Method Verification and Quality Control Procedures for Determination of phthalate

 Method Verification and Quality Control Procedures for Determination of phthalate 

Introduction

The guidance in this Article describes the Method Verification and analytical quality control requirements to support the validity of data used for checking compliance with maximum residue limits (MRLs).

The key objectives are:

1. To ensure the quality and comparability of analytical results.
2. To ensure that acceptable accuracy is achieved.
3. To ensure that false positives or false negatives are not reported.

Standards and Calibration Solutions

Identity and purity of standards:

1. “Pure” standards of analytes should be of known purity. Each pure standard must be uniquely identified, the date of receipt recorded and an expiry date allocated if the supplier has not provided this.
2. After the expiry date it must be replaced.
3. Ideally, the identity of freshly acquired pure standards should be checked if the analytes are new to the laboratory.

 

Name of Analyte	Lot   No.	purity	Date of Receipt	Date of expiry
DBP	XXXXX	99.4	30/1/2021	Aug 2023
BBP	XXXXX	98.5	30/1/2021	March 2023
BIS-2EHP	XXXXX	99.4	30/1/2021	April 2023
DNOP	XXXXX	99.2	30/1/2021	March 2023
DINP	XXXXX	99.9	30/1/2021	Aug 2023
DIDP	XXXXX	99.0	30/1/2021	MAY 2023
Benzyl benzoate (IS)	XXXXX	99.5	30/1/2021	Jan 2023

 

 Standard stock solution

 Concentration of stock solution as follows.

Sr. No.	Name of Analyte	Amount taken for stock preparation         in mg	Final Volume of Solution in ml	Concentration of stock solution mg/liter  (ppm)
1	DINP	40.41	10	4036.959
2	DIDP	14.81	10	1466.19
3	DBP	29.12	10	2894.528
4	BBP	21.24	10	2092.14
5	BIS-2EHP	19.6	10	1948.24
6	DNOP	24.3	10	2410.56

Internal standard calibration solution

Sr. No.	Name of Analyte	Amount taken for stock preparation         in mg	Final Volume of Solution in ml	Concentration of stock solution mg/liter  (ppm)
1	Benzyl benzoate	12.2	10	1213.90

 

Working stock standard:  

DBP, BBP, BIS-2EHP& DNOP calculated amount each from stock solution & diluted to 10 ml with dichloromethane. The concentration of working stock is 40 ppm. DINP & DIDP calculated amount each from stock solution & diluted to 10 ml with dichloromethane. The concentration of working stock is 500 ppm.

Preparation of linear concentrations of the standard Solution:

A series of calibration standard solution in the range of 0.4 to 10 ppm for DBP, BBP, BIS-2EHP& DNOP & 5 to 40 ppm for DIDP & DINP is prepared by transferring 0.010 ml to 0.250 ml of the working stock solution 40 ppm & 0.010 ml to 0.080 ml of working stock solution 500 ppm respectively and add 0.05 ml of IS working solution of 200 ppm before making up to mark with dichloromethane, each calibration standards containing 10 ppm of IS. 

   

Injections were done of above serial dilutions in GCMS under following conditions:

           1) Column used: - DB-5MS (30 m x 0.25 mm x 0.25 µ) 

           2) Carrier Gas: - Helium  

           3) Injection Volume: - 2.0 µl 

           4) Injection Mode- Spilt less 

           5) Injector: - 250°C

           6) Flow: - 1 ml/min.

           7)  Oven Program: - 

                    100°C (1 min)

                    100°C to 180° C (10°C/3min.)

                    180°C to 230°C (20°C/5min)

                    230°C to 250°C (10°C/5min)        

250°C to 280°C (10°C/min) (5 min)

Linearity

Linearity is the ability of a method to obtain test results that are directly proportional to the concentration of an analytes in samples within a given range.

Calibration Curve

Calibration curve is plotted with the standard concentration as per the above parameters.

Correlation coefficient should be 0.996

Sr. No.	Name of Analyte	For standard linearity R2
1	DBP	0.997
2	BBP	0.996
3	BIS-2EHP	0.998
4	DNOP	0.999
5	DINP	0.996
6	DIDP	0.995

 The Calibration curves for the DBP, BBP, BIS-2EHP, DNOP, DINP, DIDP studied met a linear least squares calibration with a correlation coefficient of R² >0.995

Repeatability & Recovery

DBP, BBP, BIS-2EHP, DNOP& DIDP, DINP

Accuracy is the measure of how close the experimental value is to the true value.

❤ Sample is spiked for 500 ppm with Mixture of DBP, BBP, BIS-2EHP, DNOP & 500 ppm 

DIDP, DINP & sample is prepared as per IS0 8124-6:2014 method and Injected in GCMS. Standard deviation and % RSD is calculated.

Above data indicates that DBP, BBP, BIS-2EHP, DNOP & DIDP, DINP analyzed met the acceptability criteria of recovery (80-120%).

 

❤ Sample is spiked with three replicate analyses at the level of 500 mg/kg for DBP, BBP, BIS-2EHP & at the level of 500 mg/kg for DIINP, DIIDP prepared as per IS 9873-9:2017 & IS 9873-6:2017 SOP and injected in GCMS. Calculate standard deviation and % RSD.

 

Above data indicates that DBP, BBP, BIS-2EHP, DNOP & DIDP, DINP analyzed met the acceptability criteria of recovery (80-120%).

 

 

❤ Sample is spiked with replicate analyses at the level of 500 mg/kg for DBP, BBP, BIS-2EHP, DNOP & at the level of 500 mg/kg for DIINP, DIIDP prepared as per CPSC-CH-C1001-09.3/EN14372:2004 and injected in GCMS. Calculate standard deviation and % RSD.

 

 

Recovery as per CPSC-CH-C1001-09.3/EN14372:2004

Above data indicates that DBP, BBP, BIS-2EHP, DNOP & DIDP, DINP analyzed met the acceptability criteria of recovery (80-120%).

 

 

LOD  &  LOQ

Limit of Detection

  ❤ Limit of Detection is the point at which a measured value is larger than the insecurity associated with it. It is the lowest concentration of analyte in a sample that can be detected but not necessarily quantified.

  ❤ In chromatography limit of detection is an amount that produces a peak with a height at least 3 times that of the baseline noise level.

  ❤ Inject Standard Mixture of DBP, BBP, BIS-2EHP, and DNOP for five replicate analyses at the level of 0.2ppm prepared as per method and injected in GCMS. Calculate S/N.

 

NAME OF ANALYTE	S/N
DBP	8.1
BBP	6.1
BIS-2EHP	8.4
DNOP	5.1

 

 

 

 

The Above data indicates that DBP, BBP, BIS-2EHP, DNOP analyzed by each mode met the acceptability criteria of LOD (S/N>3).

❤ Inject Standard Mixture of DIDP, DINP for five replicate analyses at the level of 2 ppm prepared as per method and injected in GCMS. Calculate S/N. 

Name of Analyte	S/N
DINP	4.4
DIDP	4.1

 

 

 The Above data indicates of DIDP, DINP analyzed by each mode met the acceptability criteria of LOD (S/N>3).

Limit of Quantification

      ❤ Limit of Quantification is the minimum concentration of the analyte that can be quantified with acceptable precision should apply to the complete analytical method. Variously defined but must be a value greater than the LOD.

      ❤ In chromatography limit of quantification is an amount that produces a peak with a height 10 to 20 times that of the baseline noise level.

      ❤ Inject Standard Mixture of DBP, BBP, BIS-2EHP, and DNOP for five replicate          analyses at the level of 0.4 ppm prepared as per method and injected in GCMS. Calculate S/N. 

Name of Analyte	S/N
DBP	18.8
BBP	12.4
BIS-2EHP	17.7
DNOP	14.3

 

                                          

 

 The Above data indicates that DBP, BBP, BIS-2EHP, DNOP analyzed by each mode met the acceptability criteria of LOQ (S/N>10).

 

❤ Inject Standard Mixture of DIDP, DINP for five replicate analyses at the level of 5.0 ppm prepared as per method and injected in GCMS. Calculate S/N.

 

Name of Analyte	S/N
DINP	30.2
DIDP	12.4

 

                                          

 The Above data indicates that DIDP, DINP analyzed by each mode met the acceptability criteria of LOQ (S/N>10).

 

Conclusion

Based on above satisfactory verification of method with respect to specification as per, IS-9873 part 9:2017/IS-9873 part 6:2017 & CPSC-CH-C1001-09.3/EN 14372:2004  this is to conclude that method is fit for the purpose & applied for the determination of DBP, BBP, BIS-2EHP, DNOP And DIDP, DINP. 

Done by	Checked by	Approved by
Date	Date	Date