Pesticides in Breast Milk by GCMSMS

Sophisticated Industrial Material Analytic Labs (P) Ltd DOC NO.
:
EFFECTIVE DATE: 18/12/2018
A-3/7, Mayapuri Industrial Area, Phase-II, New Delhi - ISSUE NO. : 01
ISSUE DATE : 01/12/2020
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Title : VALIDATION REPORT FOR

SIMA PESTICIDE RESIDUES IN BREAST MILK BY
GCMSMS
VALIDATION REPORT FOR PESTICIDE RESIDUES
IN BREAST MILK BY GCMSMS
SIMA
Prepared By: Checked By Reviewed & Approved By: Issued By

Sr. Analyst/Dy. Manager Technical Manager Sr. General Manager-Technical Sr. Quality Manager
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GCMSMS
CONTENT OF THIS DOCUMENT
S.No Heading Page No

1. Signature page 03
2. List of Tables and Annexure 04
3. Objective 06
4. Scope and Application 06
4.1 Application 06
5. Procedure 06
5.1 Requirements 06
5.1.1 Equipment and glassware 06
5.1.2 Reagents and Solvents 07
5.2 Preparation of reagents 07
5.3 Sample Preparation 07
5.4 Instrument condition 07
6. Statistics and Calculations 11
7. Validation Parameters 10
7.1 System suitability 12
7.2 Selectivity & Specificity 16
7.3 Linearity & Range 19
7.4 Limit of Detection 23
7.5 Limit of Quantification 25
7.6 Recovery 28
7.7 Repeatability/Reproducibility 32
8. Quality control 35
9. Conclusion 35
10. Reference 36
11. End of the method validation report 36

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SIGNATURE PAGE
ANALYST DECLARATION: We undersigned, declare that we have done the validation as per the
protocol to the best of our knowledge for which raw data has been attached.
Name Signature Date
Tarun Gupta
AUTHENTICATION: I, undersigned, declare that we have thoroughly reviewed this report and the
supporting data for the completeness, accuracy, SOP and we have critically evaluated the report
for internal consistency and to the best of our knowledge this report for material consistency and to
the best of knowledge this report accurately reflects the raw data.
Name Signature Date
Abhishek Mishra
APPROVAL & ISSUED BY : I, undersigned declare that I have reviewed this report for
anomalies and critically Evaluated the scientific validity of the statements and conclusions in this
report and to the best of my knowledge and judgment this report is scientifically valid.
Name Signature Date
Anita Singh
Aradhana Saini
(Quality Manager)

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LIST OF TABLES
Table 1 Preparation of working standard for calibration curve.
Table 2 Preparation of calibration curve standards
Table 3 GCMSMS Transition.
Table 4 System suitability (RT & Area) of Analytes.
Table 5 Specificity & Selectivity of the Analytes..
Table 6 Linearity of Analytes.
Table 7 Range of Analytes
Table 8 Limit of Detection of analyte
Table 9 Limit of Quantification of analyte.
Table 10 Recovery
Table 11 Reproducibility/Repeatability
Table 12 Limits of Recovery and Precision.
Table 13 Conclusion.

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LIST OF ANNEXURES
Annexure 1 System suitability

Annexure 2 Specificity & Selectivity
Annexure 3 Linearity & Range
Annexure 4 Limit of detection
Annexure 5 Limit of quantification
Annexure 6 Recovery
Annexure 7 Repeatability/Reproducibility

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1.0 Objective:
To validate the method for the determination of Pesticide Residues in Breast Milk by GCMSMS.
2.0 Scope and Application:
The following performance parameters were assessed during the validation:
2.1 System Suitability.

2.2 Selectivity & Specificity.
2.3 Linearity & Range
2.4 Limit of Detection
2.5 Limit of Quantification
2.6 Recovery/Accuracy
2.7 Repeatability/Reproducibility.
2.1 Application
This procedure is applicable for the determination of Pesticide Residues in Breast Milk by
GCMSMS.
3.0 Procedure
3.1 Requirements: To validate this method the following things are required for the analysis:
3.1.1 Equipments and Glassware's
3.1.1.1 GCMSMS: Agilent 7890B.

3.1.1.2 Analytical Column: HP-5MS UI 15m, 0.25µm, 25 mm ID Capillary column.
3.1.1.3 Analytical Balance.
3.1.1.4 Vortex.
3.1.1.5 Nitrogen Concentrator.
3.1.1.6 Vacuum Manifold Assembly.
3.1.1.7 Bench Top Centrifuge.
3.1.1.8 50 mL Centrifuge tubes.
3.1.1.9 Shaker.
3.1.1.10 Sonicator.
3.1.1.11 Adjustable pipettes: 10-100, 20-200 and 100-1000 µl with disposable tips.
3.1.1.12 Volumetric flasks: 10 ml.
3.1.1.13 Mobile phase filtration assembly.

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3.1.2 Reagents and Solvents
3.1.2.1 Acetonitrile HPLC Grade.

3.1.2.2 Milli-Q Water.
3.1.2.3 Anhydrous Sodium Sulfate, Analytical Grade.
3.1.2.4 Anhydrous Sodium Acetate, Analytical Grade.
3.1.2.5 Anhydrous Magnesium Sulphate, Analytical Grade.
3.1.2.6 Primary Secondary Amine (PSA) (Make: Agilent).
3.1.2.7 QUECHERS EN Salt.
3.1.2.8 D-SPE tube.
3.1.2.9 Sodium chloride.
3.1.2.10 Reference Standards.
3.2 Preparation of Reagents
3.2.1 Preparation of Stock Standards: For Pesticide Residues: Transfer 10 mg of standard

into 10 ml volumetric flask and dissolve in HPLC grade Acetonitrile & suitable solvents.
Make up to 10 mL Label with name of the standard, Concentration, date of preparation,
date of expiry.
3.2.1.1 Preparation of pesticide standard mixture solution: Pipette out 100 µL of

each pesticide standard from stock solution (1000 mg/Kg) in to 10 mL
volumetric flask and make up with acetonitrile. Label it as Mix of 10 mg/Kg.
3.2.2 Preparation of Calibration Curve
Working Volume of Final

standard Working Solvent Final volume concentration Label
concentration standard volume(µL) (mL) (mg/L)
(mg/L) ( µL )
100.0 100 900 1.000 10.0 WS3
10.0 100 900 1.000 1.0 WS2
1.0 100 900 1.000 0.1 WS1
Table 1: Preparation of working standard dilution for calibration curve:
Table 2: Preparation of standards for calibration curve:

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3.3 Sample Preparation
3.3.1 Take 2 gms sample in Tarson tube.

3.3.2 Add 2 ml milli Q water and vortex for 1 minute.
3.3.3 Add 5 ml acetonitrile and vortex for 5 minutes.
3.3.4 Add QuEChERS salt pouch and vigorously wrist shake for 1 minutes.
3.3.5 Take supernatant layer in dSPE tube for cleanup, and vortex it for 1 minutes.
3.3.6 Centrifuge at 4500 RPM for 5 minutes
3.3.7 Take 2 ml of supernatant layer in Glass tube and evaporate with Nitrogen at 40-
degree temp reconstitute with 0.8 ml Ethyl acetate.
3.3.8 Inject on GCMSMS.
3.4 Instrumental Conditions
GC System
Instrument used : Agilent GC-7890 with Auto sampler.
Column used : HP-5MS UI 15m, 0.25µm, 25 mm ID Capillary column
Column temperature: : 315 0C
Run time: : 21.433 minutes.
Inlet Program
Mode : Split less
Flow : 2.617 ml/min
Mode : Constant Pressure
Gas type : Helium
Auto Sampler Program

Sample washes :2
Sample pumps :6
Injection volume : 1.5 µl
Syringe size : 10 µl
Post injector solvent A wash :4
Post injector solvent B wash :4
Viscosity delay : 0 seconds
Plunger speed : Slow
Pre-injection dwell : 0.00 min.
Post injection dwell : 0.00 min.

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Oven Program:
Rate (ºC/min) Value (ºC) Hold Time (min) Run Time (Min)
Initial - 70 1.0 1.0
50 150 0.0 2.6

6 200 0.0 10.933
16 280 5.5 21.433
MS Conditions:
Mass Spectrometer Agilent 7000C Triple Quad

Ionization EI
Source Temperature 300°C

Tune file Last Saved
Filament 2
Solvent Delay 2
Note: Run Time may vary depending upon column/mobile phase/instrumental fluctuation etc.
Hence, run a standard and adjust run time according to RT of analyte before running whole batch.
Run Time > 1.43 * RT of latest eluting analyte.

Collision Collision Collision
Name Transition Transition Transition
S.No. Energy Energy Energy
1 Profenofos 121.0 ‐> 65.0 10 121.0 ‐> 47.0 30 128.9 ‐> 65.0 15
BHC‐alpha (benzene 216.9 ‐> 181.0 5 180.9 ‐> 145.0 15 218.9 ‐> 183.0 5
2 hexachloride)
3 BHC‐beta 216.9 ‐> 181.1 5 181.0 ‐> 145.0 15 218.9 ‐> 183.1 5
BHC‐gamma 181.0 ‐> 145.0 15 216.9 ‐> 181.0 5 218.9 ‐> 183.1 5
(Lindane, gamma
4 HCH)
5 BHC‐delta 217.0 ‐> 181.1 5 181.1 ‐> 145.1 15 219.0 ‐> 183.1 5
6 Chlorpyrifos‐methyl 124.9 ‐> 47.0 15 124.9 ‐> 78.9 5 285.9 ‐> 92.9 20
7 Dimethoate 188.1 ‐> 160.2 10 188.1 ‐> 132.1 15 160.0 ‐> 132.1 10
8 Aldrin 262.9 ‐> 192.9 35 262.9 ‐> 190.9 35 254.9 ‐> 220.0 20
9 Paraoxon 108.9 ‐> 81.0 10 108.9 ‐> 91.0 5 148.9 ‐> 119.0 5
10 Malathion 126.9 ‐> 99.0 5 157.8 ‐> 125.0 5 172.9 ‐> 99.0 15
11 Chlorpyrifos 196.9 ‐> 169.0 15 198.9 ‐> 171.0 15 313.8 ‐> 257.8 15
Heptachlor exo‐ 352.8 ‐> 262.9 15 262.9 ‐> 193.0 35 354.8 ‐> 264.9 15
12 epoxide (isomer B)
13 Chlorfenvinphos 266.9 ‐> 159.1 15 268.9 ‐> 161.0 15 322.8 ‐> 266.8 10
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14 Chlordane‐cis (alpha) 271.9 ‐> 236.9 15 372.9 ‐> 265.9 20 374.9 ‐> 265.9 20

