Procedure for plotting calibration curve:

For calibration curve in a series of 10ml volumetric flasks, 2-10ml of standard solution was pipetted out separately. The volume was completed to the mark using RO water. The absorbance was measured at wavelength 228.8nm against blank solution.

RESULTS AND DISCUSSION:

The absorption spectrum shows λ max of Lignocaine Hydrochloride at 228.8nm.

Figure 2: UV spectrum of Lignocaine Hydrochloride

Figure 3: Calibration curve for Lignocaine Hydrochloride (Conc. vs. Abs.)

Analysis of Lignocaine Hydrochloride in Gel Formulation:

10mg equivalent Lignocaine Hydrochloride gel was weighed and transferred to the 100ml volumetric flask and dissolved in RO water as a solvent. After that sonicated for 5 min and vortex for 2min. 4ml of above solution was pipetted out and transferred to the 10ml volumetric flask and make up the volume upto the mark with same solvents and analysed at 228.8nm. Calculate the % purity of Lignocaine Hydrochloride.

The proposed method was validated according to ICH Q28 R1 guidelines for validation of analytical procedure.^4-8

Linearity:

Five different concentrations of Lignocaine Hydrochloride were prepared and analysed at wavelength 228.8nm. The regression coefficient was found to be 0.9992. The absorbance was found in limit

i.e. 0-2. Hence the analysed parameter was found to be validated (table 1).

Table 1: Results of Linearity

Sr. No.	Concentration (µg/ml)	Absorbance
1	20	0.215
2	40	0.402
3	60	0.615
4	80	0.802
5	100	0.982

Table 2: Optimization parameters of Lignocaine Hydrochloride

Parameters	Method values
Maximum Wavelength	228.8nm
Beer’s Law	20-100µg/ml
Correlation Coefficient (r²)	0.9992
Regression Equation	y = 0.0097x + 0.023
Slope (m)	0.0097
Intercept (c)	0.023

Table 5: Results of Inter-day precision

Sr. No.	Concentration	Absorbance (Day1)	Absorbance (Day2)
1	(60µg/ml)	0.615	0.617
2		0.614	0.616
3		0.616	0.615
4		0.615	0.617
5		0.616	0.615
6		0.615	0.616
	SD	0.000753	0.000894
	%RSD	0.122369	0.145199

Limit of Detection (LOD):

The limit of detection was found to be 3.55µg/ml (table 6).

Limit of Quantification (LOQ):

The limit of quantification was found to be 10.75µg/ml (table 6).

Table 6: Results of LOD and LOQ

LOD	3.55 µg/ml
LOQ	10.75µg/ml

Ruggedness:

The change in analyst with same concentration and environmental condition didn’t affect the results.

Table 7: Results of Ruggedness

Concentration	Absorbance (Analyst1)	Absorbance (Analyst2)
20µg/ml	0.215	0.216
	0.216	0.214
	0.215	0.217
	0.217	0.215
	0.214	0.216
	0.216	0.215
Average	0.2155	0.2155
SD	0.001049	0.001049

Robustness:

The change in wavelength (228.8nm and 231.8nm) and concentration (30µg/ml) didn’t affect the results.

Table 8: Results of Robustness

Wavelength	228.8nm	231.8nm
Concentration	30µg/ml	30µg/ml
Absorbance	0.342	0.340
	0.341	0.343
	0.340	0.342
	0.342	0.340
	0.341	0.343
	0.343	0.340
Average	0.3415	0.3413
SD	0.001049	0.001506

Assay:

The assay was performed by using LOX 2% Jelly at concentration 40µg/ml. The % purity was found to be 99.2%.

Table 9: Results of Assay

Formulation	Labeled Amount	Amount obtained	% purity
LOX 2% Jelly	2 gm	1.98	99.2%

CONCLUSION:

An analytical UV spectrophotometric method was developed and validated thoroughly for quantitative determination of Lignocaine Hydrochloride in bulk drug and gel formulation. The presented method was found to be simple, precise, accurate, rugged, reproducible and gives an acceptable recovery of the analyte, which can be directly easily applied to the analysis of pharmaceutical gel formulation of Lignocaine Hydrochloride.

ACKNOWLEDGEMENT:

Authors are thankful to the Principal, College of Pharmacy Solapur, for providing the necessary facilities.

REFERENCES:

1. Hasan F, Ali A.(Lidocaine Hydrochloride (Antiarrhythmic).The American Society of Health-System Pharmacists. 2015; 12(3): 156-165

2. https://en.wikipedia.org/wiki/Lidocaine.

3. Tektook N, Ali A. Lidocaine Hydrochloride (Local).The American Society of Health-System Pharmacists 2015; 2(1): 123-129.

4. VaniR, SunithaM. Analytical method development and validation for the determination of Acotiamide hydrochloride using reverse phase HPLC method in bulk and tablet dosage form. World journal of pharmacy and pharmaceutical sciences. 2010; 6(10): 768-775.

5. Patel PN, Kalariya PD, Swamy CV, Gananadhamu S, Srinivas R. Quantitation of Acotiamide in rat plasma by UHPLC-Q-TOF-MS: method development, validation and application to pharmacokinetics. Biomed Chromatography. 2016; 30(3): 363- 368.

6. Klaus Florey, Lidocaine Base and Hydrochloride, Analytical Profiles of Drug Substances, Chapter 8, page 207-243.

7. Deepak v. Bageshwar, avinash s. Pawar, vineeta v. Khanvilkar, vilasrao j. Kadam. Quantitative Estimation of Mupirocin Calcium from Pharmaceutical Ointment Formulation by UV Spectrophotometry, International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 2, Issue 3, 2010.

8. ICH Q2 (R1) validation of analytical procedures: text and methodology, International Conference on Harmonization, Nov, 1996.

Name of Drug	Recovery Level in %	Concentration	Amount Recovered	% Recovery with SD
	50	20µg/ml	20.02	100.02±0.25
	100	40µg/ml	40.03	100.03±0.7
Lignocaine Hydrochloride	150	60µg/ml	59.01	99.01±0.29

Suyash Ingle*, Dr. Varsha Tegeli, Avinash Birajdar, Vinod Matole,

Sagar Adlinge, Gajanand Nangare

ABSTRACT:

INTRODUCTION:

MATERIALS AND METHOD:

Materials:

Instruments:

Experimental: