Estimation of Nevirapine Anhydrous Bulk Formulation by Using IR, RP-HPLC, GC Methods

Ch. M.M. Prasada Rao*1, Ravikumar Konda2, Narashimha Rao. R2, S. Ramanjeneeyulu1 and Gangi Reddy. P3

1Department of Pharmaceutical Chemistry, St. Ann’s College of Pharmacy, Nayunipalli Village, Vetapalem Mandal, Chirala, Andhra Pradesh, India-523187                                                                                                                                                                     2Department of  Pharmaceutical Chemistry, Siddhartha Institute of Pharmaceutical sciences, Jonnalagadda, Narsaraopet, Guntur Dist, Andhra Pradesh, India- 522601.                                                                                                                                                                     3Department of Pharmacy, Annamalai University, Chidambaram-608002, Tamil Nadu.

*Corresponding Author E-mail: chennuprasad@gmail.com, chennu_prasad@yahoo.com

 

ABSTRACT:

The non nucleoside reverse transcriptase inhibitor Nevirapine (NEV), formulating sample is analyzed with instruments Infrared spectroscopy (IR), Reverse phase High performance liquid chromatography (RP-HPLC) and Gas chromatography (GC), the type of functional groups present in the NEV sample was analyzed with IR, the concentration of Nevirapine compounds NEV A, B and impurity-1 in the formulating nevirapine sample and the assay of nevirapine was done with HPLC with retention time greater than 5 and presence of residual solvents such as Dichloro methane, Chloroform, Dimethyl formamide, O-xylene was estimated with GC, the loss on drying and residue on ignition as 0.14 and 0.04 % w/w respectively.

 

KEYWORDS: Nevirapine anhydrous, IR, RP- HPLC, GC.

 


INTRODUCTION:

Nevirapine(NEV)11-Cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2,3-e][1,4]diazepin-6-one, an important therapeutic target for treatment of HIV-1. It is a non-nucleoside reverse transcriptase inhibitor (NNRTI) class of antiretrovirals. Both nucleoside and non-nucleoside RTIs inhibit the same target, the reverse transcriptase enzyme, an essential viral enzyme which transcribes viral RNA into DNA. Unlike nucleoside RTIs, which bind at the enzyme's active site, NNRTIs bind within a pocket termed the NNRTI pocket1.

 

The structural formula of nevirapine:

 

Detailed survey of literature for Nevirapine revealed several methods based on techniques viz. HPLC-UV2-9 and Spectrophotometric10,11, LC-MS12, LC-MS-MS13,14, LC15, RPLC16, RP-HPLC17,18, ion-pair HPLC19, HPTLC20 and GC21 for its determination in pharmaceutical dosage form. The compound NEV was official in United states pharmacopiea, this article presents the analysis of NEV sample by Infrared spectroscopy, Reverse phase high performance liquid chromatography and gas chromatograph for the standard analysis in quality control of already formulating nevirapine sample as tablets at Hetero laboratories, Hyderabad.

 

MATERIALS AND METHODS:

Analysis of Nevirapine was carried at Hetero laboratories, Hyderabad. All the reagents and solutions used were HPLC and milli-q grade respectively.

 

Methods of analysis:

Infrared spectroscopy (IR):

5 mg of nevirapine formulating sample is weighed accurately and 200 mg of potassium bromide (KBr) is added into mortar and triturate and form a fine powder the disc was made with IR disc compressor. Scan was done with Perkin-Elmer FTIR at 3800 cm-1 and 650 cm-1. The spectrum was recorded and compared with standard spectrum shown in Fig 1-3.

 

Reverse phase High performance liquid chromatography (RP-HPLC):

Mobile phase was prepared as a mixture of buffer: acetonitrile (ACN) as 4: 1.

 

Preparation of standard stock solution -1:

24 mg of Nevirapine anhydrous working standard was weighed accurately and transferred into 100 ml volumetric flask, 84 ml of mixture of mobile phase: acetonitrile as 20:1 was added, diluted and made up to the mark with mobile phase.

 

Preparation of standard stock solution -2:

2.4 mg of Nevirapine related compound-A (5,11-dihydro-6H – 11-ethyl-4-methyl-dihydro[1,4] diazepin-6-one, was weighed accurately and transferred into 10 ml volumetric flask, 8 ml of mixture of mobile phase:  acetonitrile as 3:1 was added, diluted and made up to the mark with mobile phase.

 

Preparation of standard stock solution -3:

6 mg of Nevirapine related compound-B (5,11-dihydro-6H -4-methyl-dihydro[1,4] diazepin-6-one was weighed accurately and transferred into 100 ml volumetric flask, 80 ml of mixture of mobile phase: acetonitrile in the ratio of 2.2:1 was added, diluted and made up to the mark with mobile phase.

 

Preparation of standard stock solution-4:

2.4 mg of Nevirapine impurity-1 N-(2-chloro-4-methyl-3-pyridyl)-2-(cyclo proprylamino)-3- pyridine carboxamide was weighed and transfer into 10 ml volumetric flask add 8 ml of mixture of mobile phase: acetonitrile as 3:1 was added, diluted and made up to the mark with mobile phase.

 

Preparation of Resolution solution:

3 ml of standard stock solution -1,2 and 4, 6 ml of standard stock solution -3 were Pipetted out into 25 ml of volumetric flask diluted and made up to the volume with mobile phase.

 

Preparation of standard solution:

2 ml of standard stock solution -1 was pipetted out into 200ml volumetric flask, diluted and made up with the mobile phase. 5 ml of the solution was further diluted with 50 ml of mobile phase.

Preparation of test solution:

24 mg of test sample (NEV) was weighed accurately into 100ml volumetric flask, 4: 80 ml of ACN: mobile phase was added, volume is made up with mobile phase.

 

Preparation of assay solution:

24 mg of test sample (NEV) was weighed accurately into 100ml volumetric flask, 4: 80 ml of ACN: mobile phase was added, 3ml of the solution was further diluted with 25ml of mobile phase. According to USP assay preparations were prepared and analyzed for accuracy.

 

Analysis by RP-HPLC:

Mobile phase, resolution solution as blank and standard, and the formulating nevirapine sample solution of 25 µl were injected separately into Shimadzu LC 2010 HPLC system, with SUPELCOSIL LC-ABZ 150× 4.6 mm, 5 µm or equivalent to L-60 USP as column with flow rate 1 ml/min, the chromatogram was recorded up to 30 min at 220 ηm for Assay and sample solution for impurities.

 

Gas chromatograph (GC):

A standard and test solutions were prepared having 4mcg/ml of di methyl formamide (DMF) and 50mg/ml of nevirapine respectively. 1ml of standard, blank, sample and standard solutions are injected simultaneously for 6 times with 30min run time for standard solution and 20min for the remaining solutions all the chromatograms are recorded. Final standard solution is injected as online solution for braketing and %RSD is calculated. Absence of DMF in sample chromatogram shows the absence of residual solvents such as Dichloro methane, Chloroform, Dimethyl formamide, O-xylene, in nevirapine sample in Fig: 8 and 9 of sample and standard chromatogram respectively.

 

Loss on drying (LOD):

Initial and final weights of 1g nevirapine weighed sample was calculated by maintaining oven temperature 105oC +/- 2o C for 3hrs, the LOD was calculated as 0.14 %w/w, by using the formula.

                                    Loss of test sample after drying

% Loss on drying =                                                         x100

                                         Weight of test sample

 

Residue on ignition (ROI):

ROI was calculated as is 0.04 % w/w by adding 1ml of sulfuric acid to 2g of NEV sample on a crucible and temperature is increased gradually and maintained at 600oC for 30min. Initial and final weights was calculated. ROI is calculated by using the formula.

 

                                                    Weight of residue

% Residue on Ignition =                                               X 100

                                     Wt of test sample after ignition

 

RESULTS AND DISCUSSION:

The instrumental methods IR, RP-HPLC and GC were developed for nevirapine. The results of IR shows, the type of functional groups within NEV sample spectrum when compared with standard spectrum, with Regression r=0.99985. The prepared resolution solution was analyzed as standard and test solution as sample with RP-HPLC for the NEV compounds for their presence in Nevirapine sample, the concentration in test as Nev A, Nev B and Impurity-1 were 0.04, 0.02 and 0.0% respectively, with total impurity of known and unknown as 0.09%, chromatograms were analyzed for standard and sample, the results were shown in Table-1 and 2, according to USP specifications  assay of nevirapine sample is 99.8%, the absence of impurity-1 in the formulating nevirapine sample and assay chromatograms were shown in Fig:5 and 6,7 respectively. The presence of residual solvents Dichloro methane, Chloroform, Dimethyl formamide, O-xylene was estimated by GC, the chromatograms shows the absence of residual solvents when compared with standard

Table: 1 Resolution chromatogram parameters:


S. No

Name

Retention time

Height

Area

Resolution

1

Nevirapine related compound-B

5.95

241956

2482675

0.00

2

Nevirapine

7.88

300485

3990207

6.122

3

Nevirapine related compound-A

11.86

216763

4013434

9.436

4

Impurity-1

19.10

63782

1892320

11.398

 

 

Table: 2 Test chromatogram parameters:

S. No.

Name

Retention time

Height

Area

Observed   (% W/W)

Standarad specification (% W/W)

1.

Nevirapine related compound-B

5.95

1476

16213

0.02

0.15

2.

Nevirapine related compound-A

11.87

1274

25423

0.04

0.15

3.

Impurity 1

-

-

-

-

0.10

 

 


chromatogram as shown in fig: 8 and 9. Loss on drying at the end of 3hrs is 0.14 %w/w, with Residue on Ignition at 600o C is 0.04 % w/w.

 

IR spectroscopy:

Fig: 1 Nevirapine sample shows the Regression r=0.99985 with standard

 

Fig: 2 Standard spectrum:

 

Fig: 3 Comparison of both sample and stranded IR spectrums:

 

Observed groups                            Frequencies in cm-1

in NEV:

=C-H(alkenes)                                  3188.96,3011.43,3124.65,3062.17

-OH                                                     3295.51

-C-C-(aromatic ring)                       1586.64, 1568.95.

                                        1025.27, 1047.92,   

 (Substituted amines)                      1074.70, 1090.97

                                        884.74,829.67,803.64

(mono substitution)

                     789.05, 761.85.

(Di substitution)

 

RP- HPLC:

Fig: 4 Nevirapine compound A, B and impurity-1 peaks in standard Resolution solution chromatogram

 

Fig: 5: NEV sample chromatogram shows the carry of NEV compounds A, B with absence of Impurity-1 in sample

 


Estimation of Nevirapine in test chromatogram:

Area of N.R.C in test solution × weight of Nevirapine taken in standard stock solution ×weight of test sample to be taken in test solution

=                                                                                                                                                                                                                                              × 0.10

Area of standard solution × weight of Nevirapine to be taken in standard stock solution ×weight of test sample taken in test solution


 

Total impurities in test sample:

=Total known impurities+ total unknown impurities

= (0.04+0.02) +0.03 =0.09 %.

 

a) Assay chromatograms:

Fig: 6 Assay preparation -1 chromatogram:

 

S. No.

Retention time

Height

Area

Name

1

7.88

280968

3700832

Nevirapine

 

Fig: 7 Assay preparation -2 chromatogram:

 

S. No

Retention time

Height

Area

Name

1

7.88

280968

3700832

Nevirapine

 

 


% of NEV in Assay preparation:

Area of principle peak in Assay preparation-1 × weight of Nevirapine working standard taken in standard stock solution × weight of test sample to be taken in test solution × assay of working standard on dried basis

=

Area of standard solution × weight of Nevirapine working standard to be taken in standard stock solution × weight of test sample taken in test solution

 

According to USP mean average of assay variation

%assay on dried basis of assay preparation-1 + %assay on dried basis of assay preparation-2                 99.8516 + 99.8477

                                                                                                                                                             =                                                          ==    99.8% W/W

                                                                    2                                                                                                         2

                                                                                       


 

GC:

Fig: 8 Sample chromatogram:

 

Fig: 9 Comparisons of standard and sample GC chromatograms shows the absence of residual solvents

 

CONCLUSION:

Nevirapine formulating sample is analyzed for the presence of functional groups by IR, the concentration of NEV compounds A, B and impurity-1, assay of nevirapine sample was estimated with RP-HPLC, the formulating sample is having NEV compounds A and B are within limits, and the absence of residual solvents was estimated by GC, the data of analysis are within standards, with loss on drying and residue on ignition are within limits. This data is useful for standard analysis in bulk drugs as well as routine analysis in quality control of nevirapine tablet formulation.

 

ACKNOWLEDGEMENT:

The authors are grateful to Quality control G.M of Hetero laboratories, Hyderabad for his moral support and guidance.

 

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Received on 24.03.2010       Modified on 13.05.2010

Accepted on 29.05.2010      © RJPT All right reserved

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1088-1092