CDNA LABS Pvt Ltd Indore.Pesticides Samples & Standards Preparation.
Bhoopendra Singh
7248145343.
Technical Manager
CDNA LABS Pvt Ltd. Indore.
52, B.R.G Industry Malikhedi, India, Indore Madhya Pradesh, 452016, India
7.1 Pesticide for Standards Preparation
7.1.1
PREPARATION
OF PESTICIDES STANDARD STOCK SOLUTION (1000 ppm):
Weigh
equivalent to 10 mg of standard into a 10 mL calibrated volumetric flask and
dissolve in Methanol for LCMS/MS and Ethyl acetate for GC-MS/MS. Make up the
volume with the same. Label the name of the standard, concentration and
preparation and expiry date and store the solution at -21°C ±2°C or (2-8) °C for 12 months. (Note: apply purity and salt
correction to the quantity weighed for stock preparation. Apply the calculated
concentration for further dilutions).
Validity: 1 year
7.1.2
PRIMARY
WORKING STANDARDS PESTICIDES MIX (10 ppm):
Weigh equivalent to 100µl of each standard solution
into a 10mL calibrated volumetric flask
with methanol for LCMSMS & ethyl acetate
for GCMSMS Compounds. Make up the volume
with the same. Label the name of the
standard, concentration and preparation and expiry
date and store the solution at -21°C ±2°C or
(2-8) °C for 6 months.
Validity: 6 months
7.1.3
Diluents
for Working Standard
Ethyl Acetate, n-hexane, and methanol
7.1.4
PRIMARY
WORKING STANDARD (1000 ppb):
Weigh
equivalent to 1000µl of primary standard solution into a 10mL calibrated
volumetric flask with methanol for LCMSMS & Ethyl Acetate and n-hexane for GCMSMS Compounds. Make up the
volume with the same. Label the name of the standard, concentration and
preparation and expiry date and store the solution at -21°C ±2°C or (2-8) °C for
3 months.
Validity: 3 months
TABLE 3: PRIMARY WORKING STANDARD – NEAT FRESH
PREPARATION
|
Primary Working
Standard(ppm) |
Volume of Primary
Working Standard (mL) |
Volume of Diluents (mL) |
Final Volume (mL) |
Final conc. (ppb) |
Solvent for Dilution |
|
1.0 |
1 |
9 |
10 |
100 |
n-Hexane, Ethyl Acetate/ methanol |
TABLE 4: CALIBRATION STANDARDS – NEAT
FRESH PREPARATION
|
Primary Working Standard(ppb) |
Volume of Primary Working Standard (µl) |
Volume of Diluent (µl) |
Final Volume (mL) |
Final Conc. (ppb) |
Solvent for dilution |
|
1000 |
100 |
900 |
1.0 |
100 |
n-Hexane, Ethyl Acetate/ methanol |
|
1000 |
50 |
950 |
1.0 |
50 |
|
|
1000 |
25 |
975 |
1.0 |
25 |
|
|
100 |
100 |
900 |
1.0 |
10 |
|
|
100 |
50 |
950 |
1.0 |
5 |
Validity: Fresh
TABLE 5: CALIBRATION STANDARDS -
MATRIX MATCHED - FRESH PREPARATION
|
Primary Working Standard (ppb) |
Volume of Primary Working Standard (µl) |
Volume of Matrix (µl) |
Volume of Diluent (µl) |
Final Volume (mL) |
Final Conc. (ppb) |
Solvent for Dilution |
|
1000 |
100 |
800 |
100 |
1.0 |
100 |
|
|
1000 |
50 |
800 |
150 |
1.0 |
50 |
Matrix & n-Hexane,
Ethyl Acetate/ methanol |
|
1000 |
25 |
800 |
175 |
1.0 |
25 |
|
|
100 |
100 |
800 |
100 |
1.0 |
10 |
|
|
100 |
50 |
800 |
150 |
1.0 |
5 |
Note: Working standard should be prepared freshly on
every day of analysis.
TABLE 6: CALIBRATION STANDARDS -
MATRIX BASED - FRESH PREPARATION
|
Primary Working Standard (ppb) |
Volume of Primary Working Standard (µl) |
Volume of Matrix (gm) |
Final Conc. (ppb) |
Solvent for Dilution |
|
1000 |
500 |
5.0 |
100 |
|
|
1000 |
250 |
5.0 |
50 |
Matrix & n-Hexane/
Methanol |
|
1000 |
125 |
5.0 |
25 |
|
|
1000 |
50 |
5.0 |
10 |
|
|
1000 |
250 |
5.0 |
5 |
Note: Working standard should be prepared freshly on
every day of analysis.
7.2 SAMPLE
PREPARATION:
Keep the
sample in airtight container and refrigerate the sample for cooling. Blend the
sample in cold condition and avoid
heating.
7.2.1
EXTRACTION
PROCEDURE (CEREAL AND CEREAL PRODUCTS INCLUDING RICE)
7.2.1.1
Weigh 5.0 g (±0.5 g) of ground,
powder sample into a 50 ml polypropylene centrifuge tube.
7.2.1.2
Add 10 ml of cold water and shake
for 30 seconds.
7.2.1.3
Add 10 ml acetonitrile and shake
vigorously by hand for 1 minute.
7.2.1.4
Add the mixture is prepared by
taking 4 g Magnesium sulphate Anhydrous (Magnesium sulphate Anhydrous (MgSO4)),
1 g Sodium chloride(NaCl), 0.5 g Trisodium citrate dihydrate(C6H5Na3O7·2H2O) and 0.25 g Disodium hydrogen
citrate sesquihydrate (C6H6Na2O7·1.5H2O).
7.2.1.5
Shake for few seconds after each
addition to prevent lumps.
7.2.1.6
Shake vigorously for 1 min, Centrifuge
for 10 min at 4000 rpm with 10⁰c temperature.
7.2.1.7
Transfer 6 ml of cold extract
into a 15 ml centrifuge tube containing 150 mg of Primary secondary Amine (PSA),
900 mg Magnesium sulphate and 150 mg of C18. Close the tube and shake
vigorously for 30 second.
7.2.1.8
Centrifuge at 4000 rpm for 5
minutes.
LC-MS/MS
7.2.1.9
Transfer 2.0 ml of the extract
from step 7.2.1.7 and concentrate in a nitrogen concentrator at
35ºC±5ºC. Remove the residual solvent under mild stream of nitrogen and
reconstitute to 1 ml Methanol : water (1:1) after adding 15 µl of 5% v/v Formic
acid in acetonitrile. Vortex for a minute and filter through 0.22-micron PVDF
membrane filter.
7.2.1.10
Take 2 ml of the extract from
step 7.2.1.7 concentrate in a nitrogen concentrator at 35ºC±5ºC. Remove the residual solvent under mild
stream of nitrogen and re-constitute to 1 ml with Ethyl Acetate. Filter through
0.22 µm PVDF membrane filter and inject into GC-MS/MS.
7.2.2
EXTRACTION PROCEDURE (OIL, FATS
AND OIL SEEDS PRODUCTS)
7.2.2.1
Weigh 2.5 g (±0.5 g) of sample
into a 50 ml polypropylene centrifuge tube.
7.2.2.2
Add 10 ml acetonitrile and shake
vigorously by hand for 1 minute.
7.2.2.3
Shake vigorously for 1 min,
Centrifuge for 10 min at 4000 rpm with 10⁰c temperature.
7.2.2.4
Centrifuge at 5000 rpm for 10
minutes.
7.2.2.5
Transfer 6 ml of extract into a
15 ml centrifuge tube containing 150 mg of PSA, 900 mg Magnesium sulphate, 150
mg of C18 and 50mg GCB. Close the tube and shake vigorously for 30 second.
7.2.2.6
Centrifuge at 4000 rpm for 5
minutes and separate upper layer.
