RP-HPLC Assay Method Development and Validation for Lidocaine Ointment 5% USP

Figure 1: Structure of Lidocaine Hydrochloride

Lidocaine (LID) belongs to the family of narcotic drugs. It can be used as a topical anesthetic by stabilizing the nervous membrane which produces sensation of pain. It can be used to relieve the discomfort resulting from the virus Herpes which affects the skin as well as it is used in different types of minor surgery and dental treatment, childbirth and epidural anesthesia at birth, it is used particularly for the treatment of cardiac arrhythmias after having a heart attack.

[1]

Various formulations of Lidocaine are available in market. On basis of site of application formulations are classified into topical and parental. Topical formulations include gel, ointment, spray, Transdermal Patch. Parental formulations are available rather as Lidocaine-HCL or in combination with epinephrine to subside pain at site of application [2], HPLC-UV [3] thin-layer chromatography (TLC) for the Determination of Hydrocortisone Acetate and Lidocaine in a Pharmaceutical Preparation [4]

In the development of Assay method for Lidocaine Ointment USP 5% 2 trials out of 3 were performed and the 3^rd trial was finalized. First trial was failed due to to low sample concentration and the peak was failed to elute within the set run time of 20minutes..The second trial was failed to meet the system suitability parameters after the change in the concentration of reference and test solution. The third trial was passed with system suitability parameters and it was finalized for the assay method.

In the proposed work, Analytical Method development for Lidocaine ointment has been developed by chromatographic methods and to validate the proposed method with ICH and Pharmacopoeial guidelines for the intended analytical application.

The following methods have been reported for the estimation of lidocaine.

*Analytical methods development and validation of lidocaine in ointment formulation by *spectrometric method,.

*Stability indicating UPLC method for the determination of lidocaine and its degradation impurities.

*HPLC-DA stability indicating determination of Lidocaine hydrochloride and nitrofurazone in their combined topical dosage form.

*HPLC-MS/MS method for the simultaeneous determination of Nifidipine and Lidocaine in human plasma

*Development and validation of HPLC method for the simultaeneous determination of Lidocaine in topical formulation.

Review of Literature

The following methods have been reported for the estimation of Lidocaine

• Karthik.KB et al published analytical method development and validation of Lidocaine in ointment formulation by spectrophotometric method
• Shanmuga SP et al published validated and gradient stability indicating UPLC method for the determination of Lidocaine and its degradation impurities in pharmaceutical dosage form
• Shaalan RA et al published HPLC-DAD stability indicating determination of Lidocaine hydrochloride and nitrofurazone in their combined topical dosage form
• Grigoriev A et al published development of an HPLC-Ms/MS method for the simultaeneous determination of lidocaine and Nifidipine in human plasma
• Narendra M R et al published development and validation of HPLC method for the simultaeneous determination of Lidocaine and prilocaine in topical formulation.

OBJECTIVE

In the proposed work attempts have been made

• To develop new stability indicating Anaytical method for the estimation of lidocaine by chromatographic method
• To validate the proposed method in accordance with ICH and pharmacopeia guidelines intended analytical application.
• To carry out the forced degradation stability studies of lidocaine as API and in Lidocaine Ointment USP

MATERIALS AND METHODS

Formulation Details

APPARATUS AND SOFTWARE

HPLC (Agilent) computer loaded Chromeleon 6.8 software was used for all the HPLC measurements. The column was a Xterra RP C18, 150 x 4.6 x 5 μm was used. Volumetric flask with Class A, glass beakers, Measuring cylinders of Class A grade were used.Pippets of Class A grade (both bulb and graduated)were used. Balance with sensitivity of 0.01mg was used.

REAGENTS AND CHEMICALS

Lidocaine Ointment formulation was provided a from formulation department of STRIDES ACROLAB PVT.limited.

HPLC grade Acetonitrile from Spectrochem and Potassium di hydrogen, potassium Dihydrogen phosphate AR grade was purchased from Merck and HCL AR grade was purchased from SDFCL Respectively. Water used throughout the experiment was Purified HPLC grade water.

STANDARD

Lidocaine Hydrochloride (B.No/WS No-400) with a potency of 93.8% was used

SAMPLE

Lidocaine Ointment USP5% with Batch No (2004124-001) was used.

*All the materials used were within the expiry date and were stored at recommended storage condition.

STANDARD PREPARATION

PREPARATION OF LIDOCAINE HYDROCHLORIDE STANDARD SOLUTION

40.09 mg of Lidocaine reference standard were accurately weighed on weighing balance. The weighed quantity was transferred to 100ml volumetric flask.1ml of 0.1M HCL was added and sonicated for 5mins to ensure complete solubilization and diluted to volume with mobile phase .

