a
flow rate of 2-3 ml/min. Then add 10ml of purified water to the 10ml syringe
for washing the column. Expel the residual water from the column and
transfer accurate 1ml of methanol to elute aflatoxins from the column. It
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PROTOCOL DOCUMENT NO.
Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
could be back-flushed with the methanol solution for completely release of
aflatoxins from the monoclonal antibody if necessary. Collect all of the
methanol elution and dilute with 1 ml of purified water before injection into
HPLC system.
6.2.3. Preparation of system suitability solution.
Prepare a spiked sample by adding 5 mL of aflatoxin mixture stock solution to a
25g sample and repeating the procedure in 6.2.2, using 120 mL instead of
125ml of methanol: purified water (60:40, v: v).
6.2.4. Inject the blank, aflatoxin mixture stock solution and sample solutions
using the chromatographic conditions below:
Mode : LC
Mobile Phase A : 1L water containing 238 mg KBr and 700 µL
4M HNO3
Mobile Phase B : Methanol
Isocratic : A: B = 50: 50
Runtime : 15 mins
Column details : 4.6 mm × 15 cm; 5 µml packing L1
Detector : Excitation: 362 nm, Emission: 455nm, gain =
wavelength 15
Flow rate : 1.0 mL/min
Injection volume : 20 µL
Oven temperature : 40°C
Blank : 50% Methanol
6.2.5. Acceptance Criteria
The sample must contain NMT 5 ppb for aflatoxin B1 and NMT 20 ppb for the sum of
aflatoxins B1, B2, G1, and G2.
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PROTOCOL DOCUMENT NO.
Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
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PROTOCOL DOCUMENT NO.
Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
7.0 ASSAY METHOD VALIDATION PROCESS
7.1. Assay Method Validation Process
The analytical method validation process shall be carried out by analyzing
aflatoxins following the assay method test procedure described above.
7.1.1. SPECIFICITY
Specificity shall be determined by examining the response obtained from the
blank solution, aflatoxin mixture stock solution and sample solution.
Acceptance criteria:
There shall be no visible interference from the blank solution.
The principal peaks in aflatoxin mixture stock solution and sample
preparations shall be identical in physical appearance and retention.
7.1.2. PRECISION
The precision (repeatability and intermediate precision) of the system shall be
determined by carrying out 6 independent determinations at each stage .
7.1.2.1. Repeatability
Repeatability of the analytical method shall be determined by preparing six
independent assays by the same analyst under the same conditions (refer to
assay preparations in section 6.2 above). The samples shall be prepared and
injected in the HPLC system along with aflatoxin mixture stock solutions as per
chromatographic conditions given in section 6.2.4.
Acceptance criteria:
Each assay value for the six preparations must be within the acceptance
specification (NMT 5 ppb for aflatoxin B1 and NMT 20 ppb for the sum of
aflatoxins B1, B2, G1, and G2).
The %RSD for the six assay results shall not be more than 2.0%.
7.1.2.2. Intermediate Precision
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PROTOCOL DOCUMENT NO.
Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
This shall be determined by having another analyst repeat the procedure given
in section 7.1.2.1, introducing variables that may include but not limited to:
different days, independent fresh preparations of the same homogeneous
sample as analyst 1, different reagent lots, different HPLCs, different analytical
balances and different stationary phase lots.
Acceptance criteria:
Each assay value for the six preparations must be within the acceptance
specification (NMT 5 ppb for aflatoxin B1 and NMT 20 ppb for the sum of
aflatoxins B1, B2, G1, and G2).
The %RSD for results of the six assay results shall not be more than 2.0%
The percent RSD between the two average assay values for the two analysts
should not be more than 2.0%.
7.1.3. LINEARITY
The linearity of the detector response shall be demonstrated by considering six
concentrations of the aflatoxin mixture as having aflatoxin B1 & G1
concentrations as follows; blank, 0.1, 0.25, 0.5, 1.0, 5.0, 10 µg/L with respect to
the theoretical content in the aflatoxin mixture stock solution.
7.1.3.1. Preparation of linearity aflatoxin mixture stock solution.
Draw 125µL of aflatoxin mixture using the glass syringe into an umber colored
25ml volumetric flask that is at ambient temperature. Top up to the 25ml mark
with a solution of methanol: purified water (50:50). The resultant solution
should contain about 10µg/l of aflatoxin B1& G1 and about 2.5µg/l of aflatoxin
B2& G2.
Prepare the various Linearity levels as shown below:
Linearity Volume of Final volume of Aflatoxin
Level standard stock solution (ml) Concentration in
solution (ml) µg/L
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Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
Level 1 Blank to be used
Level 2 0.1 10 0.1
Level 3 0.25 10 0.25
Level 4 0.5 10 0.5
Level 5 1.0 10 1.0
Level 6 5.0 10 5.0
Level 7 Stock solution 10.0
7.1.3.2. Inject each of the solutions above into the HPLC system (use
chromatographic conditions under 6.2.4).
