Objective:
The purpose of the study is to validate the analytical method for determination of traces of API contents in Swab & Rinse samples and to establish documented evidence and provide the procedure for the same.
The scope should evaluate the acceptability of an analytical method for determination of traces of API contents in the swab and rinse samples by HPLC. This protocol should define the procedure, documentation, references, acceptance criteria and results evaluated for determination of traces of API contents in Swab & Rinse samples by HPLC.
Responsibilities:
Analytical R&D Department:
Quality Control Department:
Regulatory Affairs:
Quality Assurance Department:
Validation Team & Approval:
.jpg "Sampling for Cleaning Validation")
Training:
Training should be given to the concerned persons and training record should be maintained.
Details of Analytical Method Procedure and Solution Preparations:
Test Procedure:
The detailed test procedure is as per IP/BP/USP/IH
Description: Generic Name of the Product
Determination: By HPLC
Reagents Required :
Raw material / Working Standard/Impurities:
Name of API
Placebo
Apparatus:
Clean and Dry Class ‘A’ glassware: Volumetric flasks, Bulb Pipette, swab, test tube SS plate (10 X 10 cm SS 316L plate)
Preparation of Buffer Solution :
Mobile Phase Preparation:
Chromatographic Conditions:
Instrument :
Column :
Wavelength :
Flow rate :
Column Temperature :
Injection Volume :
Run Time :
Diluent :
Preparation of Solutions:
Blank Solution Preparation (Diluent):
Standard Solution Preparation :
Preparation of Cleaning Sample Solutions:
Soak the swab sample in 5.0 mL of diluent. Sonicate for about 10 min. Filter the solution through 0.45 μ syringe filter and inject into the HPLC system.
For Rinse Samples:
Filter the rinse samples through 0.45 μ syringe filter, discard first few mL, fill the HPLC vials and inject into the HPLC system.
Procedure:
Equilibrate the column with mobile phase for 30-45 minutes. Separately inject blank (single injection), standard solution (five Injections) and sample preparations (single injection) and record the response of the principal peak & other related peaks.
System Suitability:
1. Inject the blank solution. Disregard any peak due to the blank solution in the test solution.
2. The RSD of the peak area of API in five replicate injections of standard solution should not be more than 2.0%.
Calculations:
Calculate the amount of API present in ppm in the rinse sample using the following formula.
Where,
AT = Area of API in Rinse Sample Solution
As = Average Area of API in Standard Solution
Ws = Weight of standard taken (mg)
Ds = Dilution of standard
P = Purity of API on as is basis
Calculate the amount of API present in the swab sample using the following
Formula.
Where,
AT = Area of API in Swab Sample Solution
As = Average Area of API in Standard Solution
Ws = Weight of standard taken (mg)
Ds = Dilution of standard
V = Final volume of swab sample (5 mL)
P = Purity of API on as is basis
Acceptance Criteria:
The amount of API recovered from the effective surface area of equipment should not be more than 10 ppm to take the subsequent batches.
Analytical Method Validation Parameters:
Analytical Method for determination of Traces of API in swab & rinse samples should be validated for following parameters:
System Suitability & System Precision:
Objective
To demonstrate and verify that the system suitability parameters of the chromatographic system are adequate for the subjected analysis.
Procedure
Prepare Standard solution of API at its target concentration (10 ppm) & other solutions as per test procedure. Inject blank, five replicates of standard solution. Calculate RSD for the area of API peak from five replicates of standard solution. Record the results in observation Table-1
Observations: Table- 1 (HPLC ID )
Standard Solution (10 ppm)
| |
Sr. No.
|
Area of API
|
1
| |
2
| |
3
| |
4
| |
5
| |
Average
| |
SD
| |
RSD (%)
| |
Acceptance Criteria:
The RSD of the peak area of API in five replicate injections of standard solution should not be more than 2.0%.
Linearity & Range:
Objective
To demonstrate that the analytical method is capable to:
• Obtain test results, which are directly proportional to the concentration (amount) of analyte in the sample. (Linearity)
• Provide an acceptable degree of linearity and precision when applied to samples containing amounts of an analyte within or at the extremes of the specified range of the analytical procedure (Range).
Procedure:
Prepare a series of standard preparations (minimum five concentrations) of API over a range starting from (i.e.0.1% of standard concentration). Inject each of the standard preparation in triplicate and then take the average area for calculations. Plot a graph of concentration vs peak area to establish LOD & LOQ of API by regression function.
Plot a linearity graph of a concentration (ppm) versus average area at each level and determine the slope. The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out based on the calibration curve. Record the observation in Table-2A & Table-2B.
Determine the LOD and LOQ concentration level from the following formula.
The Limit of Detection (LOD) may be expressed as:
LOD = 3.3 σ
S
The Limit of Quantitation (LOQ) may be expressed as:
LOQ = 10 σ
S
Where σ = The residual SD of the regression line.
S= The slope of the calibration curve.
Observations: Table- 2 A (HPLC ID )
Standard Solution (10 ppm)
| |
Sr. No.
|
Area of API
|
1
| |
2
| |
3
| |
4
| |
5
| |
Average
| |
SD
| |
RSD (%)
| |
Observations: Table 2B (HPLC ID )
Standard Solution
| ||
Sr. No.
|
Concentration (ppm)
|
Average Area of API
|
1
| ||
2
| ||
3
| ||
4
| ||
5
| ||
6
| ||
7
| ||
8
| ||
9
| ||
10
| ||
Correlation Coefficient ‘r’
| ||
Slope (m)
| ||
Y intercept
| ||
Acceptance Criteria:
1. The system suitability criteria should pass as per analytical method.
2. Derive the concentration for LOD & LOQ level from linearity studies.
3. The Correlation Coefficient ‘r’ should not be less than 0.995.
Precision at LOQ Level:
Inject LOQ level in six replicates to perform the precision at LOQ level. Record the results in observation Table-2C.
Observations: Table 2C (HPLC ID )
Sr. No.
|
LOQ Level
|
Area of API
|
1
|
Injection_1
| |
2
|
Injection_2
| |
3
|
Injection_3
| |
4
|
Injection_4
| |
5
|
Injection_5
| |
6
|
Injection_6
| |
Average
| ||
SD
| ||
RSD (%)
| ||
Acceptance Criteria:
The RSD of six injections at LOQ level should not be more than 10.0 %.
Linearity from LOQ to 200%:
Procedure
To evaluate linearity, prepare standard solutions of API of six different concentrations (i.e. LOQ to 200% of target concentration). Inject each of the standard concentration in triplicate. Plot a linearity graph of concentration (ppm) versus average area at each level. Calculate the correlation coefficient, slope (m), Y intercept and record the observations in Table-2D & Table-2E as given below.
Observations: Table 2D (HPLC ID )
Standard Solution (10 ppm)
| |
Sr. No.
|
Area of API
|
1
| |
2
| |
3
| |
4
| |
5
| |
Average
| |
SD
| |
RSD (%)
| |
Observations: Table 2E (HPLC ID )
From Standard Solution
| |||
Sr.No.
|
Linearity Level (%)
|
Concentration (ppm)
|
Average Area
|
1
|
LOQ
| ||
2
|
25
| ||
3
|
50
| ||
4
|
75
| ||
5
|
100
| ||
6
|
125
| ||
7
|
150
| ||
8
|
200
| ||
Correlation Coefficient ‘r’
| |||
Slope (m)
| |||
Y intercept
| |||
Acceptance Criteria:
1. The system suitability criteria should pass as per analytical method.
2. Correlation Coefficient ‘r’ should not be less than 0.995.
Accuracy/Recovery:
Objective
To demonstrate that the analytical method is capable to yield data values close to true values. This described as follows.
• Accepted as a conventional true value. (Accuracy)
• An accepted reference value and the value found. (Recovery)
Recovery on Stainless Steel Plate (10 X 10 cm SS 316L plate):
Preparation of Stock Solution:
Preparation of Standard:
Establishment of Recovery Factor:
Prepare a standard stock solution of the drug substance in the selected solvent and spike separately 0.5 mL for 50%, 1 mL for 100% and 1.5 mL for 150% respectively on the 10 cm x 10 cm SS 316 L plate and disperse evenly.
Allow the plate to dry and perform swabbing. Transfer 5 mL of diluent to a cleaned test tube. Place a clean swab into the test tube. The swabbing should be done so as to cover the entire surface by making perpendicular vertical and horizontal strikes. Care should be taken to lift the swab at the end of each strike made on the SS plate. Collect the total swabbings into the test tube containing 5 mL of diluent and sonicate for at least 10 minutes and dilute to 10 mL with diluent. The concentration of test solution should be close as that of target standard concentration. Filter the solution through a 0.45µ syringe filter. Similarly, prepare controlled blank by dipping the swab stick in 5 mL diluent & sonicating it for 10 min. Then, inject diluent blank, controlled blank, standard and test solution into the chromatographic system.
Calculate the Amount of residue recovered “in ppm”.
Where,
AT = Area of API in Swab Sample Solution
As = Average Area of API in Standard Solution
Ws = Weight of standard taken (mg)
D1 = Dilution Factor of standard
V f = Final volume of sample
Vs = Volume of Standard spread on the 10x10 cm SS plate.
Calculate the % recovery
Rp = Amount of residue recovered “in ppm”
RT = Amount of residue added “in ppm”
[RF = Recovery Factor (RF= 100/ % Recovery of API from SS plate)]
Procedure:
Inject blank, controlled blank &standard solution and each recovery level as per the following sequence. Record the results in observation Table-3A & Table-3B.
Details
|
No. of injections
|
Blank
|
1
|
Controlled Blank
|
1
|
Standard solution
|
5
|
Recovery level -1 (50%)
|
3
|
Recovery level -2 (100%)
|
3
|
Recovery level -3 (150%)
|
3
|
Standard solution (Bracketing)
|
1
|
Observations: Table 3A (HPLC ID )
Standard Solution (10 ppm)
| |
Sr. No.
|
Area of API
|
1
| |
2
| |
3
| |
4
| |
5
| |
Average
| |
SD
| |
RSD (%)
| |
Observations: Table 3B (HPLC ID )
Standard Solution
| |||
Sr.No.
|
Replicates
|
Proposed concentration taken on plate (% of target concentration)
|
Recovery
(%)
|
1
|
1
|
50
| |
2
|
50
| ||
3
|
50
| ||
2
|
1
|
100
| |
2
|
100
| ||
3
|
100
| ||
3
|
1
|
150
| |
2
|
150
| ||
3
|
150
| ||
Average percentage recovery:
| |||
Acceptance criteria:
1. The system suitability criteria should pass as per analytical method
2. The recovery from SS plate in residue analysis should be between 70 % -130 %.
Placebo Recovery :
Objective
To demonstrate that the analytical method is capable to yield data values close to true values. This described as follows.
• Accepted as a conventional true value. (Accuracy)
• An accepted reference value and the value found. (Recovery)
Procedure
Determine the accuracy/ recovery by spiking the drug substance in placebo at LOQ, 50 %,
100 % & 150 % levels of target concentration of API (i.e. 50 % - 150% of 10 ppm) respectively. Add drug amount at LOQ, 50%, 100%, and 150% levels to the placebo respectively. Prepare each concentration in triplicate & inject once. Calculate and record the percentage recovery of API in observation Table-4A, Table-4B &Table-4C.
Observations: Table 4A (HPLC ID )
Standard Solution (10 ppm)
| |
Sr. No.
|
Area of API
|
1
| |
2
| |
3
| |
4
| |
5
| |
Average
| |
SD
| |
RSD (%)
| |
Observations: Table 4B (HPLC ID )
Level
|
Amount of
Placebo
|
Amount of std. added
X
|
Response
|
Amount Recovered
Y
|
X2
|
XY
|
LOQ level
|
--
|
--
| ||||
Formulae: n (SXY) - (SX) (SY )
% R =------------------------------------ X 100
n (SX2 ) - (SX )2
Where ‘n’= No. of experiments i.e. 3
Observations: Table 4C (HPLC ID )
Level
|
Amount of
Placebo
|
Amount of std. added
X
|
Response
|
Amount Recovered
Y
|
X2
|
XY
|
50%
|
--
|
--
| ||||
Mean
| ||||||
100%
|
--
|
--
| ||||
Mean
| ||||||
150%
|
--
|
--
| ||||
Mean
| ||||||
SX =
|
SY =
|
SX2 =
|
SXY =
| |||
Formulae: n (SXY) - (SX ) ( SY )
% R =------------------------------------ X 100
n (SX2 ) - (SX )2
Where ‘n’= No. of experiments i.e. 9
Acceptance Criteria:
1. The system suitability criteria should pass as per analytical method.
2. The average percent recovery at LOQ level should be between 80%-120%
3. Average percent recovery from 50% to 150% should be within 90% to 110% of target concentration (theoretical value).
Objective
To determine the stability of swab sample solution.
For Swab Sample Solution
Prepare the swab sample solution at 100 % level as per the test procedure in ‘Recovery on SS plate’ parameter (refer section 8.0), and inject the solution at zero hours. Place the same solution at room temperature (22ºC ± 2ºC) and inject after 2, 4, 8, 12 and 24 hrs. Calculate % recovery at different time intervals up to 24 hrs. Record the observations in observation Table-5A & Table-5B.
Observations: Table 5A (HPLC ID )
Standard Solution (10 ppm)
| |
Sr. No.
|
Area of API
|
1
| |
2
| |
3
| |
4
| |
5
| |
Average
| |
SD
| |
RSD (%)
| |
Observations: Table-5B (HPLC ID )
Swab Sample Solution
| |
Time in hrs
|
% Recovery
|
Initial
| |
2
| |
4
| |
8
| |
12
| |
24
| |
Average
| |
SD
| |
RSD (%)
| |
Acceptance Criteria:
1. The system suitability criteria should pass as per analytical method.
2. The RSD of % recovery on SS plate at different time intervals up to 24 hrs should not be more than 5.0
Filter Evaluation:
Objective
To demonstrate that the analytical method is unaffected by using filtration technique as well as by centrifuging the sample solutions.
Procedure
Prepare the sample at 100 % level as per test procedure in ‘Placebo Recovery’ parameter (refer section 8.0). Filter the sample through 0.45 µm syringe filter. Centrifuge the same sample at 2000 rpm for 15 minutes & also filter the same sample through Whatman filter paper No. 1. Record the results in observation Table-6A & Table-6B.
Observations: Table-6A (HPLC ID )
Standard Solution (10 ppm)
| |
Sr. No.
|
Area of API
|
1
| |
2
| |
3
| |
4
| |
5
| |
Average
| |
SD
| |
RSD (%)
| |
Observations: Table-6B (HPLC ID )
| Type of filter /centrifuge |
Absolute Difference
| |
| 0.45µ syringe filter | ||
| Centrifuge | ||
| Whatman No. 1 filter paper |
Acceptance Criteria:
1. The system suitability criteria should pass as per analytical method.
2. The absolute difference between % recovery of sample filtered through
0.45 µ syringe filter and unfiltered (centrifuged) sample should not be more than 2.0.
3. The absolute difference between % recovery of sample filtered through
Whatman No.1 filter and unfiltered (centrifuged) sample should not be more than 2.0.
4. The filtered samples should not show any extraneous peak as compared to unfiltered (centrifuged) samples.
Recommendations and Suggestions:
Record the recommendations or suggestions based on the interpretation of the results and reference documents where required in the validation report.
Revalidation Criteria:
Revalidation may be necessary for the following circumstances:
- Changes in the synthesis of the drug substance;
- Changes in the composition of the finished product;
- Changes in the analytical procedure.
The degree of revalidation required depends on the nature of the changes.
Annexure:
Annexure should be attached to the validation report.
Reference:
Abbreviations:
No.
|
Number
|
+
|
Plus or minus
|
i.e.
|
That is
|
RSD
RRF
%
|
Relative Standard Deviation
Relative retention factor
Percentage
|
ID
SS
WS
ppm
hrs
API
|
Identification number
Stainless Steel
Working Standard
Parts per million
Hours
Active pharmaceutical ingredient
|
Revision History:
Sr. No.
|
Revision No.
|
Effective Date
|
Change Control No.
|
Submitted By:
Dipak Dhote
Blue Cross Laboratories Ltd.
A12, MIDC, Ambad, Nashik-422010,
Maharashtra, INDIA
Email: dipak.d@bluecrosslabs.com
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Hi, i would like to ask what does Linearity to LOQ to 200% means? if my LOQ is 3ppm is it the target value, or is it referring to the safe threshold value of the cleaning validation which is 9ppm??? Thank you in advance....
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