CHAPTER 6

STAVUDINE

148

6.1. DRUG PROFILE

Stavudine is a synthetic thymidine nucleoside analogue, active against the human

immunodeficiency virus type 1 (HIV-1) approved by the FDA in June, 1994. Stavudine is

sold with the brand name ZERIT. It is in a class of drugs called reverse transcriptase

inhibitors which also includes zalcitabine (Hivid), zidovudine (Retrovir), didanosine

(Videx), and lamivudine (Epivir). Stavudine is an analog of thymidine. Stavudine does

not kill existing HIV virus and it is not a cure for HIV. But it is phosphorylated by

cellular kinases into active triphosphate. Stavudine triphosphate inhibits the HIV reverse

transcriptase by competing with natural substrate, thymidine triphosphate. It also causes

termination of DNA synthesis by incorporating into it.

Figure 6.A: Structure of Stavudine

IUPAC NAME : 1-((2R, 5S)-5-(hydroxymethyl)-2, 5-dihydrofuran-2-yl)-

5-methylpyrimidine-2, 4(1H, 3H)- Dione

FORMULA : C10H12N2O4

MOLECULAR WEIGH

: 224.2

The chemical name for Stavudine is 2', 3'-didehydro-3'-deoxythymidine.

Stavudine is a white to off-white crystalline solid. Stavudine, when used alone or in

combination with other antiviral medications, may cause serious and possibly deadly

damage to the liver and pancreas and a life-threatening condition called lactic acidosis.

Molecular Structure of Stavudine is shown in Figure: 6.A. The most severe side effects

149

with Stavudine are a decrease in blood cells, muscle pain (myopathy), pancreatitis, liver

failure and metabolic disturbance (lactic acidosis). Stavudine damages nerves and can

cause a severe peripheral neuropathy.

List of brand names of Stavidine

S.No. BRAND

NAME

FORMULATION COMBINATION AVAILABLE

STRENGTH

MANUFACTURER

1 STADINE Cap ---- 30mg Emcure

Cap ----- 40mg Emcure

2 STV Cap ---- 40mg Mcneil & Argus

3 VIROSTAV Tab ---- 30mg Ranbaxy

Tab ---- 40mg Ranbaxy

4 EMDUO

SUSP

Susp Lamivudine Emcure

5 EMDUO-40 Tab Lamivudine 40mg Emcure

6 EMDUO-E

30

Kit Lamivudine

Efavirez

30mg Emcure

7 EMDUO-E

40

Kit Lamivudine 40mg Emcure

8 EMDUO-JR Dt-tab Lamivudine Emcure

9 EMDUO-N Kit Lamivudine

Nevirapine

Emcure

10 Emtri-30 Tab Lamivudine

Nevirapine

30mg Emcure

11 EMTRI-40 Tab Lamivudine

Nevirapine

40mg Emcure

12 EMTRI-JR Dt-tab Lamivudine

Nevirapine

Emcure

13 EMTRI-

SUSP

Susp Lamivudine

Nevirapine

Emcure

14 STAVEX Tab Lamivudine

Nevirapine

Aurobindo

15 STV COMP Tab Lamivudine

Nevirapine

Mcneil & Argus

16 STV

COMP-30

Tab Lamivudine

Nevirapine

Mcneil & Argus

17 STV PLUS Cap Lamivudine Mcneil & Argus

Table 6.1

150

6.2 . LITREATURE SURVEY

Several analytical methods have been reported for the determination of Stavudine in

pure drug, pharmaceutical dosage forms and in biological samples using

spcetrophotometry liquid chromatography, electro kinetic chromatography high

performance thin layer chromatography either in single or in combined forms.

Pawan K Saini et al [1]

has described a simple, fast, precise and accurate reverse

phase high performance liquid chromatographic method for the simultaneous

determination and validation of lamivudine (3TC) and Stavudine (d4T) in tablets. A

sunfire C18, 250 × 4.6 mm, 5 mm particle size column in isocratic mode was used with

mobile phase comprising of methanol: 0.1 % w/v of ammonium acetate, adjusted to pH

3.8 with glacial acetic acid in the ratio of 15:85, v/v. The flow rate was set at 1.2 ml per

minute with UV detection at 266 nm. The retention time of 3TC, d4T was found 5.6 and

8.9 minute respectively. Linearity for lamivudine and Stavudine were found in the range

of 75-225mg/ml and 20-60mg/ml respectively. Percentage recoveries were obtained in

the range of 98.97 % to 99.71 % for lamivudine and 99.22 % to 99.59 % for Stavudine.

