“DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD
FOR THE QUANTITATIVE ESTIMATION OF STAVUDINE,
LAMIVUDINE AND NEVIRAPINE IN PHARMACEUTICAL
DOSAGE FORMS."
Wagh Jyoti.Gorakh*.,Narawate B.M., Dharmendra Ahuja, Banerjee Angshu
*MES College of Pharmacy, Sonai, Tal.Newasa, Dist.Ahmednagar, 414105
Jayoti Vidyapeeth Women’s University, Jaipur - Ajmer Express Way, Jaipur,
1
Rajasthan 303007
Email id :jjyothi2007@gmail.com.
Phone no.09823330878.
ABSTRACT
In recent times the introduction of combination therapy has reduced the morbidity and mortality of
the human deficiency (HIV) infected individuals. Stavudine (SV), Lamivudine (LV) and
Nevirapine (NV) are anti-HIV drugs (reverse transcriptase inhibitors). In present study,
an accurate, sensitive and specific reverse phase high performance liquid
chromatographic method (RP-HPLC) was developed and validated for the
simultaneous quantitative determination of the nucleoside reverse transcriptase
inhibitors .
Therefore, a high performance liquid chromatographic method was
developed and validated for the quantitative determination of 3TC, d4T and NVP in
given pharmaceutical dosage form without the necessity of sample pretreatment. The
different analytical performance parameters were determined according to
International
Conference of Harmonization
ICH Q2B
(Q2R1)
guidelines.
Chromatography was carried out by isocratic technique on reversed- phase C-18
NOVOPAK column with mobile phase based and optimized depending on polarity of
the molecules. The Linearity of calibration curves for each analyte in the desired
concentration range is good (r2>0.999) by HPLC method. The method is precise and
accurate with recoveries were well within acceptable limit. For all three drugs and
relative standard deviation <2% the proposed method is highly sensitive, precise and
accurate hence successfully applied for the reliable quantification of API content in
the commercial formulations of Lamivudine, Stavudine and Nevirapine.
Keywords: Stavudine (d4T); Lamivudine (3TC); Nevirapine (NVP); RP-HPLC.
INTRODUCTION
Stavudine (SV), Lamivudine (LV), Nevirapine (NV) are
anti HIV drugs (reverse transcriptase inhibitors). As HIV develops resistance rapidly,
HAART (highly active antiretroviral therapy) is a combination of ≥3drugs with
≥1drug penetrating the blood- brain barrier is essential to avoid resistance. Such a
combination dosage form will be adhering to effecting therapy and enhancing better
patient compliance. Hence there is need for the method development and validation
for the quantitative determination of three drugs Viz. Lamivudine, Stavudine and
Nevirapine that constitute one of the first line regimens in antiretroviral therapy.
Literature survey reveals that, several spectrometric, and
chromatographic methods have been reported for the quantitative estimation of these
drugs individually or in combination with other drugs in biological fluids and by
various techniques in mostly tablet dosage form.
.
REVIEW OF LITERATURE:
Literature survey reveals that, several methods that have been used for the quantitative
determination of Stavudine, Lamivudine and Nevirapine Individually or in combination such as
spectrometry, HPLC, HPTLC, etc or in combination with other drugs in biological fluids by
various techniques.
 Ramesh Panchagnula et al. A HPLC method and an UV spectrophotometric method were
developed and validated for the quantitative determination of three antiretroviral drugs
viz. Lamivudine, Stavudine and Nevirapine.
Condition: Column: C-18 Symmetry with mobile phase based and optimized depending
on the polarity of molecules. Detection at: 270,265and 313nm for LSN respectively.23
 Ramesh Panchagnula et al. RP- HPLC method for simultaneous determination of
Lamivudine, Stavudine and Nevirapine in antiretroviral fixed dose combinations.
Condition: column-C-18 by using Gradient elution with two mobile phase components
mobile phase A (80% of 10mM acetate buffer, pH 3.5 with glacial acetic acid and 20%
methanol) and mobile phase B (50% ACN with 50% isopropyl alcohol). Flow rate: 0.6ml
min –1 UV detection at 270nm.24
 Sockalingam Anbazhagan et al. were reported Simultaneous quantification of Stavudine,
Lamivudine and Nevirapine in tablets by UV spectroscopy; RP- HPLC and HPTLC
methods were developed.
Condition: Column- C18 ODS Hypersil in isocratic mode. Mobile phase: 20mM sodium
phosphate buffer (containing 8mM 1- octane sulfonic acid sodium salt): ACN (4:1v/v) pH
3.5using phosphoric acid. Detection at 265nm.25
METHODOLOGY:
MATERIALS:
Working reference standards of Lamivudine, Stavudine and Nevirapine were procured from
Medreich ixl R&D center, Bangalore that were equivalent to 99.02,99.62, and 98.97% of
Lamivudine, Stavudine and Nevirapine respectively.