Chlordane‐trans 271.7 ‐> 236.9 15 372.8 ‐> 265.8 15 374.8 ‐> 265.8 15
15 (gamma)
Endosulfan I (alpha 194.9 ‐> 159.0 5 194.9 ‐> 160.0 5 194.9 ‐> 125.0 20
16 isomer)
17 DDE‐o, p' 246.0 ‐> 176.2 30 248.0 ‐> 176.2 30 317.8 ‐> 248.0 15
18 DDD‐o, p' 235.0 ‐> 165.2 20 235.0 ‐> 200.2 10 237.0 ‐> 165.2 20
19 DDD‐p, p' 234.9 ‐> 165.1 20 234.9 ‐> 199.1 15 236.9 ‐> 165.2 20
20 Dieldrin 262.9 ‐> 193.0 35 262.9 ‐> 191.0 35 277.0 ‐> 241.0 5
21 Endrin 262.8 ‐> 193.0 35 244.8 ‐> 173.0 30 316.7 ‐> 280.8 5
22 DDT‐o, p' 235.0 ‐> 165.2 20 235.0 ‐> 199.1 15 237.0 ‐> 165.2 20
23 DDE‐p, p' 246.1 ‐> 176.2 30 315.8 ‐> 246.0 15 317.8 ‐> 246.0 15
24 Endosulfan sulfate 271.9 ‐> 237.0 15 273.8 ‐> 236.9 15 273.8 ‐> 238.9 15
25 Ethion 152.9 ‐> 96.9 10 124.9 ‐> 96.9 0 230.9 ‐> 175.0 10
Endosulfan II (beta 206.9 ‐> 172.0 15 194.9 ‐> 158.9 25 194.9 ‐> 124.9 25
26 isomer)
27 DDT‐p, p' 235.0 ‐> 165.2 20 235.0 ‐> 199.2 15 237.0 ‐> 165.2 20
28 Endrin aldehyde 278.9 ‐> 209.0 25 278.9 ‐> 242.9 10 249.9 ‐> 214.9 30
29 Endrin ketone 316.8 ‐> 100.8 10 316.8 ‐> 280.7 5 280.9 ‐> 244.8 10
30 Fenpropathrin 181.1 ‐> 152.1 25 125.0 ‐> 55.1 10 207.9 ‐> 181.0 5
Cyhalothrin 197.0 ‐> 141.1 10 197.0 ‐> 161.1 5 181.2 ‐> 152.1 25
31 (Gamma)
32 Permethrin II (trans) 182.9 ‐> 168.1 10 182.9 ‐> 155.1 10 162.9 ‐> 91.1 15
33 Permethrin I 183.1 ‐> 168.1 10 183.1 ‐> 165.1 10 183.1 ‐> 153.1 15
34 Cyfluthrin 162.9 ‐> 90.9 15 162.9 ‐> 127.0 5 198.9 ‐> 170.1 25
35 Cypermethrin 163.0 ‐> 91.0 10 163.0 ‐> 127.0 5 164.9 ‐> 91.0 10
Esfenvalerate 167.0 ‐> 125.1 10 181.0 ‐> 152.1 25 225.0 ‐> 119.1 20
(Fenvalerate A‐
36 alpha)
Fenvalerate II {CAS # 167.0 ‐> 125.1 5 181.0 ‐> 152.1 25 208.9 ‐> 141.1 15
37 51630‐58‐1}
38 Fenvalerate I 167.0 ‐> 125.1 5 181.0 ‐> 152.1 20 208.9 ‐> 141.1 15
39 Fluvalinate I 250.0 ‐> 55.0 20 206.0 ‐> 178.9 15 207.9 ‐> 180.9 15
40 Deltamethrin 181.0 ‐> 152.1 25 250.7 ‐> 172.0 5 252.9 ‐> 93.0 15
Table 3 : MRM Table:
4.0 Statistics and Calculations
Chromatograms have to be acquired using the computer-based Analyst software supplied by the
Agilent Technologies. The concentration of the unknown has to be calculated from the equation
using regression analysis of the reciprocal of the pesticide residue concentration as weighting
factor (1/x).The concentration of the unknown was calculated from the calibration curve using
1/X as weighting factor.
By using the below equation

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Y = mX + C
Where, Y = Analyte area
X = concentration of Analyte
m = slope of the calibration curve
C = Y-axis intercept value
Final Pesticide residues content in the sample is in µg/kg.
5.0 Validation Parameters

5.1 System Suitability.
5.2 Selectivity & Specificity.
5.3 Linearity & Range
5.4 Limit of Detection
5.5 Limit of Quantification
5.6 Recovery/Accuracy
5.7 Repeatability
5.1 SYSTEM SUITABILITY
System suitability test parameters and acceptance criteria are based on the concept that the
equipment, electronics, analytical operations, and sample to be analyzed constitute an integrated
system. System suitability testing ensures that the system is working properly at the time of
analysis. To evaluate the system suitability, inject standard with 5 replicates and calculate %
RSD of retention time and area of analyte.
Acceptance Criteria:
a) The Retention time deviation of analytes shall not be more than 2.0%.
b) The relative standard deviation for the standards peak response of 7 replicate shall not be
more than 10.0%.
System Suitability (Retention Time):
Std %
S.no. Analyte Name Rep_1 Rep_2 Rep_3 Rep_4 Rep_5 Rep_6 Average
Dev RSD
1 Profenofos
2 BHC‐alpha (benzene hexachloride)
3 BHC‐beta
4 BHC‐gamma (Lindane, gamma HCH)
5 BHC‐delta
6 Chlorpyrifos‐methyl
7 Dimethoate
8 Aldrin
9 Paraoxon
10 Malathion
11 Chlorpyrifos
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12 Heptachlor exo‐epoxide (isomer B)
13 Chlorfenvinphos
14 Chlordane‐cis (alpha)
15 Chlordane‐trans (gamma)
16 Endosulfan I (alpha isomer)
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
24 Endosulfan sulfate
25 Ethion
26 Endosulfan II (beta isomer)
27 DDT‐p, p'
28 Endrin aldehyde
29 Endrin ketone
30 Fenpropathrin
31 Cyhalothrin (Gamma)
32 Permethrin II (trans)
33 Permethrin I
34 Cyfluthrin
35 Cypermethrin
36 Esfenvalerate (Fenvalerate A‐alpha)
37 Fenvalerate II {CAS # 51630‐58‐1}
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin
System Suitability (Area)
S.no %
Analyte Name Rep_1 Rep_2 Rep_3 Rep_4 Rep_5 Rep_6 Average Std Dev
. RSD
1 Profenofos
BHC‐alpha (benzene

2 hexachloride)
3 BHC‐beta
BHC‐gamma (Lindane,

4 gamma HCH)
5 BHC‐delta
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GCMSMS
7 Dimethoate
8 Aldrin
9 Paraoxon
10 Malathion
11 Chlorpyrifos
Heptachlor exo‐epoxide

12 (isomer B)
13 Chlorfenvinphos
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
25 Ethion
27 DDT‐p, p'
28 Endrin aldehyde
29 Endrin ketone
30 Fenpropathrin
33 Permethrin I
34 Cyfluthrin
35 Cypermethrin
Esfenvalerate (Fenvalerate

36 A‐alpha)
Fenvalerate II {CAS # 51630‐

37 58‐1}
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin
Table 4: System suitability of Analyte

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5.2 SPECIFICITY AND SELECTIVITY
Specificity of a method refers to the extent to which it can determine particular analyte(s) in a
complex mixture without interference from other components in the mixture. The term ‘specificity’
refers to a method that provides responses for a number of chemical entities that may or may not be
distinguished from each other. If the response is distinguished from all other responses, the method
is said to be specific. The analysis was carried out in MRM (Multiple Reaction Monitoring) mode.
To evaluate the specificity, inject the matrix blank and observed that at the retention of the
interest of analyte not contain any peak.
S.No. Analyte RT Observation

1 Profenofos
3 BHC‐beta
5 BHC‐delta
7 Dimethoate
8 Aldrin
9 Paraoxon
10 Malathion
11 Chlorpyrifos
13 Chlorfenvinphos
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
25 Ethion
27 DDT‐p, p'
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28 Endrin aldehyde
29 Endrin ketone
30 Fenpropathrin
33 Permethrin I
34 Cyfluthrin
35 Cypermethrin
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin
Table 5 Specificity of the analytes
5.3 LINEARITY & RANGE
Calibration standard curve is the relationship between instrument response and known
concentration of the analyte. A calibration curve should be generated for each analyte in the
sample. A sufficient number of standards should be used to adequately define the relationship
between concentration and response. The number of standards used in constructing a
calibration curve will be a function of the anticipated range of analytical value and the nature of
analyte / response relationship. Concentration of the standards should be chosen on the basis of
the concentration range expected in a particular commodity.
Calibration curve w a s c o n s t r u c t e d with six different concentration range from 0.1 to 10.0
µg/Kg. Construct a calibration curve between the areas vs. concentrations of the anlaytes.
Correlation Coefficient
S.No. Analyte
(R2)
1 Profenofos
2 hexachloride)
3 BHC‐beta
BHC‐gamma (Lindane, gamma
4 HCH)
5 BHC‐delta
Chlorpyrifos‐methyl
6
7 Dimethoate
8 Aldrin
9 Paraoxon