The proposed method is precise, accurate, selective, reproducible, and rapid for the

simultaneous estimation of lamivudine and Stavudine in tablet dosage forms.

Anbazhagan S et al [2]

has developed methods for simultaneous quantification of

Stavudine (SV), lamivudine (LV) and nevirapine (NV) in tablets by UV spectroscopy,

reverse phase HPLC (RP-HPLC) and HPTLC. In the UV multi-component spectral

method, SV, LV and NV was quantified at 266, 271 and 315 nm, respectively. In the RP-

HPLC method, the drugs were resolved using a mobile phase of 20 mM sodium

phosphate buffer (containing 8 mM 1-octanesulphonicacid sodium salt): acetonitrile (4:1,

v/v) with pH adjusted to 3.5 using phosphoric acid on a C18-ODS-Hypersil (5 micron,

250 mm x 4.6 mm) column in isocratic mode. The retention time of SV, LV and NV was

2.85, 4.33 and 8.39 min, respectively. In the HPTLC method, the chromatograms were

developed using a mobile phase of chloroform: methanol (9:1, v/v) on precoated plate of

silica gel 60 F254 and quantified by densitometric absorbance mode at 265 nm. The Rf of

SV, LV and NV were 0.21-0.27, 0.62-0.72 and 0.82-0.93, respectively. Recovery values

of 99.16-101.89%, percentage relative standard deviation of <0.7 and correlation

151

coefficient (linear dynamic range) of 0.9843-0.9999 shows that the developed methods

were accurate and precise. These methods can be employed for the routine analysis of

tablets containing SV, LV and NV.

Namita Kapoor et al [3]

were described two methods for the simultaneous

determination of lamivudine (3TC) and Stavudine (d4T) in combined pharmaceutical

tablets. The first method depends on first derivative UV-spectrophotometry with zero-

crossing measurement technique. The first derivative absorbance at 280 and 300 nm were

selected for the determination of Stavudine and lamivudine, respectively. The second

method is based on the separation of both drugs by high performance liquid

chromatography using methanol: water (20:80) as the mobile phase at 0.6 ml/min on a

reverse phase column with detection at 270 nm. Both the methods showed good linearity,

reproducibility and precision. No spectral or chromatographic interferences from the

tablet excipients were found. The proposed methods were suitably applied to the assay of

commercial formulations. The procedures were rapid, simple and suitable for routine

quality control application.

M. Sarasa et al [4]

has developed a Sensitive HPLC assays for the quantification

of Stavudine (2', 3'-didehydro-3'-deoxythymidine) in human plasma and urine. The

methods are linear over the concentration range 0.025-25 and 2-150 µg/ml in plasma and

urine, respectively. An aliquot of 0.2 ml of plasma was extracted by SPE using Oasis

cartridges, while urine samples were simply diluted 1 + 99 with HPLC water. The

analytical column, mobile phase, instrumentation and chromatographic conditions are the

same for both methods. The methods have been validated separately and stability tests

under various conditions have been performed. The detection limit is 12 ng/ml in plasma

for a sample size of 0.2 ml. The bioanalytical assay has been used in a pharmacokinetic

study of pregnant women and their newborns.

Marc Schuman et al [5]

were developed and validated a reversed phase HPLC

method using photo diode array detection for the simultaneous quantification of

lamivudine, Stavudine, nevirapine, zidovudine, methyl paraben and propyl paraben in

solid and liquid drug formulations. The separation was achieved using a Waters

152

Symmetry C8 column, using a mobile phase gradient comprising 50 mM NaH2 PO4 (pH

3.8) and acetonitrile (95:5 to 45:55, v/v) and a flow gradient (0.5 to 1.0 ml/min). The

limits of detection and quantification were below 19 ng/ml and 55 ng/ml respectively. The

intra- and inter-day assay precisions were within 4.4% relative standard deviations. The

developed method was applied to 12 different generic antiretroviral medications, consisting

of tablets, capsules and solutions, produced by two Indian manufacturers and purchased by

the Central Agency of Essential Drug Procurement of Rwanda for the ESTHER project in

Rwanda. The average content of the antiretroviral agent(s) compared to the labeled

amount(s) was 101.4%. Methyl paraben and propyl paraben, added to solutions as

preservatives, were within the FDA recommended limits.

CH.Balasekharareddy et al [6]

has developed and validated a simple, rapid,

precise and accurate isocratic reverse phase stability indicating RP-HPLC method for the

simultaneous estimation of Lamivudine and Stavudine in commercial tablets. The method

has shown adequate separation for Lamivudine (RT-3.087) and Stavudine (RT-6.09)

respectively. Separation was achieved on an YMC pack, C8, 150mmX4.6mm, 5µ column

using a mobile phase consisting of buffer pH 3.5 and methanol in the ratio of 90:10v/v at

a flow rate of 1.0ml/min. the detection was carried out by PDA detector at the

wavelength maximum of 265 nm. The drugs were subjected to acid degradation, base

degradation, peroxide degradation, thermal degradation, photolytic degradation and

humidity degradation. The linearity of proposed method was investigated in the range of

5-50 µg/ml (r= 0.99989) for Stavudine and 20-220µg/ml (r= 0.99997) for Lamivudine,

respectively.

Triporn Wattananat et al [7]

has developed and validated a high performance

liquid chromatographic method with UV detection for simultaneous determination of

Stavudine and lamivudine in human plasma using solid-phase extraction for sample

clean-up. Zidovudine was used as an internal standard. Separation was performed on a

C18 column by gradient elution with a mobile phase of 10 mM acetate buffer pH 6.5 and

acetonitrile. The UV detection was set at 265 nm. The method proved to be specific,

accurate, precise and linear over the concentration ranges of 50-3,000 ng/ml for

Stavudine and 50-5,000 ng/ml for lamivudine with correlation coefficients always >0.996

for both drugs. The intra-day and inter-day precision and accuracy were less than 9.2%

153

for both analytes. The absolute recoveries of both compounds ranged from 93.3 to 97.5%.

The method was successfully applied to a bioavailability study of a combined tablet

formulation containing 30 mg of Stavudine and 150 mg of lamivudine compared with

each reference formulation concurrently administered in 26 healthy Thai male volunteers.

A. Tarinas et al [8]

has described and validated a Simple methods for the

determination of zidovudine (AZT), Stavudine (d4T), lamivudine (3TC) and indinavir

(INV) in human plasma by reversed-phase liquid chromatography (HPLC) with UV

detection. Solid-liquid extraction procedures were applied to the samples prior to

analysis. Chromatography was performed on a C-18 analytical columns and the retention

time ranged from 6.8 to 8.0 min for Stavudine, 7.5 to 9.0 min for lamivudine, 11.2 to 11.9

min for zidovudine and indinavir. Four methods were validated for specificity, inter-and

intra-assay precision and accuracy, absolute recovery and stability. Analytical curve

ranged from 10-1600 ng/ml for Stavudine, 50-3200 ng/ml for lamivudine, 0.05-5.0 µg/ml

for zidovudine and 0.1-10.0 µg/ml for indinavir. Analytes stability during sampling

processing and storage were established. Extraction recoveries are higher than 89% for

all formulations. These methods proved to be simples, accurate and precise, and are

currently in use in our laboratory for the quantitative analysis of antiretroviral products in

plasma, and for further pharmacokinetics and bioequivalence studies.

Lian Zhou et al [9]

has investigated the disposition of Stavudine, a potent and

orally active nucleoside reverse transcriptase inhibitor in six healthy human subjects.

Before dosing humans with [1-14C] Stavudine, a tissue distribution study was performed

in Long-Evans rats. Results from this study showed no accumulation of radioactivity in

any of the tissues studied, indicating that the position of the C-14 label on the molecule

was appropriate for the human study. After a single 80-mg (100 _Ci) oral dose of [1-14C]

Stavudine, approximately 95% of the radioactive dose was excreted in urine with an

elimination half-life of 2.35 h. Fecal excretion was limited, accounting for only 3% of the

dose. Unchanged Stavudine was the major drug-related component in plasma (61% of

area under the plasma concentration-time curve from time zero extrapolated to infinite

time of the total plasma radioactivity) and urine (67% of dose). The remaining

radioactivity was associated with minor metabolites, including mono- and bis-oxidized

154

Stavudine, glucuronide conjugates of Stavudine and its oxidized metabolite, and an N-

acetylcysteine (NAC) conjugate of the ribose (M4) after glycosidic cleavage. Formation

of metabolite M4 was shown in human liver microsomes incubated with 2_, 3_

didehydrodideoxyribose, the sugar base of Stavudine, in the presence of NAC. In

addition, after similar microsomal incubations fortified with GSH, two GSH conjugates,

3_GS-deoxyribose and 1_keto-2 _, 3_dideoxy-3_GS-ribose, were observed. This

suggests that 2_, 3_didehydrodideoxyribose underwent cytochrome P450-mediated

oxidation leading to an epoxide intermediate, 2_, 3_ribose epoxide, followed by GSH

addition. In conclusion, absorption and elimination of Stavudine were rapid and complete

after oral dosing, with urinary excretion of unchanged drug as the predominant route of

elimination in humans.

I.Ponnilavarasan et al [10]

described a simple reverse phase high-performance

liquid chromatographic (RP-HPLC) method was for the quantitative estimation of

antiretroviral drugs Lopinavir (LPV) and Ritonavir (RTV). The different analytical

parameters such as linearity, precision, accuracy, and specificity, limit of detection

(LOD) and limit of quantification (LOQ) were determined. Chromatography was carried

out by binary gradient technique on a reversed-phase phenomenex-Luna C column using

Ambroxol (ABM) as the internal standard. The calibration curve for each analyte in the

desired concentration range (r2 > 0.999) was found to be linear. The recovery values was

found to be 99.9 and 100.24% and relative standard deviation was <2% for LPV and

RTV respectively. The proposed method is highly sensitive, precise and accurate, which

was evident from the LOD value of 30 ηg/ml for LPV and 25 ηg/ml for RTV hence the

present method applied successfully for the quantification of active pharmaceutical

ingredient content (API) in the combined formulations of LPV and RTV.

C. Jose gnana babu et al [11]

has developed a simple, specific, accurate, precise

and sensitive reverse phase high performance liquid chromatographic method for the

quantization of Stavudine in both pure and capsule dosage form. A Phenomenex Gemini

C- 18, 5 µm column having 250×4.6 mm i.d. in isocratic mode with mobile phase

containing methanol: acetate buffer pH 6.0 (40:60). The flow rate was 1.0 ml/min and the

effluents were monitored at 265 nm. The retention time was 3.58 min. The linearity was

in the range of 25-75 mcg/ml. This method was validated for linearity, precision,

155

specificity, and limit of detection, limit of quantization, accuracy, ruggedness and

robustness. Statistical analysis proves that the method is reproducible and selective for

the estimation of the said drug.

V Reddy Panditi et al [12]

had developed a simple, accurate, precise and sensitive

First order derivative Spectrophotometric method for the estimation of Stavudine in bulk

and pharmaceutical dosage forms. The estimation of Stavudine was carried out at

maximum absorbance of 250 nm. The method was found to be linear and obeys Beer’s

law in the concentration range of 2-20 mcg / ml. The developed method was validated

according to ICH guidelines and was found to be accurate and precise. Thus the proposed

method can be successfully applied for the estimation of Stavudine in bulk and

pharmaceutical dosage forms.

S.Jayaseelan et al [13]

has developed and validated a simple, accurate, precise and

sensitive HPLC method with UV detection to separate and detect lamivudine in human

plasma using Stavudine as an internal standard. Lamivudine (3-TC) and Stavudine

(internal standard) were extracted from human plasma using

methanol protein

precipitation and were chromatographed on a Phenomenex C18 (250X4.6mm.,5mµ

particle size) column using 20µl injection volume and detection at 270nm.An isocratic

mobile phase consisting of Methanol: Water (85:15%v/v) was used to separate these

drugs. The retention times of lamivudine and I.S were 4.6 and 6.2 respectively. The

method was validated over the range of 406.10-4020.05ng/ml. The

limit

of

detection

was

200 ng/ml and

the

limit

of

quantification

was

400 ng/ml

for

3TC.

Within

and

between-

day

precisions are less than 6.5%

for

all

quality

control

samples.

The

absolute

recoveries

of

3-

TC was greater

than 90%

were

achieved.

The

described

method

can be

readily

utilized

for

analysis of

pharmaceutical

products.

Weerasak Samee et al [14]

has developed and validated a rapid and reliable

reverse-phase high performance liquid chromatographic for simultaneous determination

of lamivudine, Stavudine and nevirapine in presence of their acid-induced degradation

products. Gradient chromatography using Thermo Hypersil Gold C18 column (150 mm x

4.6 mm, 5 µm) eluted with two mobile phase components: mobile phase A comprising of

20 mM sodium phosphate buffer with pH adjusted to 3.5 with phosphoric acid and mobile

phase B (methanol) with a flow rate of 1.0 mL/min and a detection wavelength at 265

156

nm. The retention times for lamivudine, Stavudine and nevirapine were 4.6, 7.8, and 14.8

min, respectively. The calibration curves were linear (r18 > 0.9997 for all three

compounds). The R.S.D. values for intra- and inter-day precision studies were < 1.10%

and < 1.60%, respectively. The recovery of three drugs determined from a spiked sample

ranged from 98.02% to 101.56%. The method was also effective in quantitative analyses

of the marketed tablet formulations.

157

6. 3. EXPERIMENTAL

6.3.1. Instrumentation

Peak HPLC containing LC 20AT pump and variable wavelength programmable

UV-Visible detector and Rheodyne injector was employed for investigation. The

chromatographic analysis was performed on a Chromosil C18 column (250 mm × 4.6

mm, 5µm). Degassing of the mobile phase was done using a Loba ultrasonic bath

sonicator. A Denwar Analytical balance was used for weighing the materials.

6.3.2. Chemicals and Solvents

The reference sample of Stavudine (API) was obtained from Aurobindo,

Hyderabad. The Formulation VIROSTAV (Stavudine) was procured from the local

market. Methanol, Acetonitrile used was of HPLC grade and purchased from Merck

Specialities Private Limited, Mumbai, India. And orthophosphoric acid used was AR

grade purchased from local market.

6.3.3. The mobile phase

A mixture of Methanol: 0.1% orthophosphoric acid: Acetonitrile in the ratio of

40: 50:10 v/v/v was prepared and used as mobile phase.

6.3.4. The buffer solution

About 1.0 mL of orthophosphoric acid was diluted to 1000 mL with water. This

solution was mixed and pH was adjusted to 5.1 with 1% ortho phosphoric acid and

filtered through 0.45μ nylon filter.

6.3.5. Standard solution of the drug

For analysis 100 ppm standard solution was prepared, required concentrations

were obtained from 100 ppm solution by appropriate dilution.

6.3.6. Sample (tablet) solution

The formulation tablets of Stavudine (VIROSTAV - 20 mg) were crushed to give

finely powdered material. From the Powder prepared a 3 ppm solution in mobile phase

and then filtered through Ultipor N66 Nylon 6, 6 membrane sample filter paper.

158

6.4. METHOD DEVELOPMENT

For developing the method, (as described in Chapter 1 and 2) a systematic study

of the effect of various factors was undertaken by varying one parameter at a time and

keeping all other conditions constant. Method development consists of selecting the

appropriate wave length and choice of stationary and mobile phases. The following

studies were conducted for this purpose.

6.4.1. Detection wavelength

The spectrum of 10ppm solution of the Stavudine in methanol was recorded

separately on UV spectrophotometer. The peak of maximum absorbance wavelength was

observed. The spectra of Stavudine were showed maximum absorbance at 267nm.

6.4.2. Choice of stationary phase

Preliminary development trials have performed with octadecyl columns with

different types, configurations and from different manufacturers. Finally the expected

separation and peak shapes were obtained on Chromosil C18 (250 mm x 4.6 mm, 5μm)

column.

6.4.3. Selection of the mobile phase

In order to get sharp peak, low tailing factor and base line separation of the

separation of the components, a number of experiments were carried out by varying the

composition of various solvents and flow rate. To have an ideal separation of the drug

under isocratic conditions, mixtures of solvents like methanol, water and Acetonitrile

with or without different buffers indifferent combinations were tested as mobile phases

on a Chromosil C18 column. A mixture of Methanol:0.1% orthophosphoric acid

:Acetonitrile in the ratio of 40: 50:10 v/v/v was proved to be the most suitable of all the

combinations since the chromatographic peak obtained was better defined and resolved

and almost free from tailing.

6.4.4. Flow rate

Flow rates of the mobile phase were changed from 0.5 – 1.5 mL/min for optimum

separation. A minimum flow rate as well as minimum run time gives the maximum

saving on the usage of solvents. It was found from the experiments that 1.2 mL/min flow

rate was ideal for the successful elution of the analyte.

159

6.4.5. Optimized chromatographic conditions

Chromatographic conditions as optimized above were shown in Table 6.2. These

optimized conditions were followed for the determination of Stavudine in bulk samples

and in its Formulations. The chromatogram of standard (4ppm) shown in Figure 6.B.

Mobile phase MEOH: 0.1 % OPA: ACN: 40:50:10 v/v/v

Pump mode Isocratic

Mobile phase PH 5.1

Diluent Mobile phase

Column Chromosil C18 column (250 mm x 4.6

mm, 5μ)

Column Temp Ambient

Wavelength

267 nm

Injection Volume 20 μl

Flow rate 1.2 mL/min

Run time 10 min

Retention Time 6.807 min

Table 6.2: Optimized chromatographic conditions

160

Figure 6.B: Chromatogram of standard solution

161

6.5. VALIDATION OF THE PROPOSED METHOD

The proposed method was validated as per ICH guidelines (as described in

Chapter 1 and 2). The parameters studied for validation were specificity, linearity,

precision, accuracy, robustness, system suitability, limit of detection, limit of

quantification, and solution stability.

6.5.1. Specificity

The specificity of method was performed by comparing the chromatograms of

blank, standard and sample (Prepared from Formulation). It was found that there is no

interference due to excipients in the tablet formulation and also found good correlation

between the retention times of standard and sample. The specificity results are shown in

Table 6.3.

NAME OF THE SOLUTION Retention Time in Min

Blank NO PEAKS

Stavudine 6.807

Table 6.3: Specificity study

6.5.2 Linearity

Linearity was performed by preparing mixed standard solutions of Stavudine at

different concentration levels including working concentration mentioned in experimental

condition i.e. 3 ppm. Twenty micro liters of each concentration was injected in duplicate

into the HPLC system. The response was read at 267 nm and the corresponding

chromatograms were recorded. From these chromatograms, the mean peak areas were

calculated and linearity plots of concentration over the mean peak areas were constructed

individually. The regressions of the plots were computed by least square regression

method. Linearity results were presented in Table 6.4.

162

Table 6.4: Linearity results

Figure-6.C: On X axis concentration of sample, On Y axis peak area response

-50000

0

50000

100000

150000

200000

250000

300000

350000

0 0.5 1 1.5 2 2.5 3 3.5

a

r

e

a

conc. in ppm

stavudine

Level Concentration of Stavudine In ppm Mean peak area

Level -1 0.5 47842.1

Level -2 1.0 91322.2

Level -3 1.5 145921.2

Level -4 2.0 190404.5

Level -5 2.5 245574.6

Level -6 3.0 291202.2

Range: 0.5-

3.0ppm

Slope

Intercept

Correlation coefficient

98516.6285

-3692.966

0.999

163

6.5.3. Precision

Precision is the degree of repeatability of an analytical method under normal

Operational conditions. Precision of the method was performed as intraday precision,

Inter day precision.

6.5.3.1. Intraday precision

To study the intraday precision, six replicate standard solutions (2ppm) of

Stavudine were injected. The percent relative standard deviation (% RSD) was calculated

and it was found to be 0.198, which are well within the acceptable criteria of not more

than 2.0. Results of system precision studies are shown in Table 6.5.

SAMPLE CONC(PPM) INJECTION

No.

PEAKS

AREA

R.S.D(Acceptance

criteria ≤ 2.0%)

Stavudine

2

1 185329.2

0.198

2 185153.9

3 185707.3

4 185219.4

5 185969.4

6 185002.3

Table 6.5: System Precision

6.5.3.2. Inter Day precision

To study the interday precision, six replicate standard solution of Stavudine was

injected on third day of sample preparation. The percent relative standard deviation (%

RSD) was calculated and it was found to be 0.123, which are well within the acceptable

criteria of not more than 2.0. Results of system precision studies are shown in Table 6.6.

164

SAMPLE CONC(PPM) INJECTION

No.

PEAKS

AREA

R.S.D(Acceptance

criteria ≤ 2.0%)

Stavudine

2

1 186287.0

0.123 2 186407.1

3 186414.7

4 186929.5

5 186355.4

6 186503.6

Table 6.6: System Precision (Intra Day)

6.5.4. Accuracy

The accuracy of the method was determined by standard addition method. A

known amount of standard drug was added to the fixed amount of pre-analyzed tablet

solution. Percent recovery was calculated by comparing the area before and after the

addition of the standard drug. The standard addition method was performed at 50%,

100% and 150% level of 2ppm. The solutions were analyzed in triplicate at each level as

per the proposed method. The percent recovery and % RSD was calculated and results

are presented in Table 6.7 Satisfactory recoveries ranging from 99.0 to 102.0 were

obtained by the proposed method. This indicates that the proposed method was accurate.

165

Level Amount of

Stavudine spiked

(ppm)

Amount of Stavudine

recovered(ppm)

% Recovery

%RSD

50 %

1.5ppm

1.5ppm

1.5 ppm

1.48

1.47

1.47

98.66

98.0

98.0

0.387

100%

2.0 ppm

2.0ppm

2.0 ppm

1.98

1.96

1.99

99.0

98.0

99.5

0.772

150%

3.0ppm

3.0ppm

3.0ppm

2.99

2.99

2.98

99.66

99.66

99.33

0.190

Mean % of

recovery

98.86

Mean

RSD =

0.0.449

Table 6.7: Percentage Recovery and % RSD

6.5.5. Robustness

The robustness study was performed by slight modification in flow rate of Mobile

phase, pH of the buffer and composition of the mobile phase. Stavudine at 2 ppm

concentration was analyzed under these changed experimental conditions. It was

observed that there were no marked changes in chromatograms, which demonstrated that

166

the developed method was robust in nature. The results of robustness study are shown in

Table 6.8.

Condition Mean area % assay % difference

Unaltered 187147.5 100.0 0.0

Flow rate at 1.0 mL/min

Flow rate at 1.4mL/min

186885.2

186102.2

99.85

99.44

0.15

0.54

Mobile phase:

MEOH: ACN: 0.1 % OPA

45% 45% 10%

35% 55% 10%

187385.1

186920.8

100.1

99.87

0.1

0.13

pH of mobile phase at 4.9 187445.3 100.1 0.1

pH of mobile phase at 5.3 186583.0 99.69 0.31

Table 6.8: Robustness

5.6. System suitability

System suitability was studied under each validation parameters by injecting six

replicates of the standard solution 2 ppm). The results obtained were within acceptable

limits (Tailing factor ≤2 and Theoretical plate’s ≥2000) and are represented in Table 6.9.

Thus, the system meets suitable criteria.

Parameter Tailing factor Theoretical plates

Specificity study 1.32 7728

Linearity study 0.96 4809

Precision study 1.38 7831

Table 6.9: System Suitability

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6.5.7. Limit of detection and Limit of quantification

Limit of detection (LOD) is defined as the lowest concentration of analyte

that gives a detectable response. Limit of quantification (LOQ) is defined as the

lowest Concentration that can be quantified reliably with a specified level of

accuracy and Precision. For this sample was dissolved by using Mobile Phase and

injected until peak was disappeared. After 15ng/ml dilution, Peak was not clearly

observed. So it confirms that 15ng is limit of Detection and 50ng dilution is

Limit of Quantification. For this study six replicates of the analyte at lowest

concentration were Measured and quantified. The LOD and LOQ of Stavudine are

given in Table 6.10.

Table 6.10: LOQ and LOD

parameter Measured volume

Limit of Quantification 50ng

Limit of Detection 15ng

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6.6. RESULTS AND DISCUSSION

To develop a precise, accurate and suitable RP- HPLC method for the

simultaneous estimation of Stavudine different mobile phases were tried and the

proposed chromatographic conditions were found to be appropriate for the quantitative

determination. Proper selection of the stationary phase depends up on the nature of the

sample, and molecule Physico- chemical properties. The UV spectra of Stavidine showed

that the drug absorbs appreciably at 267nm was selected as the detection wave length in

liquid chromatography. Optimization of mobile phase was performed based on

asymmetric factor and peak area obtained. Different mobile phases were tried but

satisfactory separation, well resolved and good symmetrical peaks were obtained with the

mobile phase Methanol: Acetonitrile: 0.1 %OPA (40:50:10, v/v/v). The retention time of

Stavudine was found to be 6.8 min, which indicates a good base line.

The calibration curve for Stavudine was obtained by plotting the peak area ratio

versus the concentration of Stavudine over the range of 0.5-3.0 ppm, and it was found to

be linear with r2=0.999. The regression equation of Stavudine concentration over its peak

area ratio was found to be y = (-3692.966 + 98516.62 x), where x is the concentration of

Stavudine (ppm) and Y is the respective peak area. The results obtained were within

acceptable limits where capacity factor >2.0, tailing factor ≤2.0 and theoretical plates

>2000. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for

two consecutive injections was < 2.0%. The data of regression analysis of the calibration

curve was shown in table 6.4.

Precision was evaluated by carrying out six independent sample preparation of a

single lot of formulation. Sample was analyzed for five times after extracting the drug as

mentioned in assay sample preparation of the experimental section. Low values of

standard deviation denoted very good repeatability of the measurement. Thus it was

showing that the equipment used for the study was correct and hence the developed

analytical method is highly repetitive. RSD of intraday precision was found to 0.198. For

the interday precision a study carried out on consecutive days indicated a RSD of 0.123.

This indicates good method precision. Results are shown in Table 6.5 and 6.6.

169

The stability studies were evaluated by storing the solutions at ambient

temperature (20±100C) and checked in triplicate after three successive days of storage

and the data were compared with freshly prepared samples. In each case, it could be

noticed that solutions were stable for 48 hrs, as during this time the results did not

decrease below 98%. This denotes that Stavudine is stable and standard and sample

solutions for at least 2 days at ambient temperature.

The accuracy of the method was evaluated by determination of the recovery of

Stavudine with standard addition method at 50%, 100% and 150% to the proposed HPLC

method. The results showed good recoveries ranging from 99.00 to 101.45%. The mean

recovery data obtained for each level as well as for all levels combined (Table 6.7) were

within 2.0% of the label claim for the active substance with an R.S.D. < 2.0%, which

satisfied the acceptance criteria set for the study.

The system suitability parameter (Table 6.9) like capacity factor, asymmetry

factor, tailing factor and number of theoretical plates were also calculated. It was

observed that all the values are within the limits (tailing factor ≤ 2and number of

theoretical plate’s ≥ 2000). The limit of detection (table 6.10) and limit of quantization

for Stavudine was found to be 15ng and 50ng, indicates the sensitivity of the method.

The statistical evaluation of the proposed method was revealed its good linearity,

reproducibility and its validation for different parameters and let us to the conclusion that

it could be used for the rapid and reliable determination of Stavudine in tablet

formulation.

170

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