Bulk Solution Assay Test Procedure Development and validation for Esomeprazole Sodium Injection 40mg as a CPP

 

Santoshkumar R. Mulik*, Rishikesh S. Bachhav

Department of Pharmaceutical Quality Assurance, R. G. Sapkal College of Pharmacy,

Anjaneri, Nashik – 422213, Maharashtra, India.

 

ABSTRACT:

Assay analytical test procedure at the Bulk-Solution stage for Esomeprazole Sodium Injection 40mgas a Critical Processing Parameters during manufacturing developed and validated. As per current available literature and the references, Esomeprazole active substance is available in market in the five forms, i.e. Esomeprazole as Plain [Tablet]; Esomeprazole Magnesium Trihydrate [Tablet, Capsule Delayed Release]; Esomeprazole Potassium [Tablet and Capsule]; Esomeprazole Strontium [Capsule Delayed Release] and Esomeprazole Sodium [Injectable].Assay test procedure for Active and other dosage forms [Tablet, Capsule] were available based on either Potentiometric Titration or HPLC basis. Product Esomeprazole Sodium Injection 40mg is not available in official pharmacopeia. As per available reference and literature assay test method for intermediate testing and finished product is based on HPLC. Intermediate testing [In-Process Testing] required as Critical Process Parameters [CPP] to ensure the quality i.e. appropriate bulk-solution purity, before to proceed for filtration and filling of Bulk-solution in unit dosage form [Vials]. To test the bulk solution purity approximate 5-6 hours required by HPLC. Which leads to hold the Bulk solution, further it impact and may risk to Bio-Burden of bulk solution. Also it impact and reduce the productivity of line by 5-6 hours. HPLC testing required special skilled manpower and cost. Considering all above concerns Study carried for test method development based on UV-Spectroscopy, HPLC and potentiometric titration. Based on method validation performed for UV-Spectrometry, HPLC and potentiometric titration; Test procedure developed and validated based on UV-Spectrometry is found Simple, Accurate, Precise, Economical and Rapid to save time and cost with increase in productivity as a Critical Processing Parameters for Esomeprazole Sodium Injection 40mgat the Bulk-Solution stage during manufacturing.

 

 

 

1. INTRODUCTION:

Formulation:

Lyophilized Injectable Product: Esomeprazole Sodium Injection 40mg.

Category:

Non-Steroidal - Anti-inflammatory drug.

Therapeutic Use:

Proton Inhibitor in Gasteroesophageal Reflux Disease, Peptic Ulcer.

Active:

Esomeprazole Sodium.

IUPAC-Name:

Sodium5-methoxy-2-[(S)-(4-methoxy-3,5-dimethylpyridin-2-yl) methanesulfinyl]-1H-benzimidazol-1-ide

 

Molar Formula:

C₁₇H₁₈N₃NaO₃SMolar Mass: 367.4g/mol

 

Structure:

 

Figure No. 1.0: Esomeprazole Sodium

 

2. HISTORY:

Esomeprazole available in five molecule in market

1.     Esomeprazole [ Plan]: Tablet

2.     Esomeprazole Magnesium Trihydrate: Tablet and Capsule

3.     Esomeprazole Potassium: Tablet and Capsule

4.     Esomeprazole Strontium: Capsule

5.     Esomeprazole Sodium: Injectable

 

3. DRUG PROFILE:^3,25

3.1 Absorption:

The pharmacokinetic profile of Esomeprazole SodiumI.V. for Injection 40mg and 20mg

 

Pharmacokinetic Parameters of Esomeprazole Sodium I.V. for Injection 40 mg and 20 mg IUM Following I.V. Dosing for 5 days

Parameter 20mg 40mg

AUC(μmol*h/L) 5.11 16.21

Cmax (μmol/L) 3.86 7.51

t½ (h) 1.05 1.41

Values represent the geometric mean (95% CI)

 

3.2 Metabolism:^15,21,25

Esomeprazole is extensively metabolized in the liver by the cytochrome P450 (CYP) enzyme system. The major part of esomeprazole's metabolism is dependent upon the CYP2C19 isoenzyme, which forms the hydroxy and desmethyl metabolites. The remaining amount is dependent on CYP3A4 which forms the sulphone metabolite. CYP2C19 isoenzyme exhibits polymorphism in the metabolism of esomeprazole.

 

3.3 Esomeprazole Metabolism Pathway:^9,10

Esomeprazole, is a proton pump inhibitor (PPI) class drug that suppresses the final step in gastric acid production. This active metabolite then binds covalently to the potassium-transporting ATPase protein subunits, found at the secretory surface of the gastric parietal cell, preventing any stimulus. Because the drug binds covalently, its effects are dose-dependent and last much longer than similar drugs that bind to the protein non-covalently.

 

3.4 Excretion:^10,12,16,17

Esomeprazole is excreted as metabolites primarily in urine but also in feces. Less than 1% of parent drug is excreted in the urine. Esomeprazole is completely eliminated from plasma, and there is no accumulation during once daily administration. The plasma elimination half-life of intravenous Esomeprazole is approximately 1.1 to 1.4 hours and is prolonged with increasing dose of intravenous Esomeprazole.

 

3.5 Mechanism of Action:^{9,10,20,23,24}

Esomeprazole is a proton pump inhibitor that suppresses gastric acid secretion by specific inhibition of the H⁺/K⁺-ATPase in the gastric parietal cell. Esomeprazole blocks the final step in acid production, thus reducing gastric acidity

 

Table No. 01: Bill of Material

Bill of Material: BOM for 1000 ml Batch size ≈ 400 units
Material Description	UOM	Label Claim /Vial	Std. Qty. for 1000 ml batch
Esomeprazole Sodium Equivalent to Esomeprazole IH	g	42.548 mg ≈ 40.0 mg	17.019 g ≈ 16.0 g
Sodium Hydroxide IP [5%]	mg	---	640 mg
Disodium Edetate IP	mg	1.500 mg	600 mg
Water for Injection IP *	mL	Q.S.	Q.S. to 1.0 lit

 

Molecular weight of Esomeprazole Sodium [C₁₇H₁₈N₃NaO₃S]: 367.4mg is equivalent to Esomeprazole [C₁₇H₁₉N₃O₃S]: 345.4mg

 

i.e. 42.548mg ≈ 40.0mg C₁₇H₁₉N₃O₃S Factor: 367.4 / 345.4 = 1.06369

 

4. MANUFACTURING Flow:

 

Figure No. 02: Manufacturing Flow

 

5. Critical Steps of Product Manufacturing and Related Role:

5.1 Dispensing:

Dispensing of Raw material and Primary packaging materials as bill of material required for batch quantity. Quality of all in-put materials should be ensure before use and supply from valid vendor.

5.2 Preparation:

Preparation of in-put components and accessories including Cleaning, Sterilization and depyrogenation of Stoppers, Vials, Seals and Product contact machine parts and accessories including services like gas line.

5.3 Manufacturing:

Product solution manufacturing carried out as master manufacturing formula. Including monitoring of critical process parameters like Assay, pH, Temperature, Volume make-up etc.

5.4 Filtration:

After getting results of Bulk Solution, next step is 0.22 micron Filtration using sterile membrane, vessels and accessories.

5.5 Filling:

Aseptic Filling carried out under Grade-A environment with monitoring of critical parameters and Half Stoppering.

5.6 Lyophilization:

Lyophilization is carried out using a simple principle of physics sublimation. Sublimation is the transition of a substance from the solid to the vapour state, without passing through an intermediate liquid state. Lyophilization is performed at temperature and pressure condition below the triple point [Utetic Point], to enable sublimation of ice. Liquid filled - half stoppered vials loaded in lyophilizer. After formation of lyophilized powder-cake, full stoppering carried out under vacuum with sterile nitrogen blanketing and stoppered vials un-loaded and taken for final sealing with aluminum seals.

 

5.7 Leak Testing and Inspection:

100% units were inspected for required quality with random leak testing of vials.

 

5.8 Labeling and Packing:

Labeling and packing with carried out with proper label details like Label claim, manufacturer License number, Manufacturer/ marketer name and address, Batch Numbers, Manufacturing date and expiry, Storage conditions, MRP, Precautions and administration and usage instruction with leaflet.

 

5.9 Finish Product Testing:

Testing of FG performed as per specification to ensure product quality with respect to Identification, Purity, Impurity, Quality / Quantity, Sterility, Endotoxin and Particulate load etc.