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10 Malathion
11 Chlorpyrifos
Heptachlor exo‐epoxide (isomer
12 B)
13 Chlorfenvinphos
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
25 Ethion
27 DDT‐p, p'
28 Endrin aldehyde
29 Endrin ketone
30 Fenpropathrin
33 Permethrin I
34 Cyfluthrin
35 Cypermethrin
Esfenvalerate (Fenvalerate A‐
36 alpha)
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin
Table 6: Linearity of Analytes

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Working Range:
The range of an analytical procedure is its ability to obtain test results which are directly proportional to
the concentration (amount) of analyte in the sample.
The specified range is normally derived from linearity studies & depends on the intended application of
the procedure. It is established by confirming that the analytical procedure provides an acceptable
degree of linearity, accuracy & precision when applied to samples containing an amount of analyte
within or at the extremes of the specified range of the analytical procedure.
On the basis of the Recovery and repeatability exercise the range of below mentioned pesticides in
sample are given below:
S. No Name of analyte Range (ug/Kg)
1 Profenofos
3 BHC‐beta
5 BHC‐delta
7 Dimethoate
8 Aldrin
9 Paraoxon
10 Malathion
11 Chlorpyrifos
13 Chlorfenvinphos
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
25 Ethion

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27 DDT‐p, p'
28 Endrin aldehyde
29 Endrin ketone
30 Fenpropathrin
33 Permethrin I
34 Cyfluthrin
35 Cypermethrin
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin
Table 7: Range of each analyte
5.4 LIMIT OF DETECTION
The minimum concentration of the analyte that can be detected, through not quantifiable.
5.5 LIMIT OF QUANTIFICATION
The minimum concentration of the analytes that can be quantified with acceptable accuracy and
precision. To evaluate this, Samples were spiked at concentration mentioned below in table and
prepared as clause 3.3. % Recovery was calculated as:
Table 9: Limit of Quantification of Analyte.

5.6 RECOVERY
Recovery of an analyte is obtained from the known concentration added to the sample blank and
recovered from the sample blank.
Acceptance criteria: As per Codex the recoveries are within the limits (80-120%).
(Spike sample conc. – Blank sample conc.)
% Recovery = --------------------------------------------------------------- X 100
Spiking Conc.

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Recovery at LOQ
Spiked %
S.No. Analyte Rep_1 Rep_2 Rep_3 Rep_4 Rep_5 Rep_6 Average
Conc. Recovery
1 Profenofos
3 BHC‐beta
4 HCH)
5 BHC‐delta
7 Dimethoate
8 Aldrin
9 Paraoxon
10 Malathion
11 Chlorpyrifos
13 Chlorfenvinphos
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
25 Ethion
27 DDT‐p, p'
28 Endrin aldehyde
29 Endrin ketone
30 Fenpropathrin
33 Permethrin I
34 Cyfluthrin
35 Cypermethrin

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36 alpha)
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin
Recovery at 5XLOQ
Spiked %
S.No. Average
Analyte Rep_1 Rep_2 Rep_3 Rep_4 Rep_5 Rep_6 Conc. Recovery
1 Profenofos
3 BHC‐beta
4 HCH)
5 BHC‐delta
7 Dimethoate
8 Aldrin
9 Paraoxon
10 Malathion
11 Chlorpyrifos
13 Chlorfenvinphos
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
25 Ethion
27 DDT‐p, p'
28 Endrin aldehyde

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29 Endrin ketone
30 Fenpropathrin
33 Permethrin I
34 Cyfluthrin
35 Cypermethrin
36 alpha)
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin
Table 10 : Recovery
As per Validation Protocol the recoveries are within the limits (60-120%).
5.7 REPETABILITY /PRECISION:

Repeatability means Precision under repeatability conditions, i.e. conditions where independent
test results are obtained with the same method on identical test items in the same laboratory by the
same operator using the same equipment within short intervals of time.
To evaluate the repeatability, take blank samples fortified with pesticide stds at 5.0 µg/Kg, & 50.0
µg/Kg .At each level Analysis performed with 7 replicates as per the above mentioned procedure.
Calculate the % RSD and Mean concentrations of analytes.
Repetability at LOQ
STD %
S.No. Analyte Rep_1 Rep_2 Rep_3 Rep_4 Rep_5 Rep_6 Average
Dev RSD
1 Profenofos
2 hexachloride)
3 BHC‐beta
4 HCH)
5 BHC‐delta
7 Dimethoate
8 Aldrin
9 Paraoxon
10 Malathion

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GCMSMS
11 Chlorpyrifos
12 B)
13 Chlorfenvinphos
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
25 Ethion
27 DDT‐p, p'
28 Endrin aldehyde
29 Endrin ketone
30 Fenpropathrin
33 Permethrin I
34 Cyfluthrin
35 Cypermethrin
36 alpha)
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin

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GCMSMS
Repetability at LOQ X 5
S.No. Analyte Rep_1 Rep_2 Rep_3 Rep_4 Rep_5 Rep_6 Average STD Dev % RSD
1 Profenofos
2 hexachloride)
3 BHC‐beta
BHC‐gamma (Lindane,
4 gamma HCH)
5 BHC‐delta
7 Dimethoate
8 Aldrin
9 Paraoxon
10 Malathion
11 Chlorpyrifos
Heptachlor exo‐epoxide
12 (isomer B)
13 Chlorfenvinphos
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
25 Ethion
27 DDT‐p, p'
28 Endrin aldehyde
29 Endrin ketone
30 Fenpropathrin
33 Permethrin I
34 Cyfluthrin

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GCMSMS
35 Cypermethrin
Esfenvalerate (Fenvalerate
36 A‐alpha)
Fenvalerate II {CAS # 51630‐
37 58‐1}
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin
Table 11: Repetability
Acceptance Criteria: The relative standard deviation for the spiked concentration of 7 replicate
shall not be more than 32.0 % for pesticides.
6.0 Quality Control
6.1. For the linearity as per Codex Alimentarius CAC/GL 40 -1993 Rev 1-2003, For linear
calibration Regression coefficient for analytical standard solutions(r) > 0.99 and for polynomial
function (r) > 0.98.
6.2 As per Codex Alimentary CAC/GL 40 -1993 Rev 1-2003 the below % Recovery limits and
Precision are acceptable with in the laboratory criteria for method validation for pesticide
residues.
Precision Range of mean

S.No Concentration
(Repeatability) % recovery
1 < 1 µg/Kg 36 50 – 120
2 > 1µg/Kg < 0.01 mg/Kg 32 60 -120
3 >0.01 mg/Kg < 1 mg/Kg 22 70-120
4 > 0.1 mg/Kg < 1 mg/Kg 18 70-110
5 > 1 mg/Kg 14 70-110
Table 12: Limits of Recovery and Precision
7.0 Conclusion
The above stated method validation report for Pesticide Residues in Breast Milk by GCMSMS is
validated and the report conclusions are below:

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GCMSMS
S.no Comp Name Linearity % Rec LOQ

1 Profenofos
2 hexachloride)
3 BHC‐beta
4 HCH)
5 BHC‐delta
7 Dimethoate
8 Aldrin
9 Paraoxon
10 Malathion
11 Chlorpyrifos
12 B)
13 Chlorfenvinphos
17 DDE‐o, p'
18 DDD‐o, p'
19 DDD‐p, p'
20 Dieldrin
21 Endrin
22 DDT‐o, p'
23 DDE‐p, p'
25 Ethion
27 DDT‐p, p'
28 Endrin aldehyde
29 Endrin ketone
30 Fenpropathrin
33 Permethrin I
34 Cyfluthrin
35 Cypermethrin

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GCMSMS
36 alpha)
38 Fenvalerate I
39 Fluvalinate I
40 Deltamethrin
Table 13: Conclusions

8.0 References
8.1 QuEChERS method and AOAC 2007.01,

8.2 Michael S. Young, Kim Tran and Dimple Shah Waters Corporation
Technology Drive, Milford, MA 01757.
8.3 DGHS Lab Manual.
8.4 Journal of Agricultural and food chemistry 2007,55,1103 –1108.
9.0 End of the Validation Report.

Page No 26 of 26

Pesticides in Breast Milk by GCMSMS

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Sophisticated Industrial Material Analytic Labs (P) Ltd DOC NO.

Title : VALIDATION REPORT FOR

VALIDATION REPORT FOR PESTICIDE RESIDUES

IN BREAST MILK BY GCMSMS

Prepared By: Checked By Reviewed & Approved By: Issued By

Title : VALIDATION REPORT FOR

CONTENT OF THIS DOCUMENT

S.No Heading Page No

Prepared By: Checked By Reviewed & Approved By: Issued By

Title : VALIDATION REPORT FOR

Name Signature Date

Name Signature Date

Name Signature Date

Prepared By: Checked By Reviewed & Approved By: Issued By

Title : VALIDATION REPORT FOR

Table 1 Preparation of working standard for calibration curve.

Table 2 Preparation of calibration curve standards

Table 3 GCMSMS Transition.

Table 4 System suitability (RT & Area) of Analytes.

Table 5 Specificity & Selectivity of the Analytes..

Table 6 Linearity of Analytes.

Table 7 Range of Analytes

Table 8 Limit of Detection of analyte

Table 9 Limit of Quantification of analyte.

Table 12 Limits of Recovery and Precision.

Prepared By: Checked By Reviewed & Approved By: Issued By

Title : VALIDATION REPORT FOR

Annexure 1 System suitability

Prepared By: Checked By Reviewed & Approved By: Issued By

Title : VALIDATION REPORT FOR

2.0 Scope and Application:

The following performance parameters were assessed during the validation:

2.1 System Suitability.

3.1.1 Equipments and Glassware's

3.1.1.1 GCMSMS: Agilent 7890B.

Prepared By: Checked By Reviewed & Approved By: Issued By

Title : VALIDATION REPORT FOR

3.1.2 Reagents and Solvents

3.1.2.1 Acetonitrile HPLC Grade.

3.2 Preparation of Reagents

3.2.1 Preparation of Stock Standards: For Pesticide Residues: Transfer 10 mg of standard

3.2.1.1 Preparation of pesticide standard mixture solution: Pipette out 100 µL of

3.2.2 Preparation of Calibration Curve

Working Volume of Final

100.0 100 900 1.000 10.0 WS3

10.0 100 900 1.000 1.0 WS2

1.0 100 900 1.000 0.1 WS1

Table 1: Preparation of working standard dilution for calibration curve:

Table 2: Preparation of standards for calibration curve:

Prepared By: Checked By Reviewed & Approved By: Issued By

Title : VALIDATION REPORT FOR

3.3 Sample Preparation

3.3.1 Take 2 gms sample in Tarson tube.

3.4 Instrumental Conditions

Auto Sampler Program

Prepared By: Checked By Reviewed & Approved By: Issued By

Title : VALIDATION REPORT FOR

50 150 0.0 2.6

Mass Spectrometer Agilent 7000C Triple Quad

Source Temperature 300°C

Run Time > 1.43 * RT of latest eluting analyte.

Title : VALIDATION REPORT FOR