LC-MS/MS
7.2.2.7
Transfer 4.0 ml of the extract
from step 7.2.2.6 and concentrate in a nitrogen concentrator at
35ºC±5ºC. Remove the residual solvent
under mild stream of nitrogen and reconstitute to 1 ml Methanol after adding 15
µl of 5% v/v Formic acid in acetonitrile. Vortex for a minute and filter
through 0.22-micron PVDF membrane filter.
GC-MS/MS
7.2.2.8
Take 2 ml of the extract from
step 7.2.2.6 concentrate in a nitrogen concentrator at 35ºC±5ºC. Remove
the residual solvent under mild stream of nitrogen and re-constitute to 0.5 ml
with n-Hexane. Filter through 0.22 µm PVDF membrane filter and inject into
GC-MS/MS.
7.2.3
EXTRACTION PROCEDURE (FRUITS, VEGETABLE
& THEIR PRODUCTS)
7.2.3.1
Accurately weigh 10±0.1 gram of
homogenized sample into a 50ml polypropylene. Add 10 ml ethyl acetate and vortex for 1 minute.
7.2.3.2
Add
10.0 g of anhydrous sodium sulphate and vortex for 1 minute.
7.2.3.3
Centrifuge at 4000 rpm for 10 minutes with 10⁰c temperature.
GC-MS/MS
7.2.3.4
Transfer 2.0ml of extract into a
clean 2.0ml micro tube containing 120mg Sodium sulfate anhydrous, 50 mg PSA, 10
mg GCB vortex for 30 seconds and centrifuge at 4000 rpm for 5 minutes.
7.2.3.5
Analyze the solution 0.22µm PVDF
membrane filter and subject to GCMS/MS.
LC-MS/MS
7.2.3.6
Transfer 5.0ml of aliquot from
the supernatant to a 15.0ml of polypropylene tube containing 200mg Sodium sulfate anhydrous, 25
mg of PSA,20 mg GCB Vortex for 2 mins and centrifuge at 5000 rpm for 10
minutes.
7.2.3.7
Transfer 2.0 ml of supernatant into
nitrogen evaporator tube add 200µL of 10% DEG(Diethyl glycol) in methanol and vortex.
7.2.3.8
Concentrate the extract under
gentle stream of nitrogen at 35°C±2°C.
7.2.3.9
Reconstitute the residues with 1 ml methanol
and 1 ml of water containing 0.1% acetic acid (Do not use these solvents
initially, first add methanol, vortex for 30 seconds and then 0.1% acetic acid
in water) Sonicate for 1 minute and
vortex and filter through 0.22um PVDF. Perform a reagent blank without sample
and follow the above procedure.
7.2.4
EXTRACTION PROCEDURE (AYURVEDIC, HERBS,
SPICE AND CONDIMENTAL PRODUCTS)
7.2.4.1
Weigh 2.5 g (±0.5 g) of sample
into a 50 ml polypropylene centrifuge tube.
7.2.4.2
Add 10 ml of ultrapure water and
shake for 30 seconds.
7.2.4.3
Add 10 ml Acetonitrile (1% Acetic
acid )and shake vigorously by hand for 1 minute.
7.2.4.4
Add the mixture is prepared by
taking 4 g Magnesium sulphate Anhydrous (MgSO4), 1 g NaCl, 1 g Trisodium
citrate dihydrate and 0.5 g Disodium hydrogen citrate sesquihydrate.
7.2.4.5
Shake vigorously for 1 min,
Centrifuge for 10 min at 5000 rpm with 10⁰c
temperature.
7.2.4.6
Transfer 6 ml of extract into a
15 ml centrifuge tube containing 150 mg
of PSA, 900 mg Magnesium sulfate, 50 mg
of GCB, 150 mg of C18. Close the tube and shake vigorously for 30 seconds.
7.2.4.7
Shake vigorously for 2 min.
7.2.4.8
Centrifuge at 5000 rpm for 10
minutes and separate layer.
