Analytical Methods of Estimation of Oxetacaine and its quality determinations in different Pharmaceutical Formulations by Reverse Phase High Performance Liquid Chromatography (RP-HPLC)

 

Kirubakaran. N.¹*, Umashankar M. S.², Bhuvaneshwari S.³

^1,3Research Scholar, Department of Pharmaceutics, SRM College of Pharmacy,

SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Tamil Nadu 603203.

²Professor, Department of Pharmaceutics, SRM College of Pharmacy,

SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Tamil Nadu 603203.

 

ABSTRACT:

Objective: The aim of the current research was to design and develop an analytical method for the estimation of Oxetacaine and its quality control determinations of different formulation by reverse phase high performance liquid chromatography (RP-HPLC). Method: In Method I the Agilent HPLC used for the Estimation purpose. C-18 column (250 X 4.6mm i.e., 1µ) as stationary phase and 0.05% formic acid buffer: Methanol in the ratio 30:70 % v/v as mobile phase, with a flow rate of 1.0ml/min. Diluents of Methanol: water 90:10 ratio used for the study with ambient Temp conditions. In Method II the Agilent HPLC used for the Estimation purpose .C8 Column ((250 X 4.6mm i.e., 5µ) as stationary phase and ammonium phosphate buffer pH 4.8: Acetonitrile in the ratio 25:75 % v/v as mobile phase, with a flow rate of 0.9ml/min.  Diluents of Acetonitryl: water 50:50 ratios used for the study with ambient Temp conditions. Result: Method 1: Oxetacaine standard (86µg/ml concentration) peak was eluted at average of 5.629min at 3 runs with Asymmetry of 1.29813 and Peak area of 23237896 with the conditions of %RSD of max2. With qualifying of system suitability, accuracy and reproducibility. Some of Marketed suspension formulations were operated under the same instrument conditions and the results were compared with standard Oxetacaine to ensure the percentage purity of Marketed suspensions. Method 2: Oxetacaine standard (196µg/ml concentration) peak was eluted at average of 4.884 min at 3 runs with Asymmetryof1.17244 and Peak area of 360543170 with the conditions of %RSD of max 2. With qualifying of system suitability, accuracy and reproducibility. Some of Marketed suspension formulations were operated under the same instrument conditions and the results were compared with standard Oxetacaine to ensure the percentage purity of Marketed suspensions. Conclusion: The two new method of estimation of oxetacaine like using Method 1: C18 column, 0.05% formic acid buffer: Methanol in the ratio 30:70 % v/v as mobile phase @214nm and Method 2: C8 Column, ammonium phosphate buffer pH 4.8: Acetonitrile in the ratio 25:75 % v/v as mobile phase @214 achieved with simple, precise, accurate and sensitive. In addition the quality control estimating of some of random of F1, F2, F3, F4, F5, and F6 marketed oxetacaine suspensions were analysed using this Two Optimized methods.  Out of 6 formulations, 2 formulations F3, F6 determined as substandard quality on the both the methods. Hence proving these two new methods optimal for the further monograph inclusions as well as quality control predictions of oxetacaine in API as well as Finished Pharmaceutical Formulations.

 

 

 

 

 

 

INTRODUCTION: 

Oxetacaine is an anesthetic antacid used to treat gastritis, peptic ulcer disease, heartburn, esophagitis and anorexia. Oxetacaine inhibits gastric acid secretion by suppressing gastrin secretion.^1,2,3 Chemically Oxetacaine was N, N-bis-(N-methyl-N-phenyl-t-butyl-acetamide)-beta-hydroxyethylamine. The aim of the current research was to design and develop an analytical method for the estimation of Oxetacaine and its quality control determinations of different formulation by reverse phase high performance liquid chromatography                        (RP-HPLC).^{4, 5,6,7} the real Challengeis still now there is no HPLC Method for offficial IP Monograph. Only Non acquous titration method available in BP. To overcome the above various Methods tried to estimate the oxetacaine in the commercial formulations by reverse phase high performance liquid chromatography (RP-HPLC). ^8,9,10,11,12

 

Chemical structure of Oxetacaine^13,14

 

Oxetacaine to be estimated in different market formulations by the RP-HPLC by the systematic analysis protocols.^15,16,17,18 reversephase high performance liquid chromatography is the high end shopheticated technique in the modern analytical aspects with the principles of affinity and partitions.^19,20,21 the separations of active moiety by the suitable columns in the methods and appropriate detectors for the suitable wavelength by the conditioning of the flow rate of the mobile phase.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Working Standard of Oxetacaine was obtained from the manufacturers as gift sample. Acetonitrile, Methanol of HPLC grade, ammonium Phosphate AR grade were supplied by Rankem Chemicals. Water HPLC grade was used for the study.

 

Method I:

In Method I the Agilent HPLC used for the Estimation purpose. C-18 column (250 X 4.6mm i.e., 1µ) as stationary phase and 0.05% formic acid buffer: Methanol in the ratio 30:70% v/v as mobile phase, with a flow rate of 1.0ml/min. Diluents of Methanol: water 90:10 ratio used for the study with ambient Temp conditions.

 

Preparation of Formic acid Buffer solution:

0.05% of formic acid buffer solution was prepared by 50 µl of formic acid in 100ml of HPLC grade water and sonicated for 15minutes for uniform distribution.

 

 

Preparation of Oxetacaine standard:

Oxetacaine standard solution was prepared by dissolving Oxetacaine STD into Diluents (Methanol: Water 90:10 ratio) to obtained the 86µg/ml concentration

 

Preparation of Test Drug formulations:

Different Market Oxetacaine suspensions were purchased for the study and Coded F1, F2, F3, F4, F5, F6. The suspension was weighed in standardized flask of 50ml. add the  diluents up to 40ml and sonicate the sample around 10 minutes and make up the volume of 50ml with diluents. Shake few seconds and filter using 0.45µ filters. Collect the filtrate for further HPLC study.

 

Method II:

In Method II the Agilent HPLC used for the Estimation purpose. C8 Column ((250 X 4.6mm i.e., 5µ) as stationary phase and ammonium phosphate buffer pH 4.8: Acetonitrile in the ratio 25:75% v/v as mobile phase, with a flow rate of 0.9ml/min.  Diluents ofAcetonitryl: water 50:50 ratios used for the study with ambient Temp conditions.

 

Preparation of ammonium phosphate buffer pH 4.8 solution:

100mg of ammonium phosphate dissolved in 200ml of HPLC grade water and pH adjusted with 4.8 using orthophosphric acid and this buffer to be used within 24 hours for the study. Each day before staring the Experiment prepare freshly for the study.

 

Preparation of Oxetacaine standard:

Oxetacaine standard solution was prepared by dissolving Oxetacaine standard into Diluents (Acetonitryl: water 50:50 ratios) to obtained the 196µg/ml concentration

 

Preparation of Test Drug formulations:

Different Market Oxetacaine suspensions were purchased for the study and Coded F3, F6. The suspension was weighed in standardization flask of 50 ml. add the  diluents up to 40ml and sonicate the sample around 10 minutes and make up the volume of 50ml with diluents. Shake few seconds and filter using 0.45 µfilters. Collect the filtrate for further HPLC study.

 

RESULTS:

Method I: Standard Oxetacaine Solution:

Oxetacaine standard (86µg/ml concentration) peak was eluted at average of 5.629min at 3runs with Asymmetryof1.29813 and Peak area of 23237896 (Refer Table 1:  STD Method 1- Standard Oxetacaine Solution and Fig1- Std: Method I)

 

Table 1: STD Method 1-Standard Oxetacaine Solution

	Peaks	Average	Range	%RSD	status
Retention Time	5.620	5.629	5.620-5.640	0.181	P
	5.627				P
	5.640				P
Area	23399246	23237896	23768774- 23545667	1.776	P
	22768774				P
	23545667				P
Asymmetry	1.35735	1.29813	1.24812-1.35735	1.252	P
	1.24812				P
	1.28892				P

 

Table 2: STD Method II-Standard Oxetacaine Solution

	Peaks	Average	Range	%RSD	status
Retention Time	4.880	4.884	4.880-4.887	0.079	P
	4.887				P
	4.887				P
Area	364247617	360543170	355866192-364247617	1.186	P
	355866192				P
	361515701				P
Asymmetry	1.19765	1.17244	1.15765-1.19765	1.871	P
	1.15765				P
	1.16202				P

 

Method II: Standard Oxetacaine Solution:

Oxetacaine standard (196µg/ml concentration) peak was eluted at average of 4.884min at 3 runs with Asymmetry of 1.17244 and Peak area of 360543170 (Refer Table 2: STD Method II- Standard Oxetacaine Solution and Fig1- Std: Method II).

