RP UFLC METHOD DEVELOPMENT AND VALIDATION FOR NEVIRAPINE IN BULK DRUG AND DOSAGE FORMS AS AN ANTIHIV AGENT

RP-UFLC METHOD DEVELOPMENT AND VALIDATION FOR

NEVIRAPINE IN BULK DRUG AND DOSAGE FORMS AS AN

ANTI-HIV AGENT

N. K. Sathish1* and E. K. Anvar2

1

Department of Pharmaceutical Chemistry, Govt. College of Pharmacy, Bengaluru-560027, Karnataka, India.

2

Dept of Pharmaceutical Analysis, SAC College of Pharmacy, BG Nagara -571 448, Karnataka, India.

ABSTRACT

A simple, precise, rapid, accurate and reproducible RP-UFLC method has been developed and validated for the determination of Nevirapine in bulk drug and pharmaceutical dosage forms. An Eclipse plus C-8 column with a particle size of 5µm having an internal diameter of 4.6 х 250 mm was used in gradient mode. The mobile phase consisting of Methanol and Acetonitrile in the ratio of 90:10 v/v was used. The flow rate was set to 1ml/min and the detection was carried out at a wavelength of 214nm. The retention time was found to be at 2.7min and the linearity was found in the range of 2-10µg/ml with a correlation co-efficient value of 0.9996. The mean recoveries were found to be in the range of 98.51-101.80% and the % RSD reported was not more than 2. The LOD and LOQ values were found to be 0.021µg/ml and 0.063µg/ml respectively. Hence, the proposed method can be successfully applied for the quantitative determination of Nevirapine in the bulk drug and Pharmaceutical dosage forms.

KEYWORDS: Nevirapine, RP-UFLC, Validation, ICH.

INTRODUCTION

Nevirapine (NVP) is 11-cyclopropyl-4-methyl-5, 11-dihydro-6H-dipyrido [3, 2-b: 2’, 3’-e][1,4] diazepin-6-one (Figure 1). Nevirapine belongs to the class of drugs known as Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) which shows the activity against human immunodeficiency virus type 1 (HIV-1) and is already marketed for the treatment of

Volume 7, Issue 16, 1116-1126. Research Article ISSN 2277– 7105

Article Received on 17 July 2018,

Revised on 07 August 2018, Accepted on 28 August 2018

DOI: 10.20959/wjpr201816-13185

*Corresponding Author

N. K. Sathish

Department of

Pharmaceutical Chemistry,

Govt. College of Pharmacy,

Bengaluru-560027,

HIV-1. Nevirapine binds directly to Reverse Transcriptase there by blocks the RNA dependent and DNA dependent DNA polymerase activities by causing the disruption of the enzyme’s catalytic site. Nevirapine is recommended for treating HIV infections in

combination with other reverse transcriptase inhibitors such as Stavudine and Lamivudine.[1,2]

Literature survey revealed that few analytical methods such as RP-HPLC[3-10], UV-spectrophotometric[11-15] and HPLC methods have been reported for the estimation of Nevirapine in bulk and dosage forms.[16-17] LC-MS/MS method has been reported for the estimation of Nevirapine in human plasma.[18]

No reports were found for determination of Nevirapine in bulk drug and pharmaceutical dosage forms using RP-UFLC method. Hence, an attempt was made to develop a new, simple, rapid, precise, economical and accurate RP-UFLC method for the determination of Nevirapine in bulk and pharmaceutical dosage forms. The method was developed and validated as per the procedures and acceptance criteria of ICH guidelines.

EXPERIMENTATION

Reagents

Acetonitrile (HPLC grade) and methanol (HPLC grade) were procured from Rankem (New Delhi, India). A reference standard of Nevirapine was procured from Shasun Pharmaceutical Ltd, (Pandicherry, India). A commercial NEVIMUNE (Cipla) tablets containing 200 mg of NVP were procured from a local market.

Instrumentation

The RP-UFLC system consisting of binary gradient pump, Rheodine injector, PDA detector and Hamilton syringe fitted with a 20 µL loop were used. The mobile phase consisting of a mixture of Methanol and Acetonitrile in the ratio of 90:10 v / v with a flow rate adjusted to 1 mL / min was used. The elute was monitored at a wavelength of 214 nm. The column used was Eclipse plus C-8 with a particle size of 5µm having an internal diameter of 4.6 х 250mm. The chromatogram showed the peak of standard and sample at a retention time of 2.7 min. The results obtained were as per shown in the Figures 3 and 4.

Preparation of standard solution

contents were then filtered through a membrane filter of 0.45µ. The volume was made up to the mark with mobile phase (Methanol and Acetonitrile in the ratio 90:10 v/v) to get the stock solution of the concentration 1000 µg / ml. This solution was further diluted with the above mobile phase to get the working standard solution of 6 µg / ml of drug.

Preparation of sample solution

Twenty tablets (with the label claim: 200 mg) were weighed accurately and crushed to form fine powder. Aliquots of powder equivalent to the weight of one tablet were accurately weighed and transferred to 50 mL volumetric flask containing 20 ml of mobile phase. It was sonicated for about 5-10 minutes to achieve complete dissolution and then filtered through a membrane filter of 0.45µ. The volume was made up to the mark with mobile phase to get the stock solution of the concentration 1000 µg / ml. This solution was further diluted with the mobile phase to get the working standard solution of 6 µg / ml of drug.

Optimization

The successful analysis of the analyte using UFLC method relies on the optimization of chromatographic conditions like mobile phase, flow rate, solvent ratio and column detection etc. Thus for better selectivity and sensitivity, different types of columns and mobile phases were tried to achieve optimum chromatographic condition. Broad peaks with less peak response and higher retention times were observed. Finally an Eclipse plus C-8 column with an internal diameter of 4.6 mm x 250 mm having a particle size of 5µm was selected for analysis based on good peak shape, suitable retention time and selectivity. Optimum

separation with good peaks and shapes were observed in Methanol and Acetonitrile in the ratio of 90:10 v / v as the mobile phase. The analysis was performed at ambient temperature by setting the flow rate to 1 ml / min with a run time of 5 min. The eluent was monitored using PDA detector at a wavelength of 214 nm. The mobile phase with the same composition was used during the extraction of the drug from the pharmaceutical dosage forms as well.

Calibration curve

calibration curve obtained were as per shown in the Table 1 and Figure 2. The standard and sample chromatograms of Nevirapine are as per shown in the Figures 3 and 4.

METHOD DEVELOPMENT AND VALIDATION

1. Linearity

Linearity was studied by preparing the standard solution at five different concentration levels. The proposed method was checked by analyzing each calibration standard solution (2, 4, 6, 8, 10 µg / ml) for NVP. The 20 µl of each solution was injected into the RP-UFLC system. The linearity range was found to be from 2-10 µg / ml. The peak area and retention time were recorded for all the chromatograms. The calibration curve was drawn by plotting the concentration of NVP on the X- axis against the peak areas of standard NVP on the Y- axis.

2. Specificity

The specificity of the method was performed by observing the interference of any encountered ingredients present in the dosage forms. The specificity of the RP- UFLC method was determined by comparing the peak area and retention time of the standard and sample Nevirapine solutions.

3. Precision

Precision studies were carried out to ascertain the reproducibility of the proposed method. Repeatability was determined by preparing six replicates of the same concentration of the sample and the peak area was measured. Intra-day precision study was carried out by preparing drug solution of same concentration and analyzing it at three different times in a day. The same procedure was followed for three different days to determine inter-day precision. The results were reported as the % RSD. The percentage relative standard deviation (% RSD) was calculated and was found to be within the acceptable criteria of not more than 2.

4. LOD and LOQ

5. Accuracy

The Accuracy of the proposed method was performed in triplicate samples at 3 different concentrations equivalent to 50%, 100% and 150% of the active ingredient, by spiking a known amount of NVP standard with placebo. The three determinations were performed at each level and results were obtained.

6. Robustness

The robustness of the method was performed by deliberately changing the column temperature to 25oC and 18oC and the respective peak areas were measured. The method showed that there were no marked changes in the chromatographic parameters, demonstrating the robustness of the method.

RESULTS AND DISCUSSION

Method Development

A RP-UFLC was proposed as a suitable method for the estimation of NVP in bulk drug and in pharmaceutical dosage forms. In order to achieve remarkable retention time and peak asymmetry, an Eclipse plus C-8 column was selected. The selection of mobile phase and flow rates were made on the basis of peak shape, baseline drift, time required for analysis and cost of the chemicals. The mobile phase consisting of Methanol and Acetonitrile in the ratio of 90:10 v / v with a flow rate set to 1 ml / min was selected. The retention time for NVP was found at 2.7 min and the maximum absorption of the drug was seen at a wavelength of 214nm. The mobile phase with the same composition as above was used during the extraction

of the drug from the pharmaceutical dosage forms.

Method Validation

The linearity range of Nevirapine was seen in the range from 2–10 µg / ml. The linear regression equation was y = 38744x + 3471.2 with a regression coefficient value of 0.9996 as

shown in the Table - 1. The specificity studies of both standard and sample Nevirapine is shown in the Table - 2 and 3. Precision study was performed and the result obtained is shown

studies. A known amount of standard drug material was added with pre-analyzed formulation in different levels. The amount of drug recovered was calculated and the percentage recovery was found to be in the range of 98.51-101.80% and the % RSD values were calculated and is as shown in the Table - 8. The % RSD values were found to be less than 2%. The result of robustness is shown in the Table - 9.

N

NH

N

N

CH₃

O

Figure 1: Nevirapine (11-cyclopropyl-4-methyl-5, 11-dihydro-6H-dipyrido [3, 2-b: 2’,

3’-e] [1, 4] diazepin-6-one).

Figure 3: Chromatogram of Nevirapine standard.

Table 1: Calibration curve data of Nevirapine by RP-UFLC method.

S. No. Concentration (µg / ml) Retention time (min) Peak area

1. 0 0 0

2. 2 2.735 86191

3. 4 2.745 158299

4. 6 2.738 235363

5. 8 2.738 311437

6. 10 2.749 391854

Table 2: Specificity result of Standard Nevirapine.

Concentration (µg/ml) Retention time (min) Peak area

6 2.737 237929

Table 3: Specificity result of Sample Nevirapine.

Concentration (µg/ml) Retention time (min) Peak area

6 2.730 240745

Table 4: Results of precision.

Concentration (µg/ml) Peak area Statistical Analysis

6 235313

Mean :- 235455.5

SD :- 247.014

% RSD :- 0.104909

6 235287

6 235734

6 235803

6 235233

6 235363

Table 5: Results of Intra-day.

Concentration

(µg/ml) Peak area 1 Peak area 2 Peak area 3 Average %RSD

6 233950 239882 239472

6 232248 238945 237922

6 233950 238474 237929

6 232248 237127 239563

6 233139 238400 236343

6 233950 239563 240745

% RSD 0.35 0.41 0.65 0.47

Table 6: Results of inter-day precision.

Concentration (µg/ml) Peak area Day 1 Peak area Day 2 Peak area Day 3 Statistical Analysis

6 237929 238474 227826

6 228383 237929 239882

6 239882 237935 237907

6 233950 237929 239472

6 237438 228331 239039

% RSD 1.76 1.66 1.92 1.78

Table 7: Characteristic parameters of Nevirapine for RP-UFLC method.

Parameters RP-UFLC Results

Linearity range (µg / ml) 2-10

Detection wavelength (nm) 214

Mobile phase (Methanol :Acetonitrile) 90: 10 (v / v)

Retention time (min) 2.7

Regression equation (Y*) Y = 38744x + 3471.2

Slope (m) 38744

Intercept (c) 3471.2

Correlation coefficient (r2) 0.9996

Intra-day Precision (% RSD) Inter-day Precision (% RSD)

0.47 1.78

Limit of detection (µg / mL) 0.021

Limit of quantitation (µg / mL) 0.063

Accuracy 98.51-101.80 %

Table 8: Accuracy readings of Nevirapine.

Labelled claim (mg) Level of Addition (%) Amount of pure drug added (mg) % Recovery Statistical Analysis

Mean SD %RSD

200 50 25 98.51

100.35 1.679 1.673

200 50 25 101.80

200 50 25 100.74

200 100 50 99.26

99.16 0.448 0.451

200 100 50 98.68

200 100 50 99.56

200 150 75 98.54

99.13 0.600 0.605

200 150 75 99.74

200 150 75 99.11

Table 9: Results of Robustness.

Temperature 25oC

Concentration (µg/ml) Peak area Statistical Analysis

6 222846

Mean :- 225787

SD :- 3864.09

% RSD :- 1.711

6 223142

6 222551

6 225552

6 228383

6 232248

Temperature 18oC

6 235305

Mean :- 236481.3 SD :- 1758.89

6 234224

6 238400 % RSD :- 0.743

6 238216

6 237438

CONCLUSION

The proposed RP-UFLC method was found to be rapid, specific, precise and accurate for the determination of Nevirapine in bulk drug and in tablet dosage forms. The method can be successfully used for routine analysis of NVP in bulk drug and pharmaceutical dosage forms without interference.

ACKNOWOLEDGEMENTS

The authors would like to thank the Shasun Pharmaceutical Ltd, Pandicherry for providing the standard Nevirapine drug. We are grateful and thankful to Dr.B.Ramesh principal, SAC College of Pharmacy for providing the necessary facilities to carry out this research work.

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