 

6.0 OBJECTIVE of Research:

To develop and validate the analytical Assay Test procedure as a Critical Processing Parameters at the Bulk-Solution stage during manufacturing of Esomeprazole Sodium Injection 40mg for the following reasons:

·       To reduce the Assay testing cost.

·       To reduce the Testing period

·       To have assay results in minimum time More Particle approach to increase line productivity.

 

7.0 Need of Assay Test Development:

Esomeprazole Sodium Injection 40 mg is sterile dosage form. Being sterile dosage form it is necessary to handle the product process within minimum possible time. However as per current manufacturing process flow, 5-6 hours required to sample and test the bulk solution for Assay by HPLC method. Which leads to holding of bulk solution during manufacturing. It may risk to bio-burden. Hence different approach needs to introduce and develop which help to reduce testing time period and cost.

 

8. Instrumental strategies:

Now a day’s instrumental techniques of evaluation are widely accepted over the classical strategies. These methods are extremely sensitive, supplying specific and specific statistics from small pattern materials.

Instrumental methods are typically a good deal faster than chemical methods and are applicable at attention a long way too small to be amenable to determination by way of chemical methods and find wide software in industry.

 

9. Benefits of Instrumental Techniques:

9.1 Small sample may be used

9.2 Excessive sensitivity is acquired

9.3 Measurements received are reliable

9.4 The dedication may be very speedy

9.5 Even complex pattern may be dealt with easily.

 

10. METHOD Validation: ^{1, 2, 4}

After selecting the technique for analysis, method validation is important. It is very important to scientifically establish the limits and acceptance criteria to validate the analytical method of analysis.

 

A validated method is to be rugged and robust enough to measure the quantity, Purity. And It should meet the criteria’s of Specificity, Accuracy, Linearity, Range, Precision, Intermediate-Precision, Repeatability, Reproducibility, Robustness, Ruggedness, Detection Limit, Quantization Limit.

 

Different Type of Testing Methods Used:

·       UV Spectroscopy Testing

·       HPLC Basis Analysis

·       Potentiometric Titration ³

11. UV SPECTROSCOPY TESTING: ^{5, 14, 22, 23}

11.1 Materials and Methods:

11.1.1 Material Used:

Esomeprazole Sodium, Sodium Hydroxide BP, Disodium Edetate BP, Sterile Sodium chloride 0.9% w/v, Sterile Water for Injection, Water for Injection [Distilled water] andSodium Chloride Acetonitrile (HPLC grade); Disodium hydrogen orthophosphate anhydrous (AR Grade); O-Phosphoric acid (GR Grade) and 0.1 M Hydrochloric Acid.

 

 

11.1.2 Instruments used:

FTIR, UV-Spectrophotometer, pH Meter Analytical balance, Ultrasonicator, Potentiometer, HPLC, Column: Hypersil BDS C8, 250 x 4.6 mm, 5μ.; LOD Cum vacuum Oven; Photo Stability chamber; Centrifuge.

 

11.2 Determination of wavelength of maximum absorption:

ʎ_max of Bulk solution Esomeprazole Sodium for injection at 16 mcg/ml concentration is = 301nm

 

Figure No.03 and 04: UV Spectra for Esomeprazole Sodium for Injection and Disodium Edetate [Placebo]

 

Table No. 02: UV Spectra Calibration curve for Disodium Edetate [Placebo]

Slope: 0.0191 Y Intercept: -0.0013 Coefficient [r]: 0.9997

 

Fig.No.05: UV Spectra Calibration curve for Disodium Edetate [Placebo]

 

Conclusion:

Excipients not interfering the scanning of API at solution-bulk stage

 

11.3 Stability Study:

Samples prepared for repeatability study were preserved for 24 h at room temperature and analyzed on the Following day to test for short-term stability

 

Observation Table No. 03: Bulk Solution Stability Study By UV- Sample and Standard

Acceptance Criteria:

Standard and sample solution be considered stable up to 24 hours as long as the assay lies within 95.0% to 105.0%.asdifference in test results are below than 2.0 against initial value.

 

Conclusion:

The Standard and Sample solution is stable up to 24 hours as the Assay is 97.1 %.and 97.2 % respectively

Sample Preparation used for analysis should be freshly prepared.

 

11.4 Linearity and Range:¹⁹

Esomeprazole Sodium Linearity Stock solution: Weigh accurately 42.548 mg of Esomeprazole Sodium WS (equivalent to Esomeprazole 40 mg) and transfer it in to a 100 mL volumetric flask, add about 50 mL of water and shake to dissolve it, makeup volume with water and mix well.

 

Working Standard:

Esomeprazole Sodium Absorbance at301nm: 0.6105

 

Table No. 04: Linearity Study by UV

Slope: 0.0391 Y Intercept: -0.0349 Coefficient [r]: 0.998

 

Figure No.06: Linearity Graph by zero order UV-Spectrophotometer

 

Acceptance Criteria:

1. Coefficient of determination (~) should be greater than 0.995.

2. Report the values for the slope and Y- intercept

 

Optical Characteristics:

Optical characteristics and statistical data of linearity for Esomeprazole Sodium by zero order derivative method are summarized in Table no. 05.

 

 

 

Table No.05: Optical characteristics for Esomeprazole Sodium

 

Conclusion:

From this study the concentration range over which the linear response for the analyte exists can be established.

The specified range covered in the validation study is 50 % to 150 % of target concentration where in the desired linearity has been demonstrated for the estimation of analyte.

 

11.5 Accuracy/Recovery Study:

Accuracy / recovery of method determined at following levels prepare in three set of each 50%; 100% and 150 % of nominal concentration of known component.

 

Table No.06: Data for Recovery Study of Esomeprazole Sodium

 

Acceptance Criteria:

Mean recovery at each level (n=3) should be within 98.0% to102.0% Mean RSD at each level (n=3) lessthan2.0%.

 

Conclusion:

This study concludes the range of concentration over which the method is accurate.

 

11.6 Precision:

Take 2.5 mL of the sample (equivalent to 40 mg of Esomeprazole) transfer it in to a 100 mL volumetric flask, add about 50 mL of water and shake well, makeup volume with same solution and mix well. Transfer 2.0 mL from this to 50 mL volumetric flask, makeup volume with water and mix well. (Final conc. 16 mcg / mL).

 

Observation:

The observation for standard solution is tabulated below.

 

Working Standard:

Esomeprazole Sodium Absorbance at 301nm: 0.6014

 

11.6.1 Repeatability:

Table No.07: Data for Repeatability Study of Esomeprazole Sodium

 

Standard Deviation: 0.0548 Acceptance Criteria:

RSD (n=6) for assay should be NMT 2.0%.

 

Conclusion:

The study concludes that repeatability (precision) of results obtained by this method.

 

Table No. 08 and 09: Analysis on First Day and Second Day

Set No.	Sample Conc.	Absorbance at 301 nm	% Assay	% Mean	% RSD	% RSD
I	16 ppm	0.6013	99.1	99.1%	0.058 %	0.059% ≈ 0.06%
	16 ppm	0.6012	99.0
	16 ppm	0.6012	99.0
II	14 ppm	0.5258	87.4	87.33%	0.066%
	14 ppm	0.5256	87.3
	14 ppm	0.5254	87.3
III	18 ppm	0.6498	108.0	107.9 %	0.054%
	18 ppm	0.6488	107.8
	18 ppm	0.6492	107.9

 

Absorbance at 301 nm	% Assay	% Mean	% RSD	% RSD
0.6015	100.0	99.9%	0.058 %	0.059% ≈ 0.06%
0.6010	99.9
0.6013	99.9
0.5260	87.4	87.4%	0.066%
0.5262	87.5
0.5255	87.3
0.6495	108.0	107.9 %	0.053%
0.6501	108.0
0.6493	107.9

 

11.6.2 Analysis Done by: Miss. X Y Z: Second Day:

Working Standard:

Esomeprazole Sodium Absorbance at 301nm: 0.6014

 

Observation:

The observation for sample solution is tabulated below

 

Acceptance Criteria:

RSD (n=6) for assay should not be more than 2.0%.

The difference in test results between normal and change condition should be not more than 3.0

 

Conclusion:

The study proves the ruggedness of test method by variability's like different analyst and different days i.e. Intraday and Inter-day precision assures the repeatability of test-results found within 2.0 % RSD.

