75

CHAPTER 3

DEVELOPMENT AND APPLICATION OF

STABILITY-INDICATING HPLC METHOD FOR THE

DETERMINATION OF NEVIRAPINE AND ITS IMPURITIES IN

COMBINATION DRUG PRODUCT

3.1 INTRODUCTION OF DOSAGE FORM AND

LITERATURE REVIEW

Combination therapy has proven to be one of the most effective

approaches to treat HIV infection. Nevirapine is a non-nucleoside reverse

transcriptase inhibitor (NNRTI), which acts against human immuno-deficiency

virus type 1 (HIV-1). The drug is currently marketed for the treatment of adults

with HIV-1 infection. Nevirapine is recommended for treating HIV infections in

combination with other reverse transcriptase inhibitors like stavudine, zidovudine

and lamivudine (Sweetman 2009).

The analytical method has been reported for the individual nevirapine

in EP (2008) and USP (2008). Kaul et al (2004) reported the HPTLC method for

the determination of nevirapine in the pharmaceutical dosage form. Ananthan

Kumar et al (2010), Namita et al (2006) and Samee et al (2007) published method

for the estimation of nevirapine with other antiviral drugs. Some methods were

published in biological fluid samples (Ghosh et al 2011, Omary et al 2010,

Venkata Kumar et al 2010 and Vogel et al 2010). Castro et al (2011) estimated

nevirapine through stripping voltammetry. Sreevidya and Narayana (2010)

estimated this drug through spectrophotometry using tetrathiocyanatocobalt (II)

ion as a reagent.

76

3.1.1 Target of the Work

There is no stability-indicating method reported yet for the

determination of nevirapine and its impurities in the combination drug product.

To meet the requirements of pharmaceutical quality control analysis, a simple

practical method is required for this combination drug product. It is very

important to develop a simple, precise and reliable RP-HPLC method for the

simultaneous estimation of the above mentioned components. Therefore, the

focus of the study was to develop a stability-indicating RP-HPLC method for the

combination drug product by degrading the drugs together under various stress

conditions according to ICH guidelines.

3.2 EXPERIMENTAL

3.2.1 Materials and Reagents

Pharmaceutical grade of nevirapine (chemically: 1,1-Cyclopropyl-4-

methyl-5,1,1-dihydro-6H-dipyrido[3,2-b:2’,3’-e][1,4]diazepin-6-one), lamivudine

(chemically: 4-Amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]

pyrimidine-2(1H)-one) and zidovudine (chemically: 1-(3-azido-2,3-dideoxy-β-D-

erythro-pentofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione) were obtained as

gift samples from Pharma Lab (Baddi, India). Nevirapine related compound A

(chemically: 1,1-ethyl-4-methyl-5,1,1 dihydro-6H-dipyridol [3,2-b:2’,3’-

e][1,4]diazi pine-6-one), nevirapine related compound B (chemically: 4-methyl-

5,1,1dihydro-6H-dipyridol[3,2-b:2’,3’-e][1,4]diazipine-6-one), zidovudine related

compound B (chemically: 3’-chloro-3’-deoxythymidine) and lamivudine

resolution mixture containing lamivudine and diastereomer impurities were

purchased from LGC Standards (Mumbai, India). Salicylic acid (chemically: 2-

Hydroxybenzoicacid) was purchased from Aldrich (Bangalore, India). Thymine

impurity (chemically: 5-methylpyrimidine-2,4(1H,3H)-dione) was obtained from

77

Across Organics (Bangalore, India). Chemical structures are shown in Figures 3.1

to 3.9. The combination drug product containing lamivudine, zidovudine and

nevirapine was purchased from nearby pharmacies. Each tablet contains 150 mg

of lamivudine, 300 mg of zidovudine and 200 mg of nevirapine. HPLC grade

acetonitrile was purchased from Merck (India). Buffer materials and all other

chemicals were of analytical reagent grade. High purity water was manufactured

using a Millipore Milli-Q plus purification system (Bedford, MA, USA).