7.1.3.3. Plot the obtained data of response of the analyte against the
corresponding concentrations (concentration vs average peak area) for each
aflatoxin and using the appropriate statistical tools, perform regression
analysis.
Acceptance criteria:
The coefficient of determination (R Squared Value) shall be NLT 0.995.
The slope shall be statistically different from 0 and the y-intercept shall not
be statistically different from 0.
7.1.4. ACCURACY
The accuracy shall be determined by obtaining the slope of the graph obtained
in 7.1.3.3.
Acceptance Criteria:
The slope should be between 0.93 – 1.07.
7.1.5. RANGE
This shall be established by confirming that the analytical procedure provides
an acceptable degree of linearity, accuracy and precision when applied to
samples containing amounts of analyte within or at the extremes of the
specified range of the analytical procedure.
Acceptance criteria
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Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
The graph should be linear within the range of 0.1 µg/L to 10 µg/L
concentration of aflatoxin B1 in the aflatoxin mixture solution.
7.1.6. SYSTEM SUITABILITY
The system suitability shall be determined by evaluating data obtained from
the 5 readings of the aflatoxin mixture stock solutions as prepared under assay
test 6.2.1 above and by evaluating the mean recovery of aflatoxin B1 and the
total aflatoxins in the spiked sample in 6.2.3.
Acceptance criteria:
Relative standard deviation for peak area response of 5 replicate NMT 2.0%
injections of standard solutions A and B
Relative standard deviation for retention time for the 5 replicate NMT 2.5%
injection of the principal peaks in standard solution A and B
Similarity factor for the standard solutions prepared in duplicate 0.98 to 1.02
Theoretical plates for the principal peaks in the standard solution NLT 3000
The tailing factor for the principal peaks in the standard solution NMT 2.0
The mean recovery of spiked aflatoxin B1 (10 µg/L) and the total of aflatoxins
(25 µg/L) is NLT 68% and 70%, respectively. The relative standard deviation
(RSD) is NMT 10% for aflatoxin B1 and for the total of aflatoxins.
7.1.7. SOLUTION STABILITY
The standard solutions prepared at 100% in the assay procedure shall be kept
at room temperature and under refrigeration and each shall be analyzed as per
the method given in 6.2.3 above. The testing shall cease at the point when
unsatisfactory results are obtained.
Acceptance criteria:
The percentage difference from the initial peak area response shall not be
more than 2.0%.
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PROTOCOL DOCUMENT NO.
Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
8.0 CALCULATIONS
8.1. Mass fraction of each aflatoxin in ppb
Quantitation of aflatoxins is performed by measuring peak areas at each aflatoxin
retention time and comparing them with the corresponding calibration curve. If the
test portion area response is outside (higher) the calibration range, then the Test
Solution should be diluted with a mixture of methanol and water (1:1, v/v) and
reinjected into the LC column.
Plot the peak area (response, y-axis) of each of the aflatoxins against the
concentration (µg/L, x-axis) and determine the slope (S) and y-intercept (a). Calculate
the level of toxin in the sample by the following formula:
Mass fraction of aflatoxin (ppb) = ((R /S )× (V/W))
R = peak area of the sample solution
S = slope of the calibration curve
V = final volume of the injected sample solution (mL) obtained in 6.2.2
W = 1 g of test sample passed through the immunoaffinity column
The total of aflatoxins is the sum of the mass fractions of Aflatoxin G2, Aflatoxin G1,
Aflatoxin B2 and Aflatoxin B1.
Acceptance criteria: The sample must contain NMT 5 ppb for aflatoxin B1 and NMT
20 ppb for the sum of aflatoxins B1, B2, G1, and G2.
9.0 REFERENCES
9.1. USPNF 2023 Issue 3
9.2. Validation of Analytical Procedures: Sharad D. Mankumare, Ph.D., Director,
Reference Standards Laboratory & Validation Program United States
Pharmacopeia, India
9.3. Validation of Analytical Procedures: Text and Methodology Q2(R1)
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PROTOCOL DOCUMENT NO.
Analytical Method
Validation Protocol NMS/QC/PRT-M/005
Aflatoxin Testing
9.4. Guideline for Sampling and Testing for Aflatoxins: Eastern Africa Grain Council
9.5. Determination of Aflatoxins (B1, B2, G1 and G2) in Corn and Peanut Butter by
HPLC-FLD Using Pre-column Immunoaffinity Cleanup and Post-Column
Electrochemical Derivatization: Xinlei Yang, Rong An Application Engineer of
Liquid Chromatography Life Science and Chemical Analysis Group Agilent
Technologies
9.6. Determination of Aflatoxins (BI, B2, G1 and G2) in milled cereals and grains by
HPLC-FLD: Chemistry Laboratory, Uganda National Bureau of Standards
END OF DOCUMENT
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