PREPARATION OF SAMPLE SOLUTION:

800 mg of sample was accurately weighed and was transferred to 100ml volumetric flask.1ml of 0.1M HCL was added and sonicated for 5mins to ensure complete solubilization.

PREPARATION OF PLACEBO

800 mg of placebo was accurately weighed and was transferred to 100ml volumetric flask.1ml of 0.1M HCL was added and sonicated for 5mins

PREPARATION OF MOBILE PHASE:

Acetonitrile HPLC (70%) & Water (30%) were mixed and sonicated for 5 minutes.

BUFFER PREPARATION

9.703gm of Potassium Dihydrogen Orthophosphate was dissolved in 2000ml of water, adjusted the pH to 8 with 40%w/v of sodium Hydroxide solution. The solution was filtered through to 0.45 µm membrane filter & degassed for 5minutes.

Diluent Preparation:

Mobile phase was used as diluent.

OPTIMIZED METHOD

Optimized method for the Assay of Lidocaine by HPLC was finally achieved by using the following chromatographic condition

Table 1: Optimized Method

Figure 2: Chromatogram for Optimized Method

RESULTS AND DISCUSSION

1. System suitability

Standard solution of Lidocaine was injected three times into HPLC system as per test procedure. The system suitability parameters were evaluated from standard chromatograms obtained by calculating the %RSD of retention times, tailing factor, theoretical plates and peak areas from three replicate injections.

Table 2: System Suitability Parameter of Lidocaine

Figure 3: Chromatogram for System Suitability

2. Specificity by forced degradation studies

Specificity is the ability to assess unequivocally the analytes in the presence of components which may be expected to be present. Forced degradation of lidocaine ointment shall be carried out to confirm that during stability study or through out thr shelf life,any degradation product if found should not interfere with the main peak of lidocaine. The forced degradation pathway helps to identify the type of degradation pathway (oxidative, alkali hydrolysis, acid hydrolysis,water hydrolysis)for each of the degrades.

Figure 4: Chromatogram for Placebo

Figure 4: Chromatogram for Acid Stressed Sample

Figure 5: Chromatogram for Alkali Stressed

Table 3: Forced Degradation Studies

3. Precision

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurement obtained from multiple sampling of the same homogeneous sample under the prescribed conditions

System precision

The system precision is checked by using standard chemical substance to ensure that the analytical system is working properly. The retention time and area response of six determinations should be measured and calculated the relative standard deviation.

Table 4: System Precision

4. Method Precision

In method precision a homogeneous sample of single batch should be analysed six times.This indicates a method is giving consistent results of single batch.

Table 5: Method Precision

5. Intermediate Precision

The intermediate precision should be carried out to ensure that the analytical results will remain unaffected with the change in instrument ,analyst,column and day.

Table 6: Intermediate Precision

6. Stability in Analytical Solution

Evaluated the stability in analytical solution by injecting the standard preparation and sample preparation at regular interval.

standard solution at room temperature

Table 7: Stability in Analytical Solution

7. Mobile phase hold time study

Evalauted the stability of mobile phase by injecting the standard standard solution in

conjuctive (like day1,day2)days and checked the system suitability parameters.

Table 8: Mobile Phase Hold Time Study

8. Linearity and range

Six different concentration ranging 50% to 150% of lidocaine labelled claim for linearity standard solution ranging 203µg/ml to 596µg/ml. The calibration curve obtained by plotting peak area against concentration and injected under optimized chromatographic condition.

Table 9: Concentration and Peak Area of Linearity Study

Figure 6: Linearity

9. RANGE

The range of analytical method is the interval between the upper and lower level if analyte that has been demonstrated to be determined with suitable accuracy and linearity

Table 10: Range Studies

Figure 7: Range Study

10. Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted as either accepted value or an accepted reference value .This is sometimes termed trueness.

Table 10: Range Studies

Figure 9: Accuracy Study

11. Robustness

Robustness studies were carried out using slight modifications in chromatographic such as:

1) Change in column temperature ±5ºC

2) Change in flow rate ±0.2mL/min

3) Change in pH±0.2 unit

4) Change in organic phase±5%

Table 11: Robustness Studies

REFERENCES

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2. Remington Pharmaceutical Scienced.18ed 1990
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4. Instrumental Methods of analysis,Willard meritt Dean settle 7th edition pg.no 580
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