This document describes the validation of an analytical method using reverse phase high performance liquid chromatography (RP-HPLC) to quantify the amount of omeprazole in a sterile lyophilized powder injection. The validation establishes the method's accuracy, precision, specificity, linearity, range, ruggedness and robustness according to ICH guidelines. The results demonstrate the method is suitable for its intended purpose of quantifying omeprazole in the lyophilized powder injection.

1. Validation of Analytical Method of Assay
by RP-HPLC for Omeprazole 40mg/ml in
Sterile Lyophilized Powder for Injection
Prepared by: FATEN AZZAM HAMED
Presented on: February at
The honored members of jury:
Director: Dr. Sanaa AWADA
Examiner: Dr. Rania AZAR
Examiner: Mrs. Zeinab AL ZAIN

2. Validation of
Analytical
Method of Assay
by RP-HPLC for
Omeprazole
40mg/ml in
Sterile
Lyophilized
Powder for
Injection
This work was done at ARWAN Pharmaceutical Industries,
Jadra- Lebanon.

3. Index
• Introduction
• Method and Material
• Results
• Discussion
• Conclusion
• References

4. • Our aim
Introduction

5. Introduction
• Omeprazole, a proton-pump inhibitor (PPI).
• Gastroesophageal reflux disease (GERD), peptic ulcer
disease, and Zollinger– Ellison syndrome[2].
• The Lyophilized form of Omeprazole used for IV infusion
and Injection is available in different brands, but one of
the commonly used brands in Lebanon is the locally
• Figure 1: Omeprazole Sodium (5-
methoxyl group-2-{[(4-methoxyl group-
3,5-dimethyl-2-pyridine radicals)-
methyl]-sulfinyl }-1H-benzimidazole
sodium salt)

6. • Assay of Omeprazole
• Assay procedures are intended to measure
the analyte present in a given sample. In
the context of this document, the assay
represents a quantitative measurement of
the major component(s) in the drug
substance. For the drug product, similar
validation characteristics also apply when
assaying for the active or other selected
component(s).
Introduction

7. • Assay of Omeprazole
• A simple, fast, and sensitive reversed-phase high
pressure liquid chromatography (RP-HPLC)
method with PDA detector was developed for the
quantitation of omeprazole in Lyophilized powder
for injection formulation. And for any analytical
method before its implementation in the routine
test analysis, a process of validation should be
performed, documented and approved by health
authorities. This validation process is one type of
validation called analytical method validation
(AMV).
Introduction

8. • Validation of Assay method
According to ICH, method validation can
be defined as, ‘‘Establishing a
documented proof, which provides a high
degree of assurance that a specific
process will consistently produce a
desired result at its prearranged
specifications and quality characteristics.”
Introduction

9. • Validation of Assay method
• The following types analytical procedures
to be validated.[8], [15]
• Identification tests
• Quantitative tests for impurities content
• Limit tests for the control of impurities
• Quantitative tests of the active moiety in
samples of drug substance or drug
product.
Introduction

10. • Validation of Assay method
• The performance characteristics required
to validate various methods have been
mentioned in different guidelines such as
USP, ICH, FDA, EU guidelines etc. The
below table summarizes the analytical
parameters to be validated based on each
guideline:[16], [17]
Introduction

11. • Validation of Assay method
Introduction
Guideline Analytical parameters that could be validated
USP Accuracy, precision, specificity, detection of limit, quantitation limit,
linearity, range, ruggedness and robustness.
ICH Accuracy, precision, specificity, detection of limit, quantitation limit,
linearity, range, system suitability and robustness.
FDA Accuracy, precision, specificity/selectivity, detection of limit, quantitation
limit, linearity, range, system suitability, reproducibility, sample solution
stability and robustness
European Accuracy, precision, specificity, detection of limit, quantitation limit,
linearity and range.

12. • Validation of Assay method
Introduction
ICH Recommendation of validation parameters for each test
procedure[8]

13. • Validation of Assay method
• Statistical investigation of data obtained during a
method validation should be performed to reveal
validity of the analytical method. The statistics required
for the interpretation of analytical method validation
results are the computation of the following:[19]
• • Mean
• • Standard deviation
• • Relative standard deviation
• • confidence intervals
• • Regression analysis.
Introduction

14. • Validation of Assay method
• To sum up, the validation process of analytical method builds a degree of
confidence, not only for the developer but also to the user. It also
produces quality products, reduce the product cost by increasing efficacy,
few reject and longer equipment life, helps in optimization of process or
method. And helps in process improvement, technology transfer related
products validation and increased employee awareness. It eliminates
testing repetitions and leads to better time management at the end.
• The validation protocols prepared at ARWAN is used to provide
instructions and acceptance criteria for the validation of the analytical
method used for the quantification of Omeprazole in Omeprazole
Sodium lyophilized powder for injection using RP-HPLC with PDA detector.
Introduction

15. Materials and Method
• MATERIALS /EQUIPMENT
• Products to be analyzed was Omeprazole Sodium Lyophilized Powder for
injection
• Omeprazole (C17H19N3O3S), Molecular weight; 345.4 g/mol, a white to off-
white crystalline powder is a substituted benzimidazole, an irreversible
inhibitor of gastric hydrogen-potassium adenosine triphosphate pump,
which is the final common step of acid secretion in parietal cells. It is used
to treat gastric reflux esophagitis and the Zollinger- Ellison syndrome.
Omeprazole is a weak base (pKa1=4.2 and pKa2=9) freely soluble in
ethanol, methanol, and methylene chloride. It is slightly soluble in
acetone and isopropanol, very slightly soluble in water, and dissolves in
dilute solutions of alkali hydroxide. The stability of Omeprazole is a
function of pH; it is rapidly degraded in acid media, but has acceptable
stability under alkaline conditions, its boiling point is 599.991°C at 760
mmHg and its melting point is 155°C-156°C [20][14]

16. Materials and Method
• MATERIALS /EQUIPMENT
• Each vial of the Omeprazole Sodium
Sterile lyophilized powder for Solution
containing:
• Lyophilized (freeze-dried) Omeprazole
sodium equivalent to Omeprazole 40 mg
as the active ingredient and Disodium
edetate and Sodium hydroxide as the
excipients

17. Material Manufacturer Batch Number/Lot Number
Sodium Hydroxide Pellets Sigma Aldrich STBG7238
Methanol Lab Honeywell I050BS
Omeprazole Standard* Sigma Aldrich LRAB0280
Disodium Edetate Sigma Aldrich SZBF2190V
HPLC Grade Water Sigma Aldrich I070MB
Materials and Method
Table II-1: Materials Used During the Validation[22]

18. Materials and Method
Table II-2: Equipment Used During the Validation[22]
Equipment Batch Number/Lot Number
HPLC with PDA detector HPLC001
HPLC002
Digital Balance ABAL001
pH meter pH/CON001
Vortex AVAL001

19. Materials and Method
• Preparation of Solution:
• 1. Mobile Phase (MeOH: H2O 90:10)
• Methanol and water were mixed with the following proportions MeOH: H2O 90:10. Mobile phase was filtered with Nylon
filter and degassed prior to use.
• II.2.2. Diluent 1 (0.1N NaOH)
• In a 1000ml volumetric flask, 500ml of water was added; then 4g of Sodium Hydroxide pellets were weighed and
transferred into the flask to be dissolved completely with water and then the flask was completed to volume with water.
• II.2.3. Diluent 2
• Methanol
• II.2.4. Stock Standard Solution (Omeprazole Standard)
• 100 mg of Omeprazole Working Standard (WS) were weighed accurately and transferred into a 250 ml volumetric flask.
The standard was dissolved by vortexing or sonication and then dilute to volume with diluent 1 (0.1N NaOH)
• II.2.5. Standard Solution Preparation
• 5 mL of the stock standard solution was transferred into a 50ml volumetric flask. It was diluted to volume with Diluent 2
Methanol.
• Note: during dilution, 80 to 90% of the flask was filled with Methanol then vortexed to reach room temperature, then
completed to volume with Methanol.
• II.2.6. Placebo Preparation
• Accurately 150 mg of disodium edetate were weighed and transferred into a 200 mL volumetric flask; dissolved and diluted
with water; and pH of the final solution was adjusted to 11 with NaOH 4%.
• II.2.7. Placebo Test Solution Preparation
• In a 50 ml volumetric flask, transfer 2.7 ml of placebo then dilute to volume with Methanol.
• II.2.8. Sample Stock Solution Preparation
• In a 250 ml volumetric flask, 100 mg of Omeprazole Standard were accurately weighed and transferred then dissolved by
votrexing or sonication. Diluent 1 (0.1N NaOH) was added to volume.

20. Materials and Method
• Preparation of Solution:
• . Sample Solution
• II.2.1.9.1. Sample Solution 80% (prepared in triplicate):
• 4 ml of the sample stock Solution was transferred into a 50 ml volumetric flask containing about 10
ml of Methanol. 2.7 ml of placebo was added; it was then diluted to volume with Methanol.
• II.2.9.2. Sample Solution 90%:
• 4.5 ml of the sample stock solution was transferred into a 50 ml volumetric flask containing about 10
ml of Methanol. 2.7 ml of placebo was added it was then diluted to volume with Methanol.
• II.2.9.3. Sample Solution 100% (prepared in triplicate):
• 5 ml of the sample stock solution was transferred into a 50 ml volumetric flask containing about 10
ml of Methanol. 2.7 ml of placebo was added it was then diluted to volume with Methanol.
• II.2.9.4. Sample Solution 110%:
• 5.5 ml of the sample stock solution was transferred into a 50 ml volumetric flask containing about 10
ml of Methanol. 2.7 ml of placebo was added it was then diluted to volume with Methanol.
• II.2.9.5. Sample Solution 120% (prepared in triplicate)
• 3 ml of the sample stock solution was transferred into a 25 ml volumetric flask containing about 10
ml of Methanol. 1.4 ml of placebo was added it was then diluted to volume with Methanol.

21. Materials and Method
• Chromatographic HPLC Conditions
Mode RP-HPLC
Detector PDA at 301 nm
Column Merck, Purospherstar, RP-18 end-capped, 5µm 250 x 4.6mm
Column Temperature 27°C± 2°C
Flow Rate 1.5 ml/min
Injection Volume 20µl
Run Time 5 minutes
Needle Wash 10% Methanol
Auto-sampler Temperature Ambient
Retention Time of Omeprazole 2.200 minutes ± 0.11
Processing Method Parameters
Threshold: 60.000µV/sec Peak Width: 40.00 sec
Void Volume Time: 0.500 min

22. Materials and Method
• Operating Chromatographic Conditions
• Inject the diluent 2 (Methanol) as a blank
• Inject the standard solution 5 times:
• The relative standard deviation (%RSD) for
Omeprazole is NMT 2.0 %
• Inject the sequence of Sample solution
• In case of 9 or more consecutive injections of
samples, inject once the standard solution as
control every 10 samples injections and at the
end of the run.

23. Materials and Method
• Calculation
• Calculate the percentage of Omeprazole in the
sample solution using the following formula
• When Omeprazole Sodium Standard is used:
• % 𝑂𝑚𝑒𝑝𝑟𝑎𝑧𝑜𝑙𝑒 =
𝑅𝑢
𝑅𝑠
∗
Wss
𝑉𝑠
∗ 𝑃𝑠𝑠 ∗
𝐷𝐹𝑢
𝐷𝐹𝑠
∗
1
𝐿𝐶𝑢
∗
345.4
368.4
• When Omeprazole Standard is used:
• % 𝑂𝑚𝑒𝑝𝑟𝑎𝑧𝑜𝑙𝑒 =
𝑅𝑢
𝑅𝑠
∗
Ws
𝑉𝑠
∗ 𝑃𝑠 ∗
𝐷𝐹𝑢
𝐷𝐹𝑠
∗
1
𝐿𝐶𝑢

24. Materials and Method
• Assay Method Validation of Omeprazole 40mg Lyophilized Powder for
Injection
• Assay procedures are intended to measure the analyte present in a given
sample. The objective of the analytical procedure should be clearly
understood since this will govern the validation characteristics which
need to be evaluated. The recommended validation characteristics
according to ICH guidelines are as follows:
• System Suitability
• Specificity
• Linearity
• Accuracy
• Precision
• Ruggedness
• Robustness
• Stability of Solutions

25. Materials and Method
• Description of Validation Parameters and Acceptance Criteria
Parameter Criteria Specifications
System suitability Chromatograms of the Standard solution Standard solution: The % RSD of the 5 replicate injections for
Omeprazole should not exceed 2.0%
Specificity Chromatograms of the mobile phase, Diluent 2,
placebo, standards and sample solutions comparison
Results shall demonstrate that the placebo and the blank have
no interference with the peak of Omeprazole
Linearity Solutions containing Omeprazole at five different
final concentrations equivalent to about 80 %, 90 %,
100 %, 110 %, 120 % with respect to the sample
solution containing 40µg/ml of Omeprazole
Correlation factor R2≥0.995
Accuracy Average recoveries for sample solutions prepared in
triplicate levels 80 %, 100% and 120 %
Average recoveries within 98.0-102.0%
Precision/ Repeatability RSD value obtained from 3 replicates at 3
concentrations prepared under Assay accuracy
RSD value of different percentage recoveries obtained is ≤
2.0% at each level.

26. Materials and Method
• Description of Validation Parameters and Acceptance Criteria
Parameter Criteria Specifications
Ruggedness Comparative analysis The results of percent recovery should be within ±2%
Robustness Accuracy of data output Any change to the parameters should not impact the
original results by more than ±2.0%
Stability of solution Mobile phase Chromatograms should be free from any impurity or
anomalies.
RSD for retention time of known peaks should be less than
2.0% system suitability should be compliant with
acceptance criteria
Standard solution: % Recovery Recovery compared to fresh standard should be within
98.0-102.0%.
system suitability should be compliant with acceptance
criteria

27. Method and Results
• In our study, system suitability was evaluated by injecting
Diluent 2 (Methanol) one time, and 5 times for each
Standard Solution prepared throughout the validation
study. Then the %RSD of the peak areas were calculated
and compared with the acceptance level to demonstrate
the suitability and appropriateness of the analytical
method parameters and conditions.

28. Method and Results
• In our study, system suitability was evaluated by injecting
Diluent 2 (Methanol) one time, and 5 times for each
Standard Solution prepared throughout the validation
study. Then the %RSD of the peak areas were calculated
and compared with the acceptance level to demonstrate
the suitability and appropriateness of the analytical
method parameters and conditions.

29. Slide Title
Product A
• Feature 1
• Feature 2
• Feature 3
Product B
• Feature 1
• Feature 2
• Feature 3