Sample name or Batch number: LSN 060207

Label claim: Pharmaceutical dosage form (Tablet or capsule) contains
Lamivudine -150mg
Stavudine -30mg
Nevirapine -200mg
Average weight shall be considered as 720.5mg for all calculations.
Instrument used:
INSTRUMENT
SPECIFICATIONS
HPLC System
Waters HPLC 2996, an auto sampler

Pump:
515 pump for solvent delivery

Detector:
2487 UV dual wavelength detector

Software used
Millennium

Column:
Column no: ixl13- waters Novapak C18 (150mmx4.6mm: 5μm
Ultrasonicator:
ENERTECH Electronics Pvt. Ltd.
Digital pH Meter:
Systronics  pH System 362
METHOD DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE
ESTIMATION OF LAMIVUDINE, STAVUDINE AND NEVIRAPINE.

The mobile phase is selected on the basis of literature survey and first tried
with methanol: water as mentioned earlier in method development in
introduction and subsequently followed the same. The wavelength selected
was 266nm.

The chromatographic condition after optimization of the mobile phase and
other condition, the final chromatographic condition is mentioned below.
Method reference
Indian drugs, Journal of Pharmaceutical
and
Biomedical
analysis.
Analytical
Chemical Acta.
Instrument name
Waters HPLC, 515 Pump, and 2487UV
detector.
Buffer
Weigh 0.68g of Potassium dihydrogen
Phosphate and 1g of sodium octane
sulfonate in1000ml of water to this add
1ml of triethylamine and adjust pH to 3.5
with Phosphoric acid.
Mobile phase
Buffer: Methanol (65:35). The mobile
phase was filtered through 0.45μm
Millipore
filter
and
degassed
by
sonicating for 30min.
Injection volume
10 micro liters
Flow rate
Flow rate was set at 1ml/min through out
the process.
λ max determination
λ max was found to be 266nm by
scanning between 200-300nm.
Condition
The mobile phase was delivered through the
column at
the rate of
1.0ml/min. The
estimation was performed on Novapak C18
Column operated at room temperature. The
samples injected volume was 10μl; the UV
detector was set at a wavelength of 266nm
GENERAL PROCEDURE
STANDARD SOLUTION OF LAMIVUDINE, STAVUDINE AND NEVIRAPINE:
Standard Stock solution I:
An accurately weighed quantity of Lamivudine
(150mg), Stavudine (30mg) and Nevirapine (200mg) working standards was taken
into in 100ml VF and dissolved in 20ml of methanol and make up to 100ml with
solvent mixture.
Standard stock II: Dilute 10 ml of stock solution I to 100ml with solvent mixture
(150μg/ml of Lamivudine, 30μg/ml of Stavudine, 200μg/ml of Nevirapine
Preparation of sample solutions:
The formulation (LSN 060207) given by the
Medreich R&D has same ratio of Lamivudine: Stavudine: Nevirapine (15:3:20) is
analyzed in the laboratories.
Sample stock solution I:
An accurately weighed quantities of Stavudine,
Lamivudine and Nevirapine equivalent to average weight of powder taken in 100ml
VF and dissolved in 20ml of methanol and make up to 100ml with solvent mixture.
The solution was filtered using Millipore filter.
Sample stock solution II:
10ml of the stock solution I was dissolved to 100ml with solvent
mixture. [Solvent mixture used was water: methanol (50:50)].
4.1.1 Preparation of standard solution:
Weight taken: 150.4mg of Lamivudine + 202.2mg of Nevirapine + 28mg of
Stavudine
Standard stock solution I: Accurately weighed quantities of the above mentioned
weights were transferred to 100ml VF. And 20ml of methanol and make up to 100ml with
solvent mixture the solution was filtered through membrane filter (0.45μm).
Stock solution II: 10ml of the above filtered solution was diluted to 100ml with solvent
mixture 10μl of stock solution was injected and chromatograms were recorded.
Table 4.1.2: Initial assay result-1st for standard
Trial No.
Lamivudine
RT of
Stavudine
RT
Area in std
Lamivudine
Area in std
Stavudine
peak
of
Nevirapine
RT
Area in std
Nevirapine
peak
of
peak
1
3904652
3.845
891557
1.730
3588413
9.098
2
3904981
3.175
888025
1.720
3594002
8.865
3
3914642
3.658
886080
1.717
3594180
8.768
4
3917386
3.653
890788
1.719
3609997
8.932
5
3921626
3.796
890200
1.727
3615356
9.022
6
3917933
3.767
891388
1.726
3591417
8.928
Average
3913536.667
3.649
8899673
1.723
3598894.17
8.935
SD
7112.5371
0.244
2174.2671
0.0051
11010.762
0.115
RSD (%)
0.18
0.24
0.31
1.2 Preparation of Sample solution:
Sample stock solution I: 723.2mg of the sample powder equivalent to average weight of one
capsule was taken in 100mlVF and dissolved in 20ml methanol and make up 100ml with solvent
mixture and the solution is filtered through membrane filter (045μm).
Sample stock solution II: 10ml of above stock solution was diluted up to 100ml with solvent
mixture.
Table –4.1.3: Initial assay result-1st for sample
Trial
No
Lamivudine
RT of
Stavudine
RT of
Nevirapine
RT of
Area
Lamivudine
Area
Stavudine
Area
Nevirapine
peak
peak
peak
1
3803872
3.813
963459
1.732
3537237
9.093
2
3811908
3.800
963493
1.728
3551927
9.034
Average
3807890
3.8065
963476
1.73
3544582
9.06
Assay method validation:
4.2. Validation for specificity:
Table 4.2.1 specificity
Parameter
MP RT
Specificity
-
PL RT
STD RT
Inference
1.624,
There
was
no
3.653and
interference
of
8.932
for
the placebo with
Stavudine,
the
Lamivudine
peaks
principle
and
Nevirapine.
Acceptance criteria:
There should not be any peak in the blank and placebo run at the retention time of
Lamivudine, Stavudine and Nevirapine in standard run.
Conclusion: There is no peak in the blank and placebo run at the RT of Stavudine,
Lamivudine and Nevirapine.
4.3. Validation for Precision:
Table 4.3.1 precision results (s)
SI
Date
Timings
NO
Wt
of
Wt
of
Area
of
Area
of
sample
Stavudine
Stavudine
Stavudine
taken in
taken
in
in Std
in sample
grams
std
in
963192
960193
888237
887037
925714
923615
891557
963557
964191
963459
927874
963508
890206
963447
964559
953648
927382
958547
963164
962494
%
Of
Stavudine
grams
2
10.00
0.7222
14/02/07
0.0292
AM
Avg
3
4.00
0.7213
14/02/07
0.0287
PM
Avg
4
10.27
0.7323
15/02/07
AM
Avg
5
3.00
0.0279
99.70%
101.99%
102.19%
98.35%
15/02/07
0.7236
PM
0.0286
Avg
6
10.15
0.7225
16/02/07
0.0297
AM
Avg
SI
Date
Timings
NO
2
Wt
of
Wt
890877
Area
of
Area
of
%
Of
in sample
e
grams
in grams
3814818
3804878
3904699
3811908
3859758
3808393
3904652
3812812
3804843
3803872
3854747
3807942
3921702
3712922
3803992
3788762
3862847
3750842
3796961
3797857
3804868
3809882
3800914
3803866
0.1505
0.1488
PM
0.7323
0.1502
AM
Avg
3.00
0.7236
Avg
926316
in Std
AM
PM
891388
taken in std
10.27
15/02/07
964608
98.66%
taken in
Avg
5
890366
Lamivudin
0.7213
15/02/07
888025
Lamivudine
4.00
4
963336
Lamivudine
Avg
14/02/07
964011
Lamivudine
0.7222
3
964179
sample
10.00
14/02/07
of
964859
0.1496
97.58%
96.76%
95.87%
98.24%
6
10.15
16/02/07
Avg
AM
0.7225
0.1492
3924981
3921762
3796898
3917933
3860939
3919847
99.55%
Figure-10-Precision Standard Vial-3
Figure-10-Precision Sample Vial-2
Figure-10-Precision Sample Vial-4
4.4. Validation for Ruggedness (Intermediate precision):
Intermediate precision was studied from the results obtained by:
4.4.1(A)
SI No
Precision type
Day
Analyst
Column
Instrument
1
Intermediate
2nd
1st
1st
1st
2nd
1st
Precision (A)
2
Intermediate
Precision (B)
Mahesh
2nd
2nd
Nityanand
Note: The procedure of CSS preparation and for the sample preparation was followed as above
only and same followed for the Different column different analyst and different instrument of
ruggedness the assay values obtained are tabulated below.
INTERMEDIATE PRECISION A:
The repeatability of the method is estimating by analyzing samples of LSN-060207 on same
instrument by different analyst on different day. RSD for the six injection of the standard solution
was calculated and six multiple samples (from a homogeneous sample) of LSN batch no 060207
were analyzed and content of active drug are determined as mg/cap of the capsule.
INTRMEDIATE PRECISIONA: Assay values
4.4.1 (L)
SI
Condition
NO
Weight
Weight
of
of
Lamivudine
sample
taken in std
blend
solution
taken in
grams
No
of
injection
Lamivudine
Lamivudine
%
area in std
area
Lamivudine
in
sample
in
grams
1
Different
Column
2
0.7203
0.1504
Different
Analyst
0.7212
0.1493
1
3898976
3856867
2
3901752
3856952
Avg
3900364
3856909
1
3904321
3857819
2
3913108
3841248
Avg
3908714
38495335
98.04
96.81
Of
Table 4.4.1 (S)
SI
Condition
NO
Weight
Weight
of
of
Stavudine
sample
taken in std
blend
solution in
taken in
grams
No
of
injection
Stavudine
Stavudine
%
area in std
area
Stavudine
in
Of
sample
grams
1
Different
Column
2
0.7203
0.03023
Different
Analyst
0.7212
0.03012
1
954513
891275
2
953968
891089
Avg
954240.5
891182
1
958764
892265
2
958678
891009
Avg
958721
891637
93.42
92.82
Table 4.4.1 (N)
SI
Condition
NO
Weight
Weight
of
of
Nevirapine
sample
taken in std
blend
solution
taken in
grams
No
of
injection
Nevirapine
Nevirapine
%
area in std
area
Nevirapine
in
sample
in
grams
1
Different
Column
2
07203
0.19878
Different
Analyst
0.7212
0.19832
1
3589614
3532747
2
3592034
3532478
Avg
3565028
3532612.
1
3576149
3547142
2
3553907
3581024
97.28
Of
Avg
3565028
3564083
97.80
Comparison of initial assay value with ruggedness:
Table: 4.4.2 (LSN)
SI
Condition
NO
Lamivudine
Difference
Stavudine
Differenc
Nevirapin
Difference
assay (%)
with initial
assay (%)
e
e
with
assay
initial
with
assay
(%)
assay
assay
1
Different
98.04
1.33
93.42
0.67
97.28
-0.52
96.81
0.1
92.82
0.86
97.80
0.0
Column
2
Different
Analyst
Conclusion: The assay values obtained in each of the variables do not deviate by more than +1 of
initial assay values.
initial
Figure-11-Ruggedness D.C. STD Vial-3
Figure-11-Ruggedness D.C. SampleVial-1
Figure-11-Ruggedness D.A. Standard Vial-1
Figure-11-Ruggedness D.A. SampleVial-3
4.5. LINEARITY STUDIES:
Combined standard solution:
Weigh accurately 150mg, 30mg and 200mg and transfer it into a 100ml V.F. and dissolve it in 20
ml of methanol. Volume made upto 100ml with solvent mixture Linearity level-1solution (40%):
Area v/s Conc
1600000
y = 30652x + 31589
2
R = 0.9946
1400000
1375624
1200000
1178119
Area
1000000
958984
Series1
800000
Linear (Series1)
674261
600000
400000
384853
200000
0
0
10
20
30
40
50
Conc.
Figure-12 (a)-plot of Areas v/s Concentration in mcg/ ml of Stavudine at 266 nm. Linearity was
examined by the method of Least squares. Pearson’s correlation coefficient r is used.
Area v/s Conc
y = 25392x + 29071
7000000
2
R = 0.9915
6000000
5569655
4836976
Area
5000000
4000000
3861803
3000000
Linear (Series1)
2647497
2000000
Series1
Linear (Series1)
1511717
1000000
0
0
100
200
300
Conc.
Figure-12 (b) plot of Areas v/s Concentration in mcg/ ml of Lamivudine at 266 nm. Linearity was
examined by the method of Least squares. Pearson’s correlation coefficient r is used.
Area v/s Conc
6000000
5372136
5000000
y = 18368x - 38310
R2 = 0.9966
4515161
4000000
3648737
Series1
3000000
Linear (Series1)
2484639
Linear (Series1)
2000000
1420591
1000000
0
0
100
200
300
400
Lamivudine Con vs. Area
Figure-12 (c) plot of Areas v/s Concentration in mcg/ ml of Nevirapine at 266 nm. Linearity was
examined by the method of Least squares. Pearson’s correlation coefficient r is used.
Figure-14-Linearity Std -L-2
Figure-13-Linearity Std -L-3
Figure-13-Linearity Std -L-4
