INTRODUCTION:

Stavudine³is a dideoxynucleoside analog that inhibits reverse transcriptase and has in vitro activity against HIV. The chemical name of Stavudine is 1-[(2R,5S)-5- (hydroxymethyl)-2,5-dihydrofuran-2-yl]-5-methylpyrimidine-2,4-dione. Stavudine is a NRTI with activity against HIV-1. Stavudine is phosphorylated to active metabolites that compete for incorporation into viral DNA. They inhibit the HIV reverse transcriptase enzyme competitively and act as a chain terminator of DNA synthesis. The lack of a 3'- OH group in the incorporated nucleoside analogue prevents the formation of the 5' to 3' phosphodiester linkage essential for DNA chain elongation, and therefore, the viral DNA growth is terminated. Stavudine inhibits the activity of HIV-1 RT both by competing with the natural substrate dGTP and by its incorporation into viral DNA.

Figure 1: Chemical structure of Stavudine³

High Performance Liquid Chromatography (HPLC)¹

A ‘Regulatory Analytical Procedure’²is used to evaluate a defined characteristic of the drug substance or drug product. An ‘alternative analytical procedure’ is proposed by the applicant for use other than regulatory analytical procedure.

The modern methods of choice for quantitative analysis are HPLC, GC, and HPTLC, which are highly sophisticated. Chromatographic methods are commonly used in regulatory laboratories for the qualitative and quantitative analysis of drug substances, drug products, raw materials and biological samples throughout all phases of drug development, from research to quality control.

HPLC is the fastest growing analytical technique for the analysis of drugs. Its simplicity, high specificity, and wide range of sensitivity make it ideal for the analysis of many drugs in both dosage forms and biological fluids. The rapid growth of HPLC has been facilitated by the development of reliable, moderately priced instrumentation and efficient columns.

HPLC is an advanced form of liquid chromatography that is used to separate complex mixtures of molecules in chemical and biological systems. Compared to other chromatography systems, HPLC systems have higher resolution, faster cycle times and columns that can be reused without repacking or regeneration. The mobile phase of these systems can also be varied during the analysis, resulting in a gradient elution. HPLC columns have a stationary phase consisting of very small particle sizes with large surface areas that allow the application of high pressure to the solvent flow.

The advantage of a well-accepted analytical technique like HPLC is that researchers develop variations of these systems that work particularly well for their specific applications. Normal phase was the first technique developed, which uses a polar stationary phase and a non-polar mobile phase, allowing it to work well for relatively polar analytes. Reversed phase HPLC, however, is the most commonly used version. These systems have a non- polar stationary phase (i.e., treated silica) and an aqueous, moderately polar mobile phase. In reversed phase, retention times can be longer for molecules that are more non- polar, while polar molecules elute more readily. HPLC systems are widely used because of their accurate and reproducible results, high resolution, high precision, high sensitivity and ease of being automated to further reduce the attendant costs and time requirements.

MATERIAL AND METHOD:

Table No.2: DRUGS USED

Sr. No.	Drugs	Manufacturer name
1.	Stavudine	Emcure Pharma, Pune

Table No.3: REAGENT USED

Sr. No.	Chemicals	Manufacturer name	Grade
1.	Methanol	S.d. Fine chemical	HPLC
2.	Acetonitrile	S.d. Fine chemical	HPLC
3.	Water	Finar	HPLC
4.	Ammonium formate	S.d. Fine chemical	HPLC
5.	Formic acid	S.D. Fine chemical	HPLC
6.	Glacial acetic acid	S.D. Fine chemical	HPLC

Table No.4: EQUIPMENT AND APPARATUS USED

Sr. No.	Instrument Name	Model num. and software	Manufac- Turer name
1.	Hplc	Lc-10at, Lab solution	Shimadzu, japan
2.	Uv-visible spectrophoto Meter	Pharmaspec-1700, Uv probe 2.0	Shimadzu, japan
3.	Ultrasonic Bath	Frontline fs4 ultrasonic cleaner	Mumbai
4.	Ph meter	Systronic	Mumbai
5.	Electronic balance	Cp2245s analytical balance	Gottingen, germany
6.	0.45 µ hplc filter	-	Merck
7.	Whatmann filter paper no. 41	-	Merck
8.	Volumetric flask	-	Borosil
9.	Graduated pipette	-	Borosil
10.	Measuring cylinder	-	Borosil
11.	Funnel	-	Borosil
12.	Conical flask	-	Borosil
13.	Glass beaker	-	Borosil
14.	Plastic beaker	-	Borosil

EXPERIMENTAL:

PREPARATION OF SOLUTIONS:

Preparation of the mobile phase:

HPLC grade solvents were used in separate bottle of gradient pumps as mobile phase. A mixture of water and methanol in the ratio of 60:40 v/v were adjusted by gradient pump operated by LC solution software. Mixed solvents were filtered through nylon 0.45µm membrane filter and degassed by the instrument and used as mobile phase.

Preparation of standard solution (100µg/mL):

Accurately weighed 10mg Stavudine was transferred to 100mL volumetric flask, dissolved in 50mL methanol and diluted up to mark with methanol to prepare standard solution having concentration of 100µg/mL.

Preparation of sample solution:

Twenty capsules were taken and accurate quantity of capsule content equivalent to 10mg of Stavudine was weighed and transferred to 100mL volumetric flask, dissolved in methanol (60mL) and sonicated for 30 min. The solution was filtered through Whatmann filter paper No. 41 and residue was washed with methanol. The solution was diluted up to the mark with methanol.

Determination of wavelength of maximum absorbance:

The standard solution of Stavudine (5µg/mL) was scanned in the range of 200 to 400nm and the UV spectrum was recorded.

CHROMATOGRAPHIC CONDITIONS:

The chromatographic separations were performed using the final chromatographic conditions as mentioned in table 5.

Table No.5: Final chromatographic conditions

Sr. No.	Parameter	Description
1	Stationary Phase	Phenomenex^® C₁₈column with 250 mm x 4.6 mm i.d. and 5 µm particle size
2	Mobile Phase	Mixture of water and methanol in the ratio of 60:40 v/v
3	Flow Rate	1.0 mL/min
4	Detection wavelength	266 nm
5	Detector	PDA detector
6	Injector	Manual injector loop
7	Injection volume	20 µL
8	Column Temperature	Ambient
9	Run Time	06 min
10	Diluent	Methanol

METHOD VALIDATION²

Linearity and range:

Accurately measured standard solutions of Stavudine (0.1, 0.3, 0.5, 0.7, 0.9, 1.0, and 1.2mL) were transferred to a series of 10 mL of volumetric flasks and diluted to the mark with methanol. 20 µL each of these solutions were injected using manual injector loop under the final chromatographic conditions described above. Calibration curve was constructed by plotting peak area versus concentration of Stavudine and the regression equation was calculated.

Accuracy (% Recovery):

The accuracy of the method was determined by calculating recoveries of Stavudine by the standard addition method. For this known amount of standard solutions of Stavudine (50, 100, and 150 % level) were added to pre analyzed sample solutions. The amount of Stavudine was analyzed by using the regression equation of the calibration curve.

Precision:

Method precision (% Repeatability):

The precision of the instrument was checked by repeatedly injecting (n=6) standard solution of Stavudine (5µg/mL). The results were reported in terms of % CV.

Intermediate precision:

Intermediate precision was evaluated in terms of intraday and inter day precision. The intraday precision was investigated by analyzing three different standard solutions of Stavudine (3.0, 7.0 and 9.0 µg/mL). The inter day precision was investigated by analyzing three different standard solutions of Stavudine (3.0, 7.0 and 9.0µg/mL) on different days. The results were reported in terms of % CV. Method robustness was performed by applying small changes in the composition of mobile phase, analytical wavelength and flow rate. Robustness of the method was done at three different concentration levels of 3.0,7.0 and 9.0µg/mL for Stavudine. The results were expressed in terms of % Recovery ± S.D.

Robustness:

Method robustness was performed by applying small changes in the composition of mobile phase, analytical wavelength and flow rate. Robustness of the method was done at three different concentration levels of 3.0, 7.0 and 9.0µg/mL for Stavudine. The results were expressed in terms of % Recovery ± S.D.

LOD and LOQ:

The LOD and LOQ of the method were determined by using the following equations:

LOD = 3.3 σ / S AND LOQ = 10 σ / S

Where

σ = standard deviation of the response and

S = slope of the calibration curve

Specificity and selectivity:

The specificity of the method was established through resolution factor of the drug peak from the nearest resolving peak and also among all other peaks. Selectivity was confirmed through peak purity data using a PDA detector. To assess the method specificity, a placebo (without Stavudine) was prepared with the excipients as required for commercial preparation and compared with respective drug standard to evaluate specificity of the method. Representative chromatograms of placebo and standard were compared for retention time, resolution factor and purity.

System suitability:

^{The
system suitability parameters like theoretical plates (T}p^{), and asymmetry factor (As), capacity factor
(K’), resolution (R}s^{), retention time (RT) and tailing factor (T}f^{)
were calculated by Class VP LC solution software. The HPLC system was}equilibrated with the initial mobile phase composition, followed by six injections of the standard solution having same concentration. These six consecutive injections were used to evaluate the system suitability on each day of method validation. In order to establish system suitability for the instrument, six consecutive injections of Stavudine were prepared from the standard solution and analyzed.