 

Method I: Results of Test Formulations

The test samples of Marketed formulations F1, F2, F3, F4, F5, F6 Prepared samples injected in HPLC and the chromatogram output of Pear Area and Asymmetry with Retention time data in Table 3.

 

Table 3: Test formulations in Method 1

Formulations	Dilutions (g)	Peak Area	Asymmetry	Retention Time
F1	0.5 to 50 ml	4424243	1.12645	5.660
	1 to 50 ml	10867153	1.04525	5.600
F2	0.5 to 50 ml	6225790	1.12528	5.653
	1 to 50 ml	10972967	1.32556	5.620
F3	0.5 to 50 ml	2703261	1.15047	5.647
	1 to 50 ml	6270408	1.12558	5.600
F4	0.5 to 50 ml	5585066	1.27452	5.880
F5	0.5 to 50 ml	5542327	1.18611	5.893
F6	0.5 to 50 ml	4755602	1.30644	5.647

 

Method II: Results of Test Formulations

The test samples of Marketed formulations F3(am), F6(am) Prepared samples injected in HPLC and the chromatogram output of Pear Area and Asymmetry with Retention time data in Table 4.

 

Table 4: Test formulations in Method 1I

Formulations	Dilutions (g)	Peak Area	Asymmetry	Retention Time
F3(am)	1 to 50ml	38820377	1.09296	4.887
F6(am)	2 to 50ml	51068712	1.51646	4.900

 

 

Std : Method I

 

 

Std: Method II

Fig 1: Standard chromatogram

 

 

Fig 2: Samples chromatograms of Different formulations by Method I

Fig 3: Samples chromatograms of Different formulations by Method II

 

CONCLUSIONS:

The two new method of estimation of oxetacaine like using Method 1: C18 column, 0.05% formic acid buffer: Methanol in the ratio 30:70% v/v as mobile phase @214 nm and Method 2: C8 Column, ammonium phosphate buffer pH 4.8: Acetonitrile in the ratio 25:75% v/v as mobile phase @214 achieved with simple, precise, accurate and sensitive. In addition the quality control estimating of some of random of F1, F2, F3, F4, F5, and F6 marketed oxetacaine suspensions were analysed using this Two Optimized methods. Out of 6 formulations, 2 formulations F3, F6 determined as substandard quality on the both the methods. Hence proving these two new methods optimal for the further monograph inclusions as well as quality control predictions of oxetacaine in API as well as Finished Pharmaceutical Formulations.

 

ACKNOWLEDGEMENT:

The authors would like to thank SRM College of Pharmacy, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu DT, Tamilnadu, India for providing facilities and guidance to carry out the research.

 

DATA AVAILABILITY:

All datasets analyzed in the study are included in the manuscript and presented as tables.

 

COMPETING INTERESTS:

The authors declare that they have no competing interests

 

REFERENCES:

1.      Seifter J, Glassman JM, Hudyma GM. Oxethazaine and related congeners: a series of highly potent local anesthetics. Proc Soc Exp Biol Med. 1962; 109(3): 664–8. doi:10.3181/00379727-109-27300

2.      Balmforth GV, Samuel RK. Controlled Trial of Oxethazaine as an Analgesic in Duodenal Ulcer. Br Med J. 1964; 1(5379): 355-6. Doi: 10.1136/bmj.1.5379.355.

3.      Parakh RK, Patil NS. Anaesthetic antacids: a review of its pharmacological properties and therapeutic efficacy. Int J Res Med Sci. 2018; 6: 383-93. https://doi.org/10.18203/2320-6012.ijrms20180005.

4.      Sethi PD. High Performance Liquid Chromatography, New Delhi, 1st Edition, 2001

5.      Narenderan ST, Babu B, Meyyanthan SN and Gowramma B. A validated analytical method for the estimation of Oxetacaine from its pharmaceutical formulation by RP-HPLC. Int. J. Res. Dev. Pharm. L. Sci. 2017; 6(5): 2764-2768. Doi: 10.13040/IJRDPL.2278-0238.6 (5).2764-2768.

6.      Miller JM. Chromatography Concepts and Contrasts. New Jersey, 2nd Edition, 2005.

7.      Ahmed S, Rizwana I, Vani MA and Azeez Basha SA: Stability Indicating Method Development and Validation of Sucralfate and Oxetacaine in Bulk and Marketed Formulation by RP-HPLC. Int J Pharm Sci Res. 2015; 6(5): 2133-39. Doi: 10.13040/IJPSR.0975-8232.6 (5).2133-39.

8.      Sujitha Parimala S. Method development and validation of sucralfate and oxetacaine in bulk and marketed formulation by RP-HPLC. International Journal of Innovative Pharmaceutical Research. 2013; 4(4): 349-356.

9.      Elzanfaly ES, Amer EA, Galal SA, Zaazaa HE. Stability indicating methods for selective determination of Oxetacaine in the presence of its degradation products. Bulletin of Faculty of Pharmacy, Cairo University. 2018; 56(2): 199-205. https://doi.org/10.1016/j.bfopcu.2018.10.002

10.   D Nagavalli, ASK Sankar, K Anandakumar, T Vetrichelvan, M Balaji. Estimation of Metaxalone in Bulk and in Tablet Dosage Form by RP-HPLC. Research J. Pharm. and Tech. 2010; 3(2): 409-411.

11.   R.V Rele, J.M. Parab, V.V. Mhatre, C.B. Warkar. Simultaneous RP-HPLC Determination of Diclofenac Potassium and Famotidine in Pharmaceutical Preparations. Research J. Pharm. and Tech. 2011; 4(4): 638-641.

12.   Mounika P Siridevi, Hemant T Kumar, Srinivasa Y Rao, Vara Prasad K Rao. RP-HPLC Method for Quantification of Empagliflozin in Pharmaceutical Formulation. Asian J. Pharm. Tech. 2019; 9(3): 208-211. DOI: 10.5958/2231-5713.2019.00035.7

13.   Kavitha J, Bonda Vismitha, Kokilambigai KS, Seetharaman R, Lakshmi KS. Validated HPTLC Method for the Estimation of Oxetacaine in Pharmaceutical Formulations. Research Journal of Pharmacy and Technology. 2021; 14(11): 5668-2. doi: 10.52711/0974-360X.2021.00985

14.   Indian pharmacopoeia 2018; Government of India, Ministry of Health. Pharmacopoeia of India.

15.   Watson DG. Pharmaceutical analysis. 2nd Edition, 2005.

16.   H. S. Jumde, S. D. Mankar. Review on development of Analytical Method and Validation by Reverse Phase – High Performance Liquid Chromatography. Asian Journal of Pharmacy and Technology. 2022; 12(2): 179-2. DOI: 10.52711/2231-5713.2022.00030

17.   Remi. S. L, Joyamma Varkey, A. Jayakumaran Nair, Vishnu. S. Sanjeev. Application of an Eco-friendly, Cost-effective Hydrotropic solution as mobile phase for the estimation and validation of Ornidazole in bulk and Pharmaceutical Formulation by RP-HPLC. Asian J. Res. Pharm. Sci. 2021; 11(1): 15-21. DOI: 10.5958/2231-5659.2021.00003.5

18.   T. Gopi Raju, M. Gayatri Ramya, A. Rajesh, S. Kathirvel, Sanjay Kumar Gupta. ICH Guideline Practice- a Validated Stability- Indicating HPLC Method for the Determination of Efavirenz in Dosage Form. Res. J. Pharm. Dosage Form. and Tech. 2014; 6(1): 7-14.

19.   Santanu Ghosh, BB Barik. Quantization of Aceclofenac in Pharmaceutical Formulations by RP-HPLC. Research J. Pharma. Dosage Forms and Tech. 2010; 2(1): 52-55.

20.   Priyanka. R. Varade, Mahavir G. Narwade, Varsha N. Tambe. Method Development and Validation of Irbesartan by RP-HPLC Method. Research J. Science and Tech. 2020; 12(2): 131-135. DOI: 10.5958/2349-2988.2020.00016.9

21.   Akash Shelke, Someshwar Mankar, Mahesh Kolhe. Development and Validation of RP-HPLC Method for estimation of Secnidazole in API and Pharmaceutical Dosage Form. Research Journal of Science and Technology. 2021; 13(2): 100-4. DOI: 10.52711/2349-2988.2021.00015

 

 

 

 

 

Accepted on 18.05.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(12):5748-5753.