Figure-13-Linearity Std -L-5
Conclusion: The linearity range was found to be 10-50μg/ml (r =0.9946) for Stavudine,
50-250μg/ml (0.9915) for Lamivudine and 65-300μg/ml (0.9966) Pearson’s correlation coefficient
r is used.
4.6. Accuracy studies: .
Assay values for Accuracy standard Table 4.6.1
INJ.NO
Area of Stavudine
Area of Lamivudine
Area of Nevirapine
1
962648
3871803
3647376
2
958874
3880182
3647381
3
959208
3891264
3647411
4
962123
3878604
3651012
5
962024
3885145
3647809
6
958918
3891242
3647212
Avg
960632.5
3883040
3648033.5
SD
1804.501
7660.99
1472.5
Figure-14-Accuracy- A1- 1st
Accuracy values for Stavudine: Table 4.6.2 (S)
Level
Replicate
Area
mg/ tab
mg/tab
Practical
Theoretical
15
%Recovery
Average
%
of three
Spiked
1
a
498624
15.51
2
b
498358
15.50
103.3
3
c
496296
15.44
102.9
1
a
967983
30.11
100.3
2
b
967887
30.12
100.4
3
c
967891
30.12
1
a
1347086
41.90
93.3
2
b
1347286
41.91
93.4
3
c
1347079
41.90
45
93.3
93.33
150
mg/ tab
mg/tab
%Recovery
Average
%
Practical
Theoretical
of three
Spiked
99.24
50
101.38
100
93.23
150
30
103.4
100.4
50
103.2
100
100.4
Accuracy value for Lamivudine:
Table 4.6.2(L)
Level
Replicate
Area
1
a
1946538
74.43
99.24
2
b
1946588
74.44
99.25
3
c
1946573
74.43
1
a
3976824
152.07
2
b
3976768
152.08
1
a
5466871
209.02
93.23
2
b
5467657
209.04
93.24
3
c
5466877
209.04
75
99.24
101.38
150
225
101.39
93.24
Accuracy values for Nevirapine:
Table: 4.6.2(N):
Level
Replicate
Area
mg/ tab
mg/tab
Practical
Theoretical
%Recovery
1
a
1738516
94.34
94.34
2
b
1738524
94.32
94.32
3
c
1738486
94.33
1
a
3685637
199.97
2
b
3685845
199.98
1
a
5362342
290.95
96.66
2
b
5364848
291.01
96.67
3
c
5362348
290.95
100
94.33
Average
%
of three
Spiked
94.33
50
99.98
100
97.66
150
99.98
200
300
99.98
96.66
4.7. Robustness:
4.7.1. Parameters of robustness
Robustness for
Parameter
Original composition
Change in composition
A1
Buffer: Methanol (650:350)
Buffer: Methanol (663:337)
A2
Buffer: Methanol (650:350)
Buffer: Methanol (633:357)
Parameter
Original flow rate
Change in flow rate
B1
1.0ml/min
1.10ml/min
1.0ml/min
0.90ml/min
B2
Table- 4.7.2 result of standard for robustness of six injections
INJ NO.
AREA
Lamivudine
Stavudine
Nevirapine
1
3859587
926704
3573603
2
3855747
926823
3575347
3
3857086
926825
3574960
4
3853973
926838
3574257
5
3862230
927121
3574321
6
3853270
926683
3574255
Average
3856982
926832.33
3574457.16
SD
3426.2
156.33
612.164
RSD
0.088
0.016
0.017
4.8. System suitability:
Table-4.8.1: System Suitability Test
Proposed Method
System Suitability Parameters
Stavudine
Lamivudine
Nevirapine
Retention Time (TR)
1.745
3.820
9.026
Theoretical Plate (N)
9651
11766
16615
Tailing Factor (T)
1.24
1.16
1.31
Resolution Factor (Rs)
2.19
6.08
8.23
Conclusion: The result obtained is according to the level of acceptance criteria to the set
parameters.
Acceptance criteria:

The number of theoretical plates for Stavudine, Lamivudine and Nevirapine should be
more than 4000.
The resolution between the peaks should be more than 2
Conclusion

The proposed method gives a good resolution between three drugs SV, LV and NV.
 The proposed method for the quantification of SV, LV, and NV were simple, precise,
accurate, rapid and selective.

The method is linear in the concentration range reported.
 The developed method is free from interference due to the excipients present in the
formulation can be used for routine simultaneous quantitative estimation of SV, LV and
NV.

Thus the method discussed is accurate, simple, precise, selective, specific,
reproducible and money saving. The results for these three drugs were in good
agreement with label claim.
REFERENCES
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