Solution stability:

The solution stability of Stavudine in the proposed method was carried out by leaving both the test and standard solution in tightly capped volumetric flask at room temperature for 24 hours. The same sample solutions were assayed for interval of 6 hours up to the 24 hours throughout the study period. The obtained results were compared with the freshly prepared solution.

Analysis of Stavudine in capsule formulation:

Appropriate three different aliquots from sample solution were suitably diluted with mobile phase in such a way to get concentrations in a range of 1 to 12 µg/mL for Stavudine. The finally prepared solutions were analyzed under chromatographic condition as described in table no. 5. The amount of Stavudine present in sample solution was determined by fitting the area response into the regression equation of both the drugs in the method.

Comparison of the developed method with the official method:

The drug is official in I.P. The developed method was compared with the official method and the results were analyzed using t- test.

RESULTS AND DISCUSSION:

Determination of analytical wavelength:

Solution of Stavudine (5 µg/mL) was scanned between 200 and 400 nm with the help of UV spectrophotometer. From the UV spectra it was observed that the maximum response is obtained at 266 nm. Hence 266 nm was selected as the analytical ^{wavelength. The UV
spectrum is shown}in Figure 2.

Figure 2: UV spectrum of Stavudine

Method optimization:

The aim of this study was to develop a gradient RP-HPLC assay method for the analysis of Stavudine in bulk and in formulation. Since the drug is soluble in polar solvents like methanol and water, a RP-HPLC method was thought to be suitable. A ^Phenomenex
C18 ^{column (250 mm x 4.6 mm i.d., 5 µm particle size) was preferred}over other columns because it has high carbon loading with very closely packed ^{material
to give high performance over other C}18 ^{columns. Initially different
mobile}phases have been tried. The results of the trials are shown in table 6.

^{Table No.6:}Results of the initial trials for optimization of mobile phase

Mobile phase	Ratio	Result
A mixture of methanol and water	10:90	No retention of drug
A mixture of ACN and methanol	50:50	No peak observed
A mixture of water and methanol	50:50	No sharp peak observed
A mixture of water and methanol	60:40	Sharp peak and good resolution

Finally, acceptable resolution with reasonable peak shapes and high peak purity was achieved by using a mixture of water and methanol in the ratio of 60:40 v/v with flow rate of 1 mL/min at 266 nm. The method parameters were optimized for the analysis of Stavudine in capsule formulation. A representative chromatogram is shown in Figure 3, which satisfies all the system suitability criteria, better resolution of the peak from ^{solvent peak with clear base
line}separation.

Figure 3: Representative chromatogram showing peak of Stavudine (5µg/mL) at 266nm

METHOD VALIDATION²

The developed method as described above was validated for various parameters like system suitability, specificity, linearity, precision, accuracy, LOQ and LOD.

Linearity and range:

Linearity of the method was evaluated at seven concentration levels by diluting the standard solution in the concentration range of 1 - 12µg/mL for Stavudine. The results show that an excellent correlation existed between the peak area and concentration of analyte. The calibration curve was prepared by plotting the peak area versus the concentration and the regression equation was calculated. The calibration curve was repeated for five times and the average results are mentioned in table 7. Figure 4 shows the calibration curve of Stavudine for LOD and LOQ calculation. Table 8 shows the optical and regression characteristics for analysis of Stavudine by RP-HPLC method.

Table 7: Data for calibration curve for Stavudine

Conc. of Stavudine µg/mL	Peak area
1	2635
3	18586
5	45596
7	67654
9	91790
10	106786
12	133476

Figure 4: Calibration curve of Stavudine

Table 8: Optical and regression characteristics for analysis of Stavudine

Parameters	Stavudine
Concentration range (µg/mL)	1-12
LOD (µg/ml)	0.1679
LOQ (µg/ml)	0.5598
Regression Equation	Y= -12023x -14078
Correlation coefficient (R²)	0.9958

Precision Method precision:

The results of repeatability (method precision) experiment are shown in table 9. Method precision was determined by repeatedly injecting 5.0µg/mL concentration of Stavudine (n = 6). The developed method was found to be precise and the results are reported in terms of % CV.

Table 9: Method precision data of Stavudine by RP-HPLC method

Drug	Conc.	RT	%CV	Area	% CV
Stavudine	5.0 µg/mL	3.234	0.967	44365	0.902

Intermediate precision:

The results of intermediate precision experiment for both intraday and inter day are shown in table 10. Replicate analyses of three concentrations of the standard solution show good reproducibility. The results are reported in terms of % CV values.

Table 10: Intermediate precision data of Stavudine RP-HPLC method

Stavudine µg/mL	Intra-day measured mean area, % CV (n=6)	Stavudine µg/mL	Inter-day measured mean area, % CV (n=6)
3	21135, 1.2167	3	22195, 1.3434
7	70462, 0.7493	7	70497, 1.0525
9	93935, 0.5178	9	94087, 0.8273

Accuracy (% Recovery):

Good recovery of the spiked drug was obtained at each added concentration, indicating that the method was accurate. A known amount of drug (50, 100, and 150 %) was added to the pre analyzed sample solution. This solution was analyzed under the chromatographic conditions mention in table no. 5. The assay was repeated over 3 consecutive days to obtain intermediate precision data. Results of accuracy are shown in table 11.

Table 11: Accuracy (% Recovery) study of Stavudine by RP-HPLC method

Drug	Known conc. µg/mL	Added conc. µg/mL	% Recovery ± S.D.
Stavudine	4	2 (50%)	98.4725 ± 1.3521
	4	4 (100%)	99.3746 ± 1.4576
	4	6 (150%)	98.3209 ± 1.0467

Robustness:

To evaluate the robustness of the proposed method, experimental conditions were deliberately altered and the response of the drugs was recorded. The results of minor variations in composition of mobile phase, wavelength, and flow rate are shown in table 12.

Table 12: Robustness data of Stavudine (4 µg/mL) by RP-HPLC method

Parameter	Modification	% Recovery ± S.D. (n=6)
Flow rate (1 mL/min)	+ 0.1	99.1327 ± 1.1104
Flow rate (1 mL/min)	- 0.1	99.0561 ± 0.8310
Mobile phase composition	59:41	98.3473 ± 1.0124
Water : methanol (60:40 v/v)	61:39	98.6593 ± 0.9134
Wave length (266 nm)	+ 1	98.8714 ± 1.0431
Wave length (266 nm)	- 1	98.7968 ± 0.7841

LOD and LOQ:

These data show that the method is sensitive for the determination of Stavudine. The LOD and LOQ were calculated by using the equations mentioned in table no.5. The results are shown in table 13.

Table 13: LOD and LOQ for Stavudine

Slope

LOD µg/mL

LOQ µg/mL

Stavudine

(7 µg/mL)

673.0427

12023

0.1679

0.5598

Specificity and selectivity

The resolution factor for Stavudine from the nearest resolving solvent peak was > 3 in all samples. The placebo shows no detector response near retention times of 3.2 min, while the Stavudine standard displayed good resoluted peak [Figure 5] and no interference from excipients present in the formulation [Figure 6] which indicates specific nature of the method. The peak purity curve and data [Figure 7 and Table 14] of Stavudine shows that no other excipients are co-eluted with the drug and the peak is pure in nature.

Figure 5: Chromatogram showing peak of Stavudine at 266 nm in bulk drug

Figure 6: Chromatogram showing peak of Stavudine at 266 nm in formulation

Figure 7: Peak Purity Plot of Stavudine with purity 0.99999

Table 14: Peak purity data for determination of Stavudine

Drug	Peak purity Index	Single point Threshold	Min peak purity threshold
Stavudine	0.99999	0.99984	3479

System suitability:

As system suitability test was an integral part of chromatographic method development and were used to verify that the system is adequate for the analysis to be performed, the system suitability parameters for Stavudine were evaluated. The suitability of the chromatographic system was demonstrated by comparing the obtained parameter values. The obtained parameters are given in table 15 and they are found to be in concordance to the acceptance criteria.

Table 15: System suitability parameters for Stavudine by RP-HPLC method

Parameter Value for Stavudine
Retention time (Minutes)	3.234
Resolution (Rs)	4.65
Theoretical plates (TP)	6758
Tailing factor (Tf)	0.94
Asymmetric factor (Af)	1.23
Capacity factor (K’)	3.45

Solution stability:

The % CV of the assay of Stavudine during solution stability experiments were within 2%. No significant changes were observed in the content of standard drug solution during solution stability and mobile phase stability experiments when performed using the method. The solution stability and mobile phase stability experiment data confirms that the sample and standard in solvent and mobile phases used during assay determination were stable for at least 24 hours. The results of solution stability data are shown in table 16.