LC-MS/MS
7.2.4.9
Transfer 4.0 ml of the extract
from step 7.2.4.8 and concentrate in a nitrogen concentrator at
35ºC±5ºC. Remove the residual solvent
under mild stream of nitrogen and reconstitute to 1 ml Methanol: Water (1:1).
Vortex for a minute and filter through 0.22-micron PVDF membrane filter.
GC-MS/MS
7.2.4.10
Take 2.0 ml of the extract from
step 7.2.4.8 and concentrate in a nitrogen concentrator at 35ºC±5ºC.
Remove the residual solvent under mild stream of nitrogen and re-constitute to 0.5
ml with Ethyl Acetate. Filter through 0.22 µm PVDF membrane filter and inject
into GC-MS/MS.
7.2.5
EXTRACTION PROCEDURE (TEA , COFFEE
& OTHER BEVERAGES)
7.2.5.1
Weigh 2.5 g (±0.5 g) of sample
into a 50 ml polypropylene centrifuge tube.
7.2.5.2
Add 10 ml of ultrapure water and
shake for 30 seconds.
7.2.5.3
Add 10 ml acetonitrile(1% Acetic
acid) and shake vigorously by hand for 1 minute.
7.2.5.4
Transfer 6 ml of extract into a
15 ml centrifuge tube containing 150 mg
of PSA, 900 mg Magnesium sulfate, 50 mg
of GCB, 150 mg of C18. Close the tube and shake vigorously for 30 seconds.
7.2.5.5
Shake vigorously for 1 min,
Centrifuge for 10 min at 4000 rpm with 10⁰c temperature.
7.2.5.6
Centrifuge at 5000 rpm for 10
minutes.
7.2.5.7
Transfer 6 ml of cold extract
into a 15 ml centrifuge tube containing 150 mg of PSA, 900 mg Magnesium
sulphate, 150 mg of C18 and 50mg GCB. Close the tube and shake vigorously for
30 second.
7.2.5.8
Centrifuge at 5000 rpm for 5 minutes and
separate upper layer.
LC-MS/MS
7.2.5.9
Transfer 4.0 ml of the extract
from step 7.2.5.8 and concentrate
in a nitrogen concentrator at 35ºC±5ºC.
Remove the residual solvent under mild stream of nitrogen and
reconstitute to 1 ml Methanol: Water (1:1). Vortex for a minute and filter
through 0.22-micron PVDF membrane filter.
GC-MS/MS
7.2.5.10
Take 2.0 ml of the extract from
step 7.2.5.8 and concentrate in a
nitrogen concentrator at 35ºC±5ºC. Remove the residual solvent under mild
stream of nitrogen and re-constitute to 0.5 ml with Ethyl Acetate. Filter
through 0.22 µm PVDF membrane filter and inject into GC-MS/MS.
7.2.6
EXTRACTION PROCEDURE (HONEY, SUGAR
AND SUGAR PRODUCTS)
7.2.6.1
Weigh 2.0 g (±0.5 g) of ground, powder sample
into a 50 ml polypropylene centrifuge tube
7.2.6.2
Add 10 ml of ultrapure water and
shake for 30 seconds.
7.2.6.3
Add 10 ml 1% Acetic Acid and shake
vigorously by hand for 1 minute.
7.2.6.4
Shake for few seconds after each
addition to prevent lumps.
7.2.6.5
Shake vigorously for 1 min, Centrifuge for 10
min at 4000 rpm.
7.2.6.6
Transfer 6 ml of extract into a
15 ml centrifuge tube containing 75 mg
of PSA, 750 mg Magnesium sulfate, 100 mg of GCB,75 mg C18. Close the tube and
shake vigorously for 30 seconds.
7.2.6.7
Centrifuge at 4000 rpm for 5
minutes & separate upper layer.
LC-MS/MS
7.2.6.8
Transfer 2.0 ml of the extract from step 7.2.6.7
and concentrate in a nitrogen concentrator at
35ºC±5ºC. Remove the residual solvent under mild stream of
nitrogen and reconstitute to 0.4 ml Acetonitrile after adding 15 µl of 5% v/v
Formic acid in acetonitrile. Vortex for a minute and filter through 0.22-micron PVDF membrane filter.
7.2.6.9
For matrix interference, dilute
0.2 ml of extract from step 7.2.6.8 with 0.2 ml of acetonitrile and
inject into LC- MSMS. Apply the
dilution factor into calculation.
GC-MS/MS
7.2.6.10
Take 2 ml of the extract from
step 6.3.9.6 concentrate in a nitrogen concentrator at 35ºC±5ºC. Remove the residual solvent under mild stream
of nitrogen and re-constitute to 0.4 ml with Ethyl Acetate.
7.2.6.11
Filter through 0.22 µm PVDF
membrane filter and inject into GC-MS/MS.
7.2.7
EXTRACTION PROCEDURE (BAKERY
& CONFECTIONARY)
7.2.7.1
Weigh 5.0 g (±0.5 g) of ground,
powder sample into a 50 ml polypropylene centrifuge tube.
7.2.7.2
Add 10 ml of Ultra-pure water and
shake for 30 seconds.
7.2.7.3
Add 10 ml acetonitrile and shake
vigorously by hand for 1 minute.
7.2.7.4
Add the mixture is prepared by
taking 4 g Magnesium sulphate Anhydrous (MgSO4), 1 g NaCl, 1 g Trisodium
citrate dihydrate and 0.5 g Disodium hydrogen citrate sesquihydrate.
7.2.7.5
Shake for few seconds after each
addition to prevent lumps.
7.2.7.6
Shake vigorously for 1 min,
Centrifuge for 10 min at 4000 rpm with 10⁰c temperature.
7.2.7.7
Transfer 6 ml of cold extract
into a 15 ml centrifuge tube containing 150 mg of PSA, 900 mg Magnesium
sulphate and 150 mg of C18. Close the tube and shake vigorously for 30 second.
7.2.7.8
Centrifuge at 4000 rpm for 5
minutes & separate upper layer.
LC-MS/MS
7.2.7.9
Transfer 2.0 ml of the extract
from step 7.2.7.8 and concentrate in a nitrogen concentrator at
35ºC±5ºC. Remove the residual solvent under mild stream of nitrogen and
reconstitute to 1 ml Methanol : water (1:1). Vortex for a minute and filter
through 0.22-micron PVDF membrane filter.
GC-MS/MS
7.2.7.10
Take 2 ml of the extract from
step 7.2.7.8 concentrate in a nitrogen concentrator at 35ºC±5ºC. Remove
the residual solvent under mild stream of nitrogen and re-constitute to 1 ml
with Ethyl Acetate. Filter through 0.22 µm PVDF membrane filter and inject into
GC-MS/MS.
7.2.8
EXTRACTION PROCEDURE (OTHER FOOD
PRODUCTS)
7.2.8.1
Weigh 5.0 g (±0.5 g) of ground,
powder sample into a 50 ml polypropylene centrifuge tube.
7.2.8.2
Add 10 ml of ultrapure water and
shake for 30 seconds.
7.2.8.3
Add 10 ml acetonitrile and shake
vigorously by hand for 1 minute.
7.2.8.4
Add the mixture is prepared by
taking 4 g Magnesium sulphate Anhydrous (MgSO4), 1 g NaCl, 1 g Trisodium
citrate dihydrate and 0.5 g Disodium hydrogen citrate sesquihydrate.
7.2.8.5
Shake for few seconds after each
addition to prevent lumps.
7.2.8.6
Shake vigorously for 1 min,
Centrifuge for 10 min at 4000 rpm.
7.2.8.7
Transfer 6 ml of extract into a
15 ml centrifuge tube containing 150 mg of PSA, 900 mg Magnesium sulfate, 150
mg of C18. Close the tube and shake vigorously for 30 seconds.
7.2.8.8
Centrifuge at 4000 rpm for 5
minutes & separate upper layer.
LC-MS/MS
7.2.8.9
Transfer 2.0 ml of the extract
from step 7.2.8.8 and concentrate in a nitrogen concentrator at
35ºC±5ºC. Remove the residual solvent under mild stream of nitrogen and
reconstitute to 1 ml Methanol: water (1:1) after adding 15 µl of 5% v/v Formic
acid in acetonitrile. Vortex for a minute and filter through 0.22-micron PVDF
membrane filter.
7.2.8.10
For matrix interference, dilute
0.5ml of extract from step-7.2.8.8 with 0.5ml of acetonitrile and inject
into LC-MSMS. Apply the dilution factor into calculation.
GC-MS/MS
7.2.8.11
Take 2 ml of the extract from
step-7.2.8.8 concentrate in a nitrogen concentrator at 35ºC±5ºC. Remove
the residual solvent under mild stream of nitrogen and re-constitute to 1 ml
with Ethyl Acetate. Filter through 0.22 µm PVDF membrane filter and inject into
GC-MS/MS.
7.3 INSTRUMENT CONDITION:
TABLE 7: EQUIPMENT CONDITIONS FOR
LC
|
Equipment |
Liquid Chromatography-Tandem Mass
Spectrometer (LCMSMS) |
|||
|
Make & Mode |
Agilent-1260 Infinity II; 6470 LC/TQ |
|||
|
Column |
Infinity Lab Poroshell 3.0*150mm*2.7 micron, EC-C18 or Zorbax RRHD-C18,3*100mm,1.8 micron |
|||
|
Liquid
Chromatography-Tandem Mass Spectrometer (LCMSMS) CONDITIONS |
||||
|
Flow rate |
0.4 mL/min |
|||
|
Injection volume |
10.0 µl |
|||
|
Purge flow |
5.0 mL/min |
|||
|
Mobile Phase A |
5mM Ammonium Formate +0.01% Formic Acid in water |
|||
|
Mobile Phase B |
5mM Ammonium Formate + 0.01% Formic Acid in methanol |
|||
|
Column Oven temperature |
40°C |
|||
|
Sampler Oven temperature |
8°C |
|||
|
Run Time |
23 minutes |
|||
|
Mode |
Gradient |
|||
|
|
Time (minute) |
A % |
B% |
|
|
|
0 |
95.00 |
5.00 |
|
|
|
3.0 |
70.00 |
30.00 |
|
|
|
16 |
0.00 |
100.00 |
|
|
|
20.00 |
95.00 |
5.00 |
|
|
|
21.00 |
95.00 |
5.00 |
|
|
Post run time |
1 minutes |
|||
|
Mode |
ESI Positive, Negative |
|||
|
Gas Temperature ⁰C |
200s |
|||
|
Gas flow (l/min) |
9 |
|||
|
Nebulizer (psi) |
35 |
|||
*Gradient Programming changes as per
matrix
TABLE 8: EQUIPMENT CONDITIONS FOR
GC-MSMS
|
Equipment |
Gas
Chromatograph- Tandem Mass Spectrometer (GC-MS/MS) |
||
|
Make & Mode |
Agilent-7890; 7000
GC/TQ |
||
|
Column |
HP5-MS UI-(30 m X
0.25 mm x 0.25 µm) |
||
|
Injection Volume |
1 µl |
||
|
Oven Program |
Temp Rise/ min |
Temp °C |
Hold Time |
|
Initial |
|
60 |
2 min |
|
Ramp 1 |
25 |
150 |
0 min |
|
Ramp 2 |
3 |
200 |
0 min |
|
Ramp 3 |
8 |
280 |
10 min |
|
Maximum Temperature
(°C) |
325°C |
||
|
Run time |
42.267 minutes |
||
|
Post run |
310 (°C) |
||
|
MSMS Conditions |
|
||
|
Acquisition Mode |
MRM |
||
|
Injection Mode |
Solvent Vent |
||
|
Liner & Liner
volume |
Pulse Split less,
900 µl |
||
|
Total Flow |
54.616 ml/min |
||
|
Ion Source |
Electron ionization |
||
|
Purge Flow |
3 ml/min |
||
|
Equipment |
Gas Chromatograph- Tandem Mass Spectrometer
(GC-MS/MS) |
||
|
Inlet flow (column
1) |
~ 1 mL/min, 2.0 psi
(during backflush) |
||
|
PUU flow (column 2) |
column 1 flow + 0.2
mL/min |
||
|
Carrier Gas |
Helium |
||
|
Collision Gas |
Nitrogen |
||
|
Quadrupole
temperature(°C) |
Q1 and Q2 = 180 |
||
|
Initial
Temperature(°C) |
60 |
||
|
Transfer line Temperature(°C) |
280 |
||
|
Ion Source
Temperature (°C) |
300 |
||
TABLE 9: MRM TABLE FOR LC-MS/MS AND
GC-MS/MS PARAMETER
Refer to
ANNEXURE-II
7.4 BATCH
SEQUENCE AND QUALITY CONTROL ACCEPTANCE CRITERIA
|
S.NO. |
QUALITY CONTROL POINTS |
CRITERIA |
RUN |
|
1 |
Solvent
Blank |
Analyte
Free |
1 |
|
2 |
Calibration
Standards- 5 levels |
R2
³ 0.9900 |
Each
1 |
|
3 |
Reagent
Blank |
Analyte
Free |
1 |
|
4 |
Samples
1 to 10 |
- |
Each
1 |
|
5 |
QC
Recovery |
70%
to 120% Refer
Validation data (or) as prescribed in the standard |
1 |
|
6 |
Sample
11 to 20 |
- |
Each
1 |
|
7 |
QC
Recovery |
70%
to 120% Refer
Validation data (or) as prescribed in the standard |
1 |
|
8 |
Calibration
Check Standard @ LOQ |
80%
or 120% (or) compound specific as per validation data |
1 |
7.5 NOTE
7.5.1
Perform Reagent Blank at
the time of material inward to verify its suitability for testing.
7.5.2
Organize the batch
sequence based on product nature.
7.5.3
Batch runs can be altered
based on the response of calibration standard response at LOQ level.
7.5.4
Minimum 3 calibration
standards including zero level with linear response is required for
quantification and calibration curve covers the concentration of analytes in
sample.
7.5.5
Suitable equipment
conditions can be optimized for the brands and models and after the preventive maintenance.
7.6 CALCULATIONS & DATA PROCESSING
7.6.1 CALCULATION:
Conc. mg/kg= =
Results with Recovery Correction: Calculated
concentration X Recovery factor
(Note: Report the values in mg/Kg in
two decimals: 0.00)
Were,
DF: Dilution Factor
V: Total Volume of Extract
W: Sample Weight
Results with Recovery Correction: Calculated
concentration X Recovery factor
(Note: Report the values in mg/Kg in
two decimals: 0.00, 00.00, 000.0)
7.6.2 FORMULAE:
REFERENCE MATERIAL – SALT & PURITY CORRECTION FORMULA
Table-10 SALT & PURITY
CORRECTION FORMULA
|
(Molecular weight of Compound with salt –
Salt Mass) X = ------------------------------------------------------------------------x
Reference Material Weight(mg) x Purity
Molecular weight of
Compound with salt
|
|
Purity
= x/100 where x = percentage of compound |
|
Final concentration in solution Y
(mg/L) = X *1000/V where V is the makeup volume. |
7.6.3
DATA
PROCESSING:
Acquired
chromatograms using the computer-based Agilent Mass Hunter software, Process the data
by peak area method. The concentration of the unknown is calculated from the
following equation using regression analysis of calibration standard.
y = mx + c
Where, x = concentration of analyte
m = slope of calibration curve
y = peak area of analyte
c = y-axis intercept of the calibration
curve
8.0 QUALITY CONTROL
& ACCEPTANCE
CRITERIA
Add 0.50 ml of 100.0 mg/L Standard Mixture to
5 g of sample and follow the procedure as specified in cl. 7.2.1
or
Suitable
concentration at or around LOQ level can be taken for spike and recovery for
the analytes
having LOQ > 0.005 mg/kg
or 0.010 mg/kg
Calculate the recovery and
compare against the standard method validation study.
Table-11: ACCEPTANCE
CRITERIA
|
RT with respect to
standard |
± 0.20min (generalized) |
CONFIRMATION &
REPORTING OF RESULTS
8.2.1 Repeat the analysis of the
extract for any peak distortion or any ambiguity in the first
analysis. Follow Standard addition to the sample extract to
confirm the analyte as an
additional measure for enhanced/suppressed
peak responses.
8.2.2 Co-Relation
Coefficient should be greater than or equal to 0.99.
8.2.3 Run Spike recovery in
matrix sample after every 10 sample & it should be in the range of 70- 120%
8.2.4
Recovery of known sample should be
70 to 120 % of spike Concentration. (If required)
8.2.5 Ensure calibration curve
range covers the analyte concentration in test sample and
minimum 3 levels can be selected for calibration curve and do
not force through zero.
8.2.6 Relative difference of
individual results can be <30% of Mean. If the variation is more for the
Results close to Reporting Limit, take additional caution before reporting).
8.2.7 Run the analysis in a
non-interfering background and check the analytes presence due to breakdown/metabolites/isomeric
compounds. Also check the pseudo positive/pseudo negative responses in the test
sample due to matrix background (or) noise & drift due to other machine
maintenance factors.
8.2.8 Standard
Check should be 70 to 120 % of Standard value.
8.2.9 If sample result is at ML
(Maximum limit) or near to ML, calculate new ML with t, g value calculation for
confirmation of result with MOU.
g (Guard
band value) =t*u
where,
t=statistical table (attached with annexure 1)
u=standard uncertainty
Final decision limit will be =P-g
Where,
P=Maximum limit
G=Guard band value
From above,
we can conclude that, sample comes under acceptance zone or rejection zone.
9.0 PRECAUTIONS AND
SAFETY INSTRACTION(S)
9.1
Always wear appropriate PPEs when
using hazardous materials. Some examples of Protective equipment are a facemask
with filter, rubber gloves and a lab coat should be used where splashing may
occur. avoid breathing vapours by working in fume hoods.
9.2
Main power supply should not be
switched off while MS is ON to avoid vacuum break.
9.3
Change milli-Q water of seal wash
and solvent reservoir bottle containing water on very two day to prevent
clogging of solvent reservoir filter and LC column due to fungal growth.
9.4
Read back voltages/gas flow
values should match applied values before starting flow.
9.5
Failure to flow desolation gas
during operation can cause damage the source.
9.6
Do not open and touch source
block without bringing down the source temperature.
9.7
Never perform cone cleaning
operation before source temperature has come down to around 50ºC.
9.8
Please ensure that both the drain
ports at the rear of the nitrogen generator are led to a suitable connection or
container. It should be noted that the generator will liberate considerable
amounts of water from these ports (approx. 1-2 litres weekly). If a container
is used it should be emptied at regular intervals.
9.9
Read gas flow values should match
applied values before starting flow.
9.10
Failure to flow desolvation gas
during operation can cause damage to the source.
9.11
Do not open and touch the source
block without bringing down the source
temperature.
9.12
Never perform cone cleaning
operation before source temperature has come
down to around 50°C.
9.13
Refer to material safety data
sheets (MSDS) for further information on the safe
handling of all chemical and
reagents used in this method.
9.14
Always use filtered mobile phase.
9.15
Weekly clean Syringe and injection
wash vail
9.16
Proper purging should be done to
remove air bubble. There should not be
pressure fluctuation.
9.17
Before starting a run, column
must be equilibrated at least for 20 minutes
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