Figure 3.1 Chemical structure of nevirapine

(MF: C15H14N4O, MW: 266)

Figure 3.2 Chemical structure of lamivudine

(MF: C8H11N3O3S, MW: 229)

78

Figure 3.3 Chemical structure of zidovudine

(MF: C10H13N5O4, MW: 267)

Figure 3.4 Chemical structure of nevirapine related compound A

(MF: C13H12N4O, MW: 240)

Figure 3.5 Chemical structure of nevirapine related compound B

(MF: C12H10N4O, MW: 226)

79

Figure 3.6 Chemical structure of lamivudine diastereomer

(MF: C8H11N3O3S, MW: 229)

Figure 3.7 Chemical structure of salicylic acid

(MF: C7H6O3, MW: 138)

Figure 3.8 Chemical structure of thymine

(MF: C5H6N2O2, MW: 126)

80

Figure 3.9 Chemical structure of zidovudine related compound B

(MF: C10H13Cl N2O4, MW: 261)

3.2.2 Instrumentation

The Waters HPLC system consisting of 2695 binary pump plus auto

sampler, a 2996 photo diode array and a 2487 UV detector (Waters Corporation,

Milford, USA) was used for the development and validation.

3.2.3 Preparation of Standard Solution

A standard solution of nevirapine at the target concentration of 240

µg/mL, chosen for this study, was prepared by transferring 24 mg of standard

solution into a 100 mL volumetric flask containing about 60 mL of diluents. The

solution was sonicated for 30 min or until the solid completely dissolved keeping

the water in the sonicator at an ambient temperature. Once dissolved, the

volumetric flask was filled to mark with diluents. This standard solution was used

for the assay determination of nevirapine.

A stock solution of related compound A and related compound B at

0.01 mg/mL was prepared in a 100 mL volumetric flask, filled to volume with

diluent and thoroughly mixed. Next, a diluted 2.5 mL of the resulting solution

was prepared in a 100 mL volumetric flask, filled to volume with diluent and

thoroughly mixed. This solution was used for the determination of related

81

compound A and related compound B. This solution corresponding to 0.1 % of

the target concentration of nevirapine (240 µg/mL).

3.2.4 Preparation of Sample Solution

Twenty tablets were weighed and crushed to fine powder. The powder,

equivalent to 24 mg of nevirapine, was weighed in a 100 mL volumetric flask and

around 60 mL of diluent was added. After sonication for 30 min, the solution was

cooled and made up to the mark with diluents. The solution was centrifuged and

the supernatant was used for the analysis. This sample preparation is used for the

estimation of nevirapine, related compound A and related compound B.

3.3 RESULTS AND DISCUSSION

3.3.1 Optimization of Chromatographic Method

The main objective of the chromatographic method is to separate

nevirapine from its related compound A, related compound B and other sample

matrices. By increasing the acetonitrile concentration, sample matrices like

lamivudine, zidovudine and their impurities (Salicylic acid, lamivudine

diasteriomer impurity, thymine and zidovudine related compound B) were eluted

in the column void. The optimized chromatographic method is shown in Table

3.1. The developed LC method was found to be specific and selective for

nevirapine and its impurities (Related compound A and related compound B in

combination drug product). The peak shape of the nevirapine, related compound

A and related compound B was found to be symmetrical. The representative

chromatogram is shown in Figure 3.10.

82

Table 3.1 Optimized chromatographic method

Buffer 0.05 M mono basic ammonium phosphate buffer (pH

4.5 adjusted with dilute sodium hydroxide solution)

Mobile phase Buffer:Acetonitrile (7:3, v/v)

Diluent Mixture of mobile phase and acetonitrile

(9:1, v/v)

Column Supelcosil ABZ, 150 mm x 4.6 mm, 5 micron

Column oven

temperature 30°C

Detection

wavelength 220 nm

Injection volume 50 µL

Flow rate 1.2 mL/min

Figure 3.10 Typical HPLC overlay chromatograms of normal and impurity

spiked samples (NV-nevirapine, RCB-related compound B

and RCA-related compound A)

3.3.2 Method Validation

The developed HPLC method was validated according to ICH and

FDA guidelines in terms of precision, ruggedness, linearity, specificity,

selectivity, robustness, LOD, LOQ and accuracy.

83

3.3.2.1 System Suitability

Six injections of standard solution were used for system suitability

check. System suitability was analyzed in terms of USP tailing factor (< 2.0) and

USP theoretical plate counts (> 5000) of the components. The resolution between

close eluting impurity of related compound B and nevirapine should not be less

than 2.0. System suitability results are shown in Table 3.2.

Table 3.2 System suitability results

Parameter Nevirapine Related

compound B

Related

compound A

Retention time

Relative retention time

USP resolution

USP tailing factor

USP theoretical plates

8.77

-

2.82

1.02

6921

6.26

0.71

-

0.92

7570

13.99

1.59

-

1.12

8575

3.3.2.2 Specificity and Selectivity

Specificity is the ability of the method to measure the analyte response

in the presence of its potential impurities and other sample matrices. Stress

studies were performed in the combination drug product to provide stability-

indicating property and specificity for the proposed method. Intentional

degradation was attempted to a stress condition of heat (80°C), humidity (85 %),

light (254 nm), acid (0.1 N HCl), base (0.1 N NaOH) and peroxide (3 % H2O2) to

evaluate the proposed method’s ability to separate nevirapine from its degradation

impurities and sample matrices. For heat, light and humidity studies, the study

period was 2 days, whereas for the acid, base and oxidation, it was 6 hours. Peak

purity test was carried out for nevirapine, related compound A and related

84

compound B peak by using a PDA detector in stress samples. Assay studies were

carried out for stress samples against the qualified nevirapine standard. Assay was

also calculated for nevirapine samples by spiking the nevirapine related

compound A and related compound B at the specification level (0.1 %). To prove

the proposed method’s selectivity, all individual compounds of nevirapine,

nevirapine related compound A, nevirapine related compound B and other

compounds, lamivudine and its impurities (Salicylic acid and diasteriomer

impurity), zidovudine and its impurities (Thymine and zidovudine related

compound B) were injected. The impurity details were obtained from USP

(2008).

Degradation was not observed in the sample when subjected to stress

conditions like humidity, light and heat. Minor degradation was observed in base

hydrolysis. One unknown degradation impurity was formed at the retention time

of 6.8 min, with more than 1.3 USP resolution from close-eluting impurity of

related compound B. Major degradation was observed in acid hydrolysis

(Unknown degradation impurity RTs are 4.87 min, 6.76 min, 21.55 min, and

31.16 min) and peroxide oxidation (Unknown degradation impurity RTs are 4.86

min, 6.75 min, and 3.72 min). The forced degradation chromatograms are shown

in Figure 3.11. Peak purity test results confirmed that nevirapine, related

compound A and related compound B peak are homogenous and pure in all the

analysed stress samples. More than 1.2 resolutions were found in all degradation

impurities. The assay of nevirapine is unaffected in the presence of related

compound A, related compound B and other sample matrices, which confirms the

stability-indicating power of the method. The specificity of the developed LC

method for nevirapine was revealed in the presence of its impurities (Related

compound A and related compound B). The summary of forced degradation

studies are given in Table 3.3.

85

Table 3.3 Forced degradation study results

Condition % Assay of

Nevirapine

% Mass Balance ( %

Assay + % Impurity)

Normal sample

Acid Hydrolysis (0.1 N HCl)

Base Hydrolysis (0.1 N NaOH)

Oxidation (3 % H2O2)

Thermal (80°C)

UV (254 nm)

Humidity (85 %)

99.8

94.2

98.9

95.4

99.8

99.8

99.1

99.9

99.3

99.2

99.3

99.9

99.9

99.2

86

Figure 3.11 Typical HPLC chromatograms of tablet under stress

conditions: Normal, acid, base and peroxide. (NV- nevirapine,

RCB- related compound B and RCA- related compound A)

87

The individual injections of nevirapine, related compound A, related

compound B, lamivudine, zidovudine, thymine, zidovudine related compound B,

lamivudine diastereomer and salicylic acid further prove the method’s selectivity.

Diluent was injected as a blank and no interference was found at the peak RT of

nevirapine, related compound B and related compound A, which prove the

developed method’s specificity. The overlay chromatograms of individual

injection and blank are shown in Figure 3.12.

Figure 3.12 Typical HPLC overlay chromatograms of blank (A), nevirapine

(B), related compound A (C), related compound B (D),

lamivudine (E), lamivudine resolution mixture containing

lamivudine and its diasteriomer impurity (F), salicylic acid (G),

thymine (H), zidovudine related compound B (I) and

zidovudine (J)

88

3.3.2.3 Precision

The precision of the assay method was evaluated by carrying out six

independent assays of the nevirapine test sample against a qualified standard and

calculating the % RSD of the assay. The precision of the related substances was

checked by performing six individual preparations of nevirapine spiked with 0.1

% of nevirapine related compound A and related compound B with respect to the

nevirapine analyte concentration (240 µg/ mL). The % RSD for the nevirapine

related compound A and related compound B was calculated. The intermediate

precision of the method was also performed using different analysts, different lot

columns and different instruments in the same laboratory. The % RSD of assay of

nevirapine during the method precision and intermediate precision study was

within 0.5 % and the % RSD for the area of related compound A and related

compound B in related substances method precision and intermediate precision

study was within 5 %, confirming good precision of the method. Precision data

results are shown in Tables 3.4 and 3.5.

Table 3.4 Method precision results

Injection Nevirapine (%) Related

compound A (%)

Related

compound B (%)

1

2

3

4

5

6

Mean

SD

% RSD

100.1

100.1

100.2

100.1

99.2

100.1

99.97

0.38

0.38

0.13

0.12

0.13

0.13

0.13

0.12

0.13

0.01

4.08

0.13

0.14

0.13

0.13

0.13

0.14

0.13

0.01

3.87

89

Table 3.5 Intermediate precision results

Injection Nevirapine (%) Related

compound A (%)

Related

compound B (%)

1

2

3

4

5

6

Mean

SD

% RSD

99.3

99.1

99.5

99.8

99.0

99.1

99.3

0.30

0.31

0.12

0.12

0.13

0.13

0.13

0.12

0.13

0.01

4.48

0.14

0.14

0.13

0.13

0.13

0.14

0.14

0.01

4.06

3.3.2.4 Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD and LOQ for related compound A and related compound B were

determined by the slope method, where a series of dilute solution with a known

concentration was injected. Limit of detection was measured as the lowest

amount of the analyte, where a significant response could be detected which is

different from that of a blank. Limit of detection and limit of quantification were

approved by calculations based on the standard deviation of the response (σ) and

slope (S) of the calibration curve at the levels of approaching the limits according

to equation LOD = 3.3 (σ/S) and LOQ = 10 (σ/S). Precision study was also

carried out at the LOQ level by injecting six individual preparations of related

compound A and related compound B and calculating the % RSD of the area.

LODs for related compound A and related compound B were 0.008 %

and 0.003 % respectively. Limit of quantification (LOQ) was measured as the

lowest amount of analyte that could be reproducibly quantified above the baseline

90

noise, for which six injections resulted in an RSD ≤ 5 %. A practical LOQ giving

a good precision was 0.02 % for both the related compound A and related

compound B. The results of LOQ level precision are shown in Table 3.6.

Table 3.6 LOQ level precision for impurities

Peak Area

Injection Related compound A Related compound B

1

2

3

4

5

6

Mean

SD

% RSD

8511

8423

8911

8255

8476

8423

8499.8

219.8

2.6

10200

9822

9911

9652

10111

9855

9925.2

200.2

2.0

3.3.2.5 Linearity

Linearity test solution for the nevirapine assay method was prepared at

five concentration levels from 50 % to 150 % of assay analyte concentration, i.e.,

120, 180, 240, 300 and 360 µg/mL. The peak area versus concentration data was

treated by least squares linear regression analysis. Linearity test solution for the

related substances was prepared by diluting the stock solution to the required

concentrations. The solution was prepared at six concentration levels from LOQ

to 300 % (0.72 µg/mL) of the specification level (LOQ, 0.05 %, 0.1 %, 0.15 %,

0.2 % and 0.3 %).

91

The linearity calibration plot for the assay method was obtained over

the calibration ranges tested, i.e., 120 to 360 µg/mL, and correlation coefficient

obtained was greater than 0.999. The results show that an excellent correlation

existed between the peak area and the concentration of the analyte. Linear

calibration plot for the related substance method was obtained over the calibration

range tested, i.e., LOQ (0.02 %) to 0.3 % for related compound A and related

compound B. The correlation coefficient obtained was greater than 0.999. The

results show that an excellent correlation existed between the peak area and the

concentration of related compound A and related compound B. This linearity was

represented by a linear regression equation. Linearity graphs are shown in Figures

3.13 to 3.15.

YNV = 164488x + 24200 (r=0.9996)

YRCA = 172756x - 555.6 (r=0.9996)

YRCB = 201923x + 500.95 (r=0.9999)

Nevirapine Linearity Graph

y = 164488x + 24200

R2 = 0.9996

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

0 50 100 150 200 250 300 350 400

Concentration (ppm)

Peak

Are

a

Figure 3.13 Linearity graph for nevirapine

92

Related Compound A Linearity Graph

y = 172756x - 555.6

R2 = 0.9996

0

20000

40000

60000

80000

100000

120000

140000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Concentration (ppm)

Peak

Are

a

Figure 3.14 Linearity graph for related compound A

Related Compound B Linearity Graph

y = 201923x + 500.95

R2 = 0.9999

0

20000

40000

60000

80000

100000

120000

140000

160000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Concentration (ppm)

Peak

Are

a

Figure 3.15 Linearity graph for related compound B

93

3.3.2.6 Accuracy

The recovery experiments were carried out by spiking the already

analyzed samples of the tablet with five different concentrations of standard

nevirapine, i.e., 110 %, 120 %, 130 %, 140 % and 150 %. The rcentages of

recoveries were calculated. The accuracy of the related substance was carried out

in a triplicate at LOQ, 0.05 %, 0.1 %, 0.15 %, 0.2 % and 0.3 % of the nevirapine

analyte concentration (240 µg/ml). The percentage recoveries for impurities were

calculated.

The percentages recovery of nevirapine in tablet sample ranged

between 98.2 % and 101.5 %. The percentage recoveries of impurities in tablet

samples were varied from 96.2 % to 104.3 %. Recovery results are shown in

Table 3.7.

94

Table 3.7 Accuracy results

Compound Level

(%)

Amount added

(µg/mL)

Recovery

(%)

% RSD

(n = 3)

Nevirapine

Related

compound A

Related

compound B

110

120

130

140

150

0.02

0.05

0.10

0.15

0.20

0.30

0.02

0.05

0.10

0.15

0.20

0.30

264

288

312

336

360

0.05

0.12

0.24

0.36

0.48

0.72

0.05

0.12

0.24

0.36

0.48

0.72

99.1

98.2

98.5

100.1

101.5

97.2

98.2

97.5

100.1

102.3

96.2

102.4

100.5

96.8

103.3

104.3

99.2

0.2

0.5

0.8

1.1

1.3

4.8

3.2

4.1

3.9

2.8

3.1

3.9

3.5

3.3

4.4

4.2

3.9

95

3.3.2.7 Robustness

By introducing small changes in chromatographic parameters, the

effects of the results were examined. The flow rate of the mobile phase was 1.2

mL/min. To study the effect of flow rate, flow was changed by 0.1 unit (From 1.1

to 1.3 mL/min) and the effect of the column temperature was studied at 28°C and

32°C instead of 30°C. The effect of solvent concentration was studied at ± 5 %

from the nominal concentration. The pH of the buffer solution was studied at 4.3

and 4.7 instead of 4.5.

In all deliberately varied chromatographic conditions, i.e., flow rate,

column temperature, organic solvent and pH, the resolution between the critical

pairs of nevirapine and related compound B was greater than 2. The assay of

nevirapine was obtained well within the limit, i.e., between 98 % and 102 %,

illustrating the robustness of the method. The robustness results are shown in

Table 3.8.

96

Table 3.8 Robustness results

Variations

Resolution between

nevirapine and related

compound B

Assay of nevirapine

(%)

Flow rate

1.2 mL/min (original)

1.1 mL/ min

1.3 mL/min

pH

4.5 (original)

4.3

4.7

Column temperature

30°C (original)

28°C

32°C

Organic composition

Buffer:Acetonitrile

(70:30, v/v) (original)

Buffer : Acetonitrile

(75:25, v/v)

Buffer : Acetonitrile

(65:35, v/v)

2.84

3.10

2.65

2.84

2.81

2.87

2.84

2.79

2.81

2.84

3.59

2.22

99.30

98.25

99.51

99.30

100.10

100.52

99.30

99.11

98.75

99.30

99.82

100.20

97

3.3.2.8 Application of the Developed Method

The method was applied for the assay of nevirapine and its impurities

in market sample tablets (Tablet contains 150 mg lamivudine, 300 mg zidovudine

and 200 mg nevirapine manufactured by Hetero). Nevirapine content was 99.3 %.

The percentage of related compound A and related compound B was 0.03 % and

0.04 % respectively.

3.3.2.9 Conclusion

RP-HPLC method was developed and validated for the determination

of nevirapine and its impurities. The developed method is precise, accurate,

linear, selective and specific. The method was validated showing satisfactory data

for the method validation parameters tested. The developed method is stability-

indicating and can be conveniently used by the quality control department to

determine the related substances and assay of nevirapine in regular samples and

stability samples.