Table 16: Solution stability data for Stavudine

Peak Area Stavudine 6µg/mL
Time (Hr)	Standard	Sample	% sample stability
00 45595 45563 00.00
06	45568	45518	99.90
12 45439 45487 99.83
18	45331	45476	99.81
24 45187 45459 99.77

Comparison of developed method with the official method:

The assay results in terms of AUC calculated as assay % were compared with each other for both the developed method and the standard method after 100% spiking with a known concentration of the standard drug solution. Six replicate readings were taken for each method. The results were compared by using t-test. At 5% probability the tabulated value was 2.57. The calculated value was obtained as 0.9828. Hence it is deduced that there is no significant difference between the accuracy of the two methods.

CONCLUSION:

A validated RP-HPLC analytical method has been developed for estimation of Stavudine in bulk and in formulation. The proposed method is very fast, simple, accurate, precise, specific, and has ability to separate drug from excipients. The method is suitable for routine analysis of Stavudine in capsule formulation. The simplicity of the method allows for application in laboratories that lack sophisticated analytical instruments. Prime importance was given to develop less time consuming and simple RP-HPLC method that requires no sample pre-treatment and is quite economical for routine analyses. The proposed method developed meets the system suitability criteria, peak integrity and resolution for the drugs. Detection and quantification limits achieved describe that the method is quite sensitive. High recoveries and acceptable % CV values confirm that the proposed method is accurate and precise. The analytical results demonstrate the ability of the developed method to assay the drug in the presence of its excipients. Also the data for precision show that developed method is precise. Assay results found from the study show that the method can be successfully applied for the estimation of Stavudine in capsule formulation. Also by applying statistical technique it is concluded that there is significant difference between the developed method and the official method. Hence the proposed method is recommended for routine analysis of Stavudine in bulk and in capsule formulation.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. N. J. Patel, Principal of Shree S. K. Patel College of Pharmaceutical Education and Research for providing all facilities for my research work and Emcure Pharma, Pune for providing drug samples Stavudine to carry out this work.

REFERENCES:

1. The Merck Index, An Encyclopedia of Chemicals, Drugs and Biological, Published by Merck research laboratories, Merck and Co., Inc., Whitehouse Station, N.J., USA, 14th Edition, 2006.

2. Validation of Analytical Procedures: Text and Methodology Q2(R1); International Conference on Harmonization: ICH Harmonized Tripartite Guideline; International Conference on Harmonization of Technical requirements for registration of pharmaceuticals for human use; Current step 4 version; November 2005.

3. Drugbank: DB00649 (Stavudine).htm; The Merck index online SM, an encyclopedia of chemicals, drugs and biological, Published by Merck research laboratories, Merck and Co., Inc., Whitehouse Station, N.J., USA, 14th edition, 2006.

4. Indian Pharmacopoeia, Vol-II and Vol III, Published by the controller of publications, Delhi, India, 2010.

5. British Pharmacopoeia, Vol. II, Her Majesty's Stationary Office, London, 2003.

6. María Sarasa, Neus Riba, Laura Zamora and Xavier Carné, Determination of Stavudine in human plasma and urine by high-performance liquid chromatography using a reduced sample volume; Journal of Chromatography B: Biomedical Sciences and Applications; 746, 2, 15 September 2000,183-189.

7. D. M. Burger, H. Rosing and R. Van Gijn, P. L. Meenhorst, O. Van Tellingen, J.H. Beijnen, Determination of Stavudine, a new antiretroviral agent, in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection; Journal of Chromatography: Biomedical Application, 584, 2, 23 December 1992, 239-247.

Received on 18.10.2019 Modified on 10.11.2019

Research J. Pharm. and Tech. 2020; 13(1):15-21.

DOI: 10.5958/0974-360X.2020.00003.7

Development and Validation of RP-HPLC Method for Estimation of Stavudine in Bulk and in Capsule Formulation

Figure 1: Chemical structure of Stavudine3

MATERIAL AND METHOD:

Table No.2: DRUGS USED

EXPERIMENTAL:

PREPARATION OF SOLUTIONS:

Preparation of the mobile phase:

Preparation of standard solution (100µg/mL):

Preparation of sample solution:

Determination of wavelength of maximum absorbance:

CHROMATOGRAPHIC CONDITIONS:

Accuracy (% Recovery):

Precision:

Intermediate precision:

Robustness:

LOD and LOQ:

Specificity and selectivity:

System suitability:

Solution stability:

Analysis of Stavudine in capsule formulation:

Comparison of the developed method with the official method:

RESULTS AND DISCUSSION:

Determination of analytical wavelength:

Figure 2: UV spectrum of Stavudine

Table No.6: Results of the initial trials for optimization of mobile phase

METHOD VALIDATION2

Linearity and range:

Figure 4: Calibration curve of Stavