Development and Validation of UV Spectrophotometric Method of Pirenzepine Dihydrochloride in API and Stress Degradation Studies

 

Archana Rajmane1, Rahul Trivedi2, Tanaji Nandgude3

1Research Scholar, B. R. Nahata College of Pharmacy, Mandsaur University, Mandsaur, 458001, MP, India.

2Associate Professor, B. R. Nahata College of Pharmacy, Department of Pharmacology,

Mandsaur University, Mandsaur MP, India.

3Associate Professor, Dr. D. Y. Patil Institute of Pharmaceutical Science and Research,

Pimpri, Pune, 411018, Maharashtra, India.

*Corresponding Author E-mail: archurajmae@gmail.com, rahul.trivedi@meu.edu.in, tanajinandgude@gmail.com

 

ABSTRACT:

A simple, accurate, precise spectrophotometric method was developed for the detection of Pirenzepine dihydrochloride (PZC). The optimum condition for the analysis of the drug was studied. PZC was subjected to stress degradation under different conditions like acidic, alkali, neutral, oxidation, photolytic, and thermal degradation as per recommended by the International Conference on Harmonization (ICH). The samples thus prepared were used for degradation studies by with the developed method. The lambda max i.e. absorption maxima found at 281nm and calibration curve linear over the range of 0-20µg/ml. The standard regression equation and correlation coefficient fond to be y = 0.04x + 0.056 R˛ = 0.998 respectively. Percent   RSD found to be less than one. The accepted limits of accuracy (recovery) were found to be 90% - 120% and all observed data are within required range which indicates good recovery value. and LOD and LOQ found to be 0.165 and 0.500 respectively by simple UV spectroscopy.

 

KEYWORDS: Pirenzepine dihydrochloride, UV Spectrophotometric Method, accuracy, Precision, Stress Degradation studies.

 

 


INTRODUCTION:

 

 

Pirenzepine dihydrochloride chemically known as 5,11Dihydro-11-[(4-methyl-1piperazinyl) acetyl]-6H-pyrido[2,3-b][1,4]benzodiazepine 6-one dihydrochloride monohydrate.

 

It is an M1 selective antagonist, is used in the treatment of peptic ulcers, as it reduces gastric acid secretion and reduces muscle spasm. It is in a class of drugs known as muscarinic receptor antagonists - acetylcholine being the neurotransmitter of the parasympathetic nervous system which initiates the rest-and-digest state,1 in an increase in gastric motility and digestion; whereas pirenzepine would inhibit these actions and cause decreased gastric motility leading to delayed gastric emptying and constipation. It has no effects on the brain and spinal cord as it cannot diffuse through the blood–brain barrier. Analysis is an important component in the formulation development of any drug molecule. A suitable method has to be available for the analysis of drug (s) in the bulk and finished dosage form. The literature survey reveals that Pirenzepine dihydrochloride was analyzed by the HPTLC2, RP-HPLC method. but Ultraviolet (UV) estimation of Pirenzepine dihydrochloride has not been reported in bulk or in pharmaceutical formulation. In This study, UV/Visible absorption spectrophotometric method for the estimation of Pirenzepine dihydrochloride in bulk drug has been developed. Method Were Validated by performing accuracy, precision, sensitivity, linearity, Limit of Detection and Limit of Quantification ((LOD and LOQ), Ruggedness As per the ICH Guidelines.3-8

 

MATERIAL AND METHODS:

Chemicals and Reagents:

Analytically pure sample of Pirenzepine Dihydrochloride With purity greater than 99% was obtained as gift sample from clear synth lab, Mumbai, India. All Other chemicals and solvents used were of analytical grade.

 

Instruments:

A Double beam UV-Visible Spectrophotometer (Shimadzu UV-1900) with a spectral bandwidth of 1nm, wavelength accuracy of 0.5nm and a pair of 1cm quartz cells were used to Measure the absorbance of the resulting solutions. An electronic analytical weighing balance (0.1mg sensitivity, Shimadzu AY 220), and a sonicator (Sonica, model 2200 MH) were used in this study.

 

Selection of solvent:

1gm of Pirenzepine Dihydrochloride was weighed and solubility was checked in 10ml water, ethanol, 0.1N NaOH and 0.1 N HCl. The drug was found to be freely soluble in ethanol, water and practically poorly soluble in 0.1N NaOH and 0.1NHCl. Therefore methanol and water was selected as diluents and Pirenzepine Dihydrochloride was also found to be stable in water and ethanol for 48 hours in stability studies

 

Preparation of standard stock solutions:

Transfer 10mg of pure Pirenzepine Dihydrochloride In 10ml volumetric flask containing 5ml of water and ethanol in 70:30 proportion as diluent and then sonicated for 15 minutes and final make up the volume up to 10ml with solvent. the final strength was1000µg/ml

 

Preparation of Working Standard Solution:

From above standard stock solution 5ml was further diluted to 50ml with solvent followed by sonication for 5 minutes. The final strength was 100µg/ml. The stock was used to prepare various concentrations from 2-20 µg/ml by dilution with solvent.

 

Selection of Wavelength for Analysis of Pirenzepine Dihydrochloride:

 Appropriate volume 1ml of working stock solution of Pirenzepine dihydrochloride Was transferred into 10ml volumetric flask, diluted with solvent up to the mark to give a concentration 10µg/ml. The resulting solution was scanned between 200-400nm. Absorbance of these solutions was recorded at 281nm against water and ethanol (70:30) as blank using UV-visible spectrophotometer

 

Validation of UV Spectroscopic method:

The method developed was validated for the following parameters according to the ICH Guidelines Q2 (R1): Validation of Analytical Procedures: Text and Methodology.

 

Preparation of standard calibration curve:

From above std. working solution of Pirenzepine dihydrochloride (100μg/ml), pipette out aliquots 0.2 to 2 ml of Pirenzepine Dihydrochloride and transferred to series of 10ml volumetric flasks and final volume made up to mark with water and ethanol (70:30) as diluent to form solutions of 2 to 20μg/ml of Pirenzepine dihydrochloride. These Solutions were then scanned at 281nm against diluent as blank. Then the calibration curve was plotted as absorbance vs concentration.9

 

Precision:

The intra-day and inter-day precisions of developed methods was measured by estimating thrice corresponding response on present day and on three different days, over a period of one week and the results were reported in terms of relative standard deviation10

 

Repeatability:

By analyzing six samples of same drug concentrations (10μg/ml) the repeatability was determined. From the resulting absorbance SD and RSD were calculated11

 

Accuracy:

The accuracy of the developed method is calculated by comparing closeness of the observed value to the standard value for the sample. Recovery study was performed by addition level of 80, 100 and 120% for test solution and absorbance of each measured in triplicate12

 

Limit of Detection and Limit of Quantification ((LOD and LOQ):

It is the lowest concentration of analyte in the sample that can be detected but not necessarily quantified. LOD and LOQ were calculated with help of response along with its standard deviation as per ICH guidelines. LOD and LOQ were calculated using formula13

 

LOD = 3.3 *σ/s

 

The smallest possible quantity of analyte that can be measured quantitatively.

 

LOQ = 10 *σ/s

Where σ is the standard deviation   and S is the slope 

 

Degradation Studies:

The ICH guidelines allowed stability testing of new drug substances and products that required stress testing to be carried out to elucidate the inherent stability characteristics of the active substance. The aim of this

study was to perform the stress degradation studies on Pirenzepine dihydrochloride using the method developed.14-15

 

Stress Degradation by Hydrolysis under Acidic Condition:

A stock solution of Pirenzepine dihydrochloride was prepared by dissolving 10 mg of the drug in 10ml of ethanol and water (30:70) to produce 1000μg/ml of the solution. To 1ml of the stock solution, 1ml of 1 N HCL was added in a 10ml volumetric flask and the volume was make upto the mark using solvent. Three sample prepared and these volumetric flask was kept under normal conditions for 1hr. 2hr, and 4hr. then make the solution neutral by adding 1m NaOH solution and absorbance were checked. sample were tested in triplicate

 

Stress Degradation by Hydrolysis under Alkaline Condition:

A stock solution of Pirenzepine dihydrochloride was prepared by dissolving 10 mg of the drug in 10ml of ethanol and water (30:70) to produce 1000μg/ml of the solution. To 1ml of the stock solution, 1ml of 1 N NaOH was added in a 10ml volumetric flask and the volume was make upto the mark using solvent. Three sample prepared and these volumetric flask was kept under normal conditions for 1hr.2hr, and 4hr. then make the solution neutral by adding 1m HCl solution and absorbance were checked. sample were tested in triplicate

 

Dry Heat -Induced Degradation:

To three amber colored 10ml volumetric flasks sample solution containing 1ml aliquot of Pirenzepine dihydrochloride was transferred and flasks were kept on water bath for 1 hr, 2 hr, 4 hr at 60°C ± 2°C then diluted with solvent. All three samples were tested in triplicate

 

Oxidative Degradation:

To 1.5ml of the stock solution of Pirenzepine Dihydrochloride (1000μg/ml), 1ml of 30% w/v of hydrogen peroxide was added in a 10ml volumetric flask and the volume was made up to the mark with ethanol and water (30:70). The volumetric flask was kept at room temperature for 1hr., 2hr. and 4hr. dilutions were made from the stock solution to achieve the required concentration (6μg/ml). The solution was further analyzed with the help of a UV-VIS spectrophotometer16All three solution were then tested in triplicate

 

Photolytic Degradation:

To a clear volumetric flasks, 1ml sample was added and then exposed to direct UV light for 1, 2 and 4 Hour. All three solution were then tested in triplicate.17

RESULTS AND DISCUSSION:

Determination of absorption maxima (λ max):

The standard stock solution of Pirenzepine Dihydrochloride Having the concentration1000μg/ml was further diluted to 100μg/ml with water and ethanol (70:30). The absorbance of solution was scanned in the range of 200-400nm. The λmax was found to be 281nm as shown in figure 1.

 

 

Fig. -1 Absorption Maxima of Pirenzepine dihydrochloride

 

Linearity:

The linearity study of the Pirenzepine was performed by plotting different concentrations of standard solution against their respective absorbance as shown in and figure 2.3 and 4The calibration curve was found to be linear having R2 value 0.998in the concentration range of 0-20μg/ml.

 

 

Fig: 2standard Calibration Curve of Pirenzepine dihydrochloride Linearity 1 at 281nm

 

 

Fig: 3 Standard Calibration Curve of Pirenzepine dihydrochloride Linearity 2at 281nm

 

Fig: 4 Standard Calibration Curve of Pirenzepine dihydrochloride Linearity 3at 281nm

 

 

Fig: 5 Standard Calibration Mean Curve of Pirenzepine dihydrochloride at 281nm

 

Precision:

Intraday precision:

The intraday precision was determined by analyzing the drug in particular concentration for three times on the same day taking the time intervals of 4 h at 8:00 am, 12:00 noon and 4:00 pm respectively.

 

Inter-day precision:

Precision was determined by measuring values of precision for 03 consecutive days. The values of relative standard deviation (%RSD) were in the range of 0.153 -0.454 % respectively. This indicates the reproducibility of the method. Results were shown in table 2. Results of precision shows that the current method is reliable and repeatable. Thus, the methodology can be applied for the determination of Pirenzepine in different formulations and bulk in treatment of Peptic Ulcer treated by Pirenzepine.18

 

Repeatability:

The repeatability of the developed method was validated by taking the absorbance of six samples of the same concentration (10 μg/ml) The SD and %RSD was in the given limits as shown in table 2. The Repeatability of the methodology is significant for routine and frequent result analysis of drugs in API. Result Conforms that results remains unchanged on repetition of developed methods.19

 

Accuracy:

Accuracy was determined by means of recovery experiments, by the determination of % mean recovery of sample at three different levels (80-120%). At each level, three determinations were performed. Percent mean recovery was calculated as shown in Table3 at 281 nm. The accepted limits of recovery are 90% - 120% and all observed data are within the required range which indicates good recovery values. Hence the accuracy of the method developed shown in Table 4.

 

Ruggedness:

Ruggedness was determined by carrying out analysis by two different analysts and the respective percentage recovery was noted and the results were indicated as % RSD found as follows

 

Analyst 1: Percentage recovery of Pirenzepine Dihydrochloride at 281nm is 104.276 ± 1.005

Analyst 2: Percentage recovery of Pirenzepine dihydrochloride at 281nm is 106.229 ± 1.580


 

Table 01: Results for Intra-day and Inter-day precision of Pirenzepine

Drug

Conc. (µg/ml)

Intra-day Mean Abs.

Absorbance

± S.D.

%RSD

Inter-day Mean Abs.

Absorbance

± S.D.

% RSD

 

Pirenzepine

10

0.470

± 0.002

0.425

0.471

± 0.002

0.424

15

0.651

± 0.001

0.153

0.653

± 0.001

0.153

20

0.859

± 0.003

0.349

0.660

± 0.003

0.454

Mean %RSD

0.309

0.343

*Each value represents mean ± S.D. of three observations

 


Table no. 2: Data showing Repeatability of Absorbance

Sr. No.

Conc. (µg/ml)

Absorbance

Mean ± S.D.

% R.S.D

1

 

 

10

0.470

0.471±0.002

0.224

2

0.471

 

 

3

0.471

 

 

4

0.472

 

 

5

0.471

 

 

S.D. = Standard Deviation, R.S.D. = Relative Standard Deviation

 

Table 3: Accuracy Study of Pirenzepine Hydrochloride At 281nm

Level

Amount added

Percentage recovery

% RSD

80%

12 (µg/ml)

106.439 ± 0.758

0.712 12 (µg/ml)

100%

15 (µg/ml)

105.960 ± 0.763

0.720 15 (µg/ml)

120%

18 (µg/ml)

99.663 ± 0.771

0.774 18 (µg/ml)

 

Summary of Validation Parameters:

Table 4: Summary of validation parameters

Sr. No.

Parameters

Results

1

Absorption maxima (nm)

281 nm

2

Linearity range (μg/ml)

0-20 µg/ml

3

Standard Regression Equation

y = 0.04x + 0.056

4

Correlation coefficient (R2)

= 0.999

5

Specificity

A 10 µg /ml solution of Pirenzepine Dihydrochloride in water and ethanol (70:30) as a solvent at UV detection of 281 nm will show an absorbance value of 0.471±0.002

6

Precision RSD Repeatability (n=6)

Intra-day(n=3)

Inter-day(n=3)

 

 0.224

 0.309

0.343

9

LOD

0.165 µg /ml

10

LOQ

0.500 µg /ml

 

Summary of Stress Degradation Study:

The effect of Acid/Base hydrolysis, Oxidation, Photo Degradation, Heat-induced degradation on the spectra was observed. On acid hydrolysis Pirenzepine dihydrochloride do not showed any significant degradation or no additional peak of sample after 1hr, 2hr and 4hr at different process. In other cases there was minor shift in peak that was not significant

 

Table 5: Summary of Stress Degradation Study

Degradation condition

Observed peak-281nm

Time (Hr)

Reported peak

1 N HCl 1ml.

1

281nm

2

281

4

281

0.1 N NaOH 1ml

1

281

2

281

4

282

30% w/v of hydrogen peroxide 1ml

1

281

2

281

4

280

Dry heat 70o C

1

281

2

280

4

277

Photostability chamber

1

281

2

280

4

278

 

CONCLUSION:

A simple, accurate, precise and cost-effective UV-spectroscopic method has been developed for the estimation of Pirenzepine Dihydrochloride. The proposed method is successfully applied for estimation of drug in any formulation. The method can be used for the routine quality control analysis of Pirenzepine Dihydrochloride. In force degradation studies as Acid/Base hydrolysis, Oxidation, Photo Degradation, Heat-induced degradation the spectra for acid degradation of Pirenzepine dihydrochloride do not showed any significant degradation or no additional peak of sample after 1hr, 2hr and 4hr at different process

 

REFERENCE:

1.      Humeida El-Obeid et.al. Analytical Profile of Pirenzepine dihydrochloride, Academic Press, New York 1987;16(1): 447-483.

2.      J. Tu. et. al. HPLC determination of pirenzepine dihydrochloride in rabbit aqueous humor. J Chromatogr B Analyt Technol Biomed Life Science.2005. August; 822(1-2):300-3. DOI: 10.1016/j.jchromb.2005.03.026.

3.      Galatage ST et.al. Determination Of Amphotericin B In Bulk And Pharmaceutical Formulation By Spectrophotometer. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2020May;11(3):31-37.

4.      ICH Harmonized Tripartite Guideline Validation of Analytical Procedures: Text and Methodology, Q2(R1), Current Step 4Version, Parent Guidelines on Methodology,1996.

5.      Patil SM, Galatage ST, Patil AB. Development Of Validated UV Spectrophotometric Method For Estimation Of Fosfestrol In Bulk and Pharmaceutical Dosage Form. World Journal of Pharmaceutical Research. 2013;6(3):2345-2353.

6.      Hiremath R. et.al. Development and Validation of UV Spectrophotometric Area Under Curve (AUC) Method for Sildenafil Citrate in Pharmaceutical Formulation. Research Journal of Pharmacy and Technology. 2016;9(3):235-237.

7.      Sumithra M, Yuvanesh P, Mistry A. Analytical method development and validation of ambroxol hydrochloride by UV spectroscopy and forced degradation study and detection of stability. Research Journal of Pharmacy and Technology. 2016; 9(7):794-800.

8.      Pravallika KE et.al. Development and validation of UV spectrophotometric methods for the estimation of vortioxetine hydrobromide in bulk and pharmaceutical dosage forms. Research Journal of Pharmacy and Technology.(2017); 10(11): 3928-3932.

9.      Sushma G.et.al New first derivative spectrophotometric methods for the determination of Sitagliptin - An antidiabetic agent. Research Journal of Pharmacy and Technology.2020;13(6): 2838-2842.

10.   Bandgar SA, Jadhav NR. Validated UV Spectrophotometric method for Estimation of Simvastatin in Bulk and Pharmaceutical Formulation. Research Journal of Pharmacy and Technology. 2019; 12(12): 5745-5748.

11.   Manideep G. et.al. Development and Validation of A UV Spectroscopic Method to Estimate Etoricoxib in Bulk and Tablet Formulation. Research Journal of Pharmacy and Technology 2018; 11(2):758-760.

12.   Kumar S. et.al. UV Spectrophotometric Method Development and Validation for the Estimation of Gliclazide in Bulk and Pharmaceutical Dosage Form. Asian Journal of Pharma Analysis 2016; 6(3): 143-146.

13.    Raut ID, Doijad RC, Mohite SK. Validated Spectrophotometric method for estimation of Paclitaxel in Bulk and Pharmaceutical Formulation. International Journal of Pharmacy. 2017; 7(1): 68-72.

14.   Sreejith KR, Rajagopal PL, Premaletha K. Analytical Method Development and Validation of Anastrozole in Pure and Tablet Dosage Form by UV Spectroscopy Research Journal of Pharmacy and Technology. 2017;10(4), 1015-1019.

15.   Mulsa N.et. al. Development of the UV Spectrophotometric Method of Phenytoin Sodium in API and Stress Degradation Studies. Inventi Rapid: Pharm Analysis and Quality Assurance. 2013; (1): 1-5.

16.   Kaul N.et.al. The ICH Guidance in Practice: Stress Degradation Studies on Stavudine and Development of a Validated Specific Stability-Indicating HPTLC Assay Method, Journal of Chromatographic Science. 2005; (43):406-415.

17.    Hussain QS, Bukhari K. Method Development and Stress Degradation Profile of 5-Benzyl-1,3,4-Oxadiazole-2-Thiol Studied by UV Spectroscopy. Pharmaceutical Chemistry Journal. 2018; (52):278–283

18.   Patil SM, Galatage ST, Choudhari AU. Delopement Of UV Spectrophotometric Method For Estimation Of Letrozole In Pure And Pharmaceutical Dosage Form. Indo American Journal of Pharmaceutical Research. 2018 May;8(04):1080-1085.

19.   Galatage ST, Trivedi R, Bhagwat DA. Characterization of camptothecin by analytical methods and determination of anticancer potential against prostate cancer. Future Journal of Pharmaceutical science. 2021May7;104:1-9. doi.org/10.1186/s 43094- 021- 00236-0.

 

 

 

Received on 12.02.2021           Modified on 17.06.2021

Accepted on 26.10.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(6):2503-2507.

DOI: 10.52711/0974-360X.2022.00418