 

11.7 Robustness:

Take 2.5 mL of sample (equivalent to 40mg of Esomeprazole) transfer it into a 100mL volumetric flask, add 50mL of water and shake well, makeup volume with same solution and mix well. Transfer 2.0mL from this to 50mL volumetric flask, makeup volume with water and mix well.

a) Change in wavelength in ± 1 nm

b) Decrease in the wavelength by -1 nm i.e. 301 to 300

 

Table No. 10 and 11: Analysis at increase in wavelength – Sample and Decrease in wavelength - Sample

 

 

Acceptance Criteria:

RSD (n=3) for assay should not be more than 2.0% The absolute difference in assay between normal and change condition should be not more than 3.0.

 

Conclusion:

The study proves the reliability of test method for minor changes in the conditions i.e. Increase in the wavelength by +1 nm i.e. 301 to 302

 

 

Acceptance Criteria:

RSD (n=3) for assay should not be more than 2.0%.

The absolute difference in assay between normal and change condition should be not more than 3.0

 

Conclusion:

The study proves the reliability of test method for minor changes in the conditions

Increase in the wavelength by +1 nm i.e. 301 to 300.

 

11.8 LOD and LOQ:

Limit of Detection (LOD) and Limit of Quantization (LOQ): Table No. 12:

Formula	Standard Deviation	Slope	Results
LOD = 3.3 σ/S	0.0016	0.0391	0.135 %
LOQ = 10 σ/S	0.0016	0.0391	0.409 %

 

11.9 Specificity: [Selectivity]:

Specificity of an analytical method it is ability to measure the analyte accurately and specifically in the presence of components that may expect to be present in the sample matrix. Excipients and impurities were not interacting with the standard drug, hence method is specific.

 

Experiment: Standard Preparation:

Weigh accurately 43.18mg of Esomeprazole Sodium WS (equivalent to Esomeprazole 40 mg) and transfer it in to a 100mL volumetric flask, add about 50mL of water and shake to dissolve it, makeup volume with water and mix well. Transfer 2.0mL from this to 50mL volumetric flask, makeup volume with water and mix well.

 

Placebo Solution:

Take 30.30mg of EDTA, diluted to 50mL of water; adjust pH 11.2 with 0.1 M NaOH mix well.

 

Test Preparation:

Take 2.5 mL of the sample (equivalent to 40mg of Esomeprazole) transfer it in to a 100mL volumetric flask, add about 50 mL of water and shake well, makeup volume with same solution and mix well. Transfer 2.0mL from this to 50 mL volumetric flask, makeup volume with water and mix well.

 

Observation:

The observation for standard solution and sample solution are tabulated below.

 

Table No. 13: Placebo Interference

 

Figure No. 07: Overlay Spectrum for standard and sample solution Data Evaluation:

 

Acceptance Criteria:

Placebo and Diluent should not exhibit any absorption at known components. There should no interference from placebo and blank with analyte and report absorption maxima.

 

Conclusion:

Placebo and diluents does not exhibit any absorption at known components.

The spectra obtained from Esomeprazole Sodium bulk-solutions were identical with that obtained from standard containing an equivalent concentration of Esomeprazole Sodium. This showed that there was no any interference from Excipients. Therefore, it could be said that developed method is highly selective.

 

11.10 Analysis of Injectable Formulation for Purity i.e. Assay at Solution-bulk stage:

Standard Used for activity: 100.0% Standard Deviation: 0.0016 Slope: 0.0391

Absorbance of Solution at 301 nm: Standard: 0.6374[As] and Bulk Sample Solution: 0.6424 [At]

 

Table No. 14: Analysis of Injectable Formulation for Purity

 

                                    At       W        2.0        100        50     45.4         P  

% Esomeprazole = ------ x ------x------- x ----------x------x-----------x  ------x  2.5  x 100

                                   As    100      50         V x LC    2.0      367.4       100

 

 

                                 0.6424        42.548       2.0           100        50        345.4      100  

% Esomeprazole = ------------ x ----------x------- x ------------x------x-----------x  ------x  2.5  x 100   

                                  0.6374         100        50x2.5     40           2.0    367.4        100

i.e. Results    = 100.78 %

 

Conclusion:

Testing bulk-solution sample of Esomeprazole sodium for injection by validated method found satisfactory results and within acceptance criteria.

 

Table No. 15: Summary Results of UV Spectroscopy Validation

 

12.0 HPLC ANALYSIS:^6,7,8,11,13

12.1 Preparation of Buffer:

Dissolve 1.40 g of disodium hydrogen Ortho-phosphate anhydrous [Na₂HPO₄] in 1000.0mL of water, adjust pH to 7.6 (±0.1) with 10% v/v ortho-phosphoric acid.

 

12.2 Mobile Phase:

Mix buffer solution and Acetonitrile in ratio (73:27 v/v) degas it with ultrasonic bath, filter it through 0.45 μ N-66 Nylon membrane filter.

 

Figure No. 08: Mobile Phase Chromatogram

 

12.3 Preparation of standard stock and working standard of Drug Solution:

·       Preparation of Standard Solution:

Weigh accurately about 42.548mg of working standard of Esomeprazole sodium (equivalent to 40mg of Esomeprazole) and transfer into a 200mL volumetric flask. Add 170.0mL mobile Phase, keep the flask in ultrasonic minutes to dissolve the drug completely and make up the mark with mobile phase. (Conc. of Esomeprazole is 200 mcg/ml) bath for 5 to 10 min. Pipette out 5ml of above solution in 10ml volumetric flask and dilute up to the mark with mobile phase. (Conc. of Esomeprazole is 100mcg/ml) bath for 5 to 10 min.

 

·       Sample Preparation:

Pipette out 2.5ml sample solution (equivalent to 40mg of Esomeprazole) in 200ml dry volumetric flask, add 170ml of Mobile Phase, Sonicate it for 5 to 10 minutes and make up the mark with Mobile Phase. (Conc. of Esomeprazole is 200mcg/ml) Pipette out 5ml of above solution in 10ml volumetric flask and dilute up to the mark with mobile phase. (Conc. of Esomeprazole is 100mcg/ml)

 

·       Procedure for Injection:

Separately inject equal volume of about 20 Micro liters of Standard Preparation in replicate and sample preparation in duplicate into the equilibrated HPLC system and record the Chromatograms and measure the response of the major peak due to Esomeprazole Sodium.

 

Table No. 16: Stability of analytical solution at room temperature for Standard and Bulk Sample

Sr. No.	Time	Area of Esomeprazole	% Correlation	% Label Claim of Esomeprazole	% Correlation
1.	Initial Sample	1827029	----	100.7	----
2.	5 Hours Sample	1821382	99.7	100.5	99.8
3.	18 Hours Sample	1816579	99.4	100.1	99.4
4.	26 Hours Sample	1813721	99.3	100.4	99.7

Acceptance criteria:

Correlation of old sample solution and standard solution against initial assay is between 98.0 to 102.0%.

 

Conclusion:

Standard and sample solutions are stable up to 26 hours at room temperature.

 

Table No. 17: Summary Results of Forced Degradation Studies

Sr. No.	Name	Condition	RT	Purity Angle	Purity Threshold	Purity Criteria	% Assay	% Degradation
1.	Acid degradation	0.01M HCI 70°C/60min	7.489	0.094	1.030	Pass	100.0	--
2.	Base degradation	0.1M NaOH 70°C/60min	7.488	0.459	1.059	Pass	93.6	7.1
3.	Peroxide degradation	1.0% v/v H2O2 Room Temp/120min	7.487	0.066	1.044	Pass	74.9	25.8
4.	Thermal degradation	105°C 24 hours	7.512	0.084	1.028	Pass	98.1	2.6
5.	Photolytic degradation	1.2 million Lux hours	7.512	0.100	1.031	Pass	98.5	2.2
6.	Humidity degradation	25°C/92% RH 24 hours	7.519	0.108	1.037	Pass	96.1	4.6

 

12.4 Validation of Analytical Method:

The developed analytical method was further subjected to validation in pursuance of ICH Q2 (R1) guidelines. The parameters evaluated were system suitability, specificity, linearity, precision, accuracy and robustness, limit of detection (LOD) and limit of quantitation (LOQ).

 

12.5 System Suitability:

Relative Standard deviation for five replicate injection of standard solution should NMT 2%

Number of Theoretical Plate NLT 2000 Tailing Factor: NMT 2

 

Table No. 18: Table for System Suitability

 

 

Acceptance Criteria:

The USP Theoretical plate should not be less than 2000. The USP Tailing should not be more than 2.0. The relative standard deviation of five replicate injections should not be more than 2.0%.

 

Conclusion:

The above results indicate that the system is suitable for all sample sequence and conditions outlined in the method.

 

 

12.6 Summary of Data of HPLC Method Validation:

Table No. 19: Summary of Data of HPLC Method Validation

Sr. No.	Validation Parameter	Acceptance Criteria	Results
1.0 1.1   1.2   1.3	Specificity Selectivity   Placebo interference   Forced degradation	Results should be comparable with respect to retention time. Peak purity should pass for Esomeprazole peak. Mobile phase and placebo should not show any peak at the Retention time of Esomeprazole peak. Esomeprazole peak should be homogeneous and there should be no co-elutic peaks. Peak purity should pass.	Retention time of Esomeprazole peak in sample preparation is comparable with respect to retention time of Esomeprazole peak in standard preparation. Peak purity passes for Esomeprazole peak in standard and sample preparations. No interference was observed at the retention time of Esomeprazole peak. Peak purity passes for Esomeprazole peak in all Degradation conditions.
2.0	Linearity and Range	Correlation coefficient should not be less than 0.999	Correlation coefficient is 0.99989
3.0	Accuracy (Recovery)	Mean recovery should be in the range of 98.0% to 102.0%. The RSD should not be more than 2.0%.	The mean recovery is 100.4 % and RSD is 0.678 %.
4.0 4.1 4.2	Precision System precision Method precision	RSD should not be more than 2.0%. RSD should not be more than 2.0%.	RSD is 0.170 %. RSD is 0.098 %.
5.0	Ruggedness	Overall RSD for twelve results obtained from two different analyst should NMT 2.0%	Overall RSD for twelve results obtained from two different analysts are 0.285 %.
6.0	Stability in analytical solution at room temperature	Correlation of room temperature sample solution and standard solution at different time interval against initial assay is between 98.0 % to 102.0 %	Standard and sample solutions are stable for 26 hours at room temperature.
7.0	Robustness	System suitability should meet as per the test method at each variable condition. Overall RSD should not be more than 2.0 % for the results obtained at control and variable conditions.	The test method is robust for all variable conditions.
8.0	System suitability	The USP theoretical plates should not be less than 2000. The USP tailing should not be more than 2.0. The relative standard deviation for five replicate Injection of standard solution should not be more than 2.0%.	Complies   Complies
9.0	LOD	Limit of Detection (LOD) (μg/mL) LOD = 3.3 σ / s	0.598
10.0	LOQ	Limit of Quantitation (LOQ) (μg/mL) LOQ = 10 σ /s	1.814

 

12.7 Summary of Report:

The test method was validated for Specificity, Linearity and Range, Precision, Accuracy, Ruggedness, Stability of Analytical Solution, Robustness and System Suitability was found to meeting the predetermined acceptance criteria. The validated method is Specific, Linear, Precise, Accurate, Robust and Rugged for determination of assay for Esomeprazole in Esomeprazole sodium for Injection. Hence this method can be introduced into routine use and stability for the assay test of Esomeprazole in Esomeprazole sodium for Injection at solution-bulk stage. However time and cost analysis will matter

 

13. POTENTIOMETRIC TITRATION METHOD:

13.1 Preparation of Standard Stock Solution:

Standard stock solution of Esomeprazole Sodium was prepared by accurately weighing 0.300g of Esomeprazole Sodium to 50 ml volumetric flask with WFI and shake well and sonicated to dissolve it. [6000 ppm Esomeprazole Sodium] Titrate this with 0.1 M hydrochloric acid. Determine the end-point by potentiometric titration.

 

Factor:

1 mL of 0.1 M hydrochloric acid equivalent to 36.4 mg of Esomeprazole Sodium

 

Formula:

300mg x 1ml x100/ 36.4 x 8.24 ml

 

 

 

 

Table No. 20: Potentiometric Reading for Standard

 

CONCLUSION:

Standard solution found comply within acceptance criteria i.e. less than 2.0 % RSD against theoretical potency.

 

13.2 Placebo Interference:

Procedure:

Prepare placebo solution for Esomeprazole sodium for Injection. Placebo should be in proportion to Standard stock solution and also with bulk formulation. i.e. 52.85 mg of Disodium Edetate add in 235ml WFI, adjust pH with 5% Sodium Hydroxide with help of 56.4 mg [59.89 mg NaOH / 1.2 ml Qs. WFI] at pH11.40 to 11.60. Make up volume q. s. to 250 ml in volumetric flask. Take 50 ml Placebo and titrate this with 0.1 M hydrochloric acid. Determine the end-point by potentiometric titration.

 

Table No. 21: Potentiometric Reading for Placebo

 

13.3 Sample Preparation:

Take 17.63 mL of the bulk sample dilute to Q.S. 50 ml in volumetric flask and mix well. to get the concentration equivalent to 6000 mcg/ml i.e. 6000 ppm of Esomeprazole sodium. Titrate this with 0.1 M hydrochloric acid. Determine the end-point by potentiometric titration.

 

Table No. 22: Potentiometric Reading for Sample

 

Conclusion:

Sample of bulk solution found non-complied to acceptance criteria. %RSD was observed 20.31 i.e. more than 2.0%

 

13.4 Summary Report of Potentiometric Titration Method:

Potentiometric Titration method was suitable and found working as per EU monograph for active ingredients [API] - Esomeprazole sodium. However it's not found working at bulk-solution stage, it may be due to the interference of added excipient in the bulk solution. As Placebo Interference not complied during study, hence method validation study by Potentiometric Titration was dropped / discontinued for Esomeprazole sodium at solution – bulk stage.

 

14. RESULTS AND DISCUSSION:

14.1 Summary:

In the present research work, a successful attempt was made for determination of Esomeprazole Sodium in injectable dosage form at Solution-bulk stage by UV-Visible Spectrophotometric, HPLC and Potentiometric titration. The method developed by experimentation, based on literature survey. The simplicity, rapidity, reproducibility and economy of the proposed method completely fulfill the objective of this research work.

 

14.1.1 In UV- spectrophotometer Water [WFI] used as solvent and detection done at 301 nm.The % RSD for all parameters found within 2. The result showed that proposed method is suitable, accurate, precise and rapid determination of Esomeprazole n its Solution-bulk stage of injectable dosage form.

 

14.1.2 The HPLC method developed and validated for estimation of Esomeprazole Sodium. The mobile phase was consisting of Buffer Solution: Acetonitrile (73:27 v/v). Detection done at 280nm. The method found simple, accurate, precise, reproducible and robust. The % RSD was found within limit. The result showed that proposed method suitable, accurate and precise determination of Esomeprazole in its Solution-bulk stage of injectable dosage. However Time required for testing is more and Analysis cost is more than as compared to UV- spectrophotometer test method.

 

14.1.3 The potentiometric titration method as per [EU 9.7] was studied for Active ingredient i.e. Esomeprazole Sodium found complied. Same method studied and tried its implementation and validation to estimation of Esomeprazole Sodium in Solution-Bulk stage of formulation with 0.1 M hydrochloric acid with Solution-bulk of with other Excipients. However detection done through potentiometrically end-point determination was not observed as its shows the interference due to Excipients used in injectable formulation. Hence potentiometric titration method not found suitable for estimation of Esomeprazole Sodium in Solution-Bulk stage of formulation.

 

14.2 CONCLUSION:

After research work, it has been conclude that Esomeprazole Sodium for injection in its Solution-bulk stage of injectable dosage form can be determine with UV-Spectroscopy method as method is validated and proved as Simple, Accurate, Precise, Economical and Rapid to save time and cost with increase in productivity of manufacturing line.

Implementation of the UV-Spectroscopy based method will help to:

 

·       Reduce the Assay testing cost.

·       Reduce the Testing period assay results in minimum time

·       Less sample quantity required

·       More Practical approach to increase and enhance line productivity

·       Reduce the risk related bio-burden of bulk - solution of product.

·       More accurate in compression with respect to Potentiometer Titration

·       Time period required less for sample preparation and testing.

·       Test method based on UV-Spectroscopy is accurate equally to HPLC test method results.

 

15. ACKNOWLEDGEMENTS:

The author would like to thank the Management and Staff of R. G. Sapkal College of Pharmacy, Anjaneri, Nashik, Maharashtra, India – 422213 for providing the facilities to perform research work.

 

16. CONFLICT of INTEREST:

Authors declare no conflicts of interest.

 

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Received on 20.12.2020       Modified on 22.02.2021 Accepted on 25.03.2021      ©A and V Publications All right reserved Research J. Science and Tech. 2021; 13(3):177-192. DOI: 10.52711/2349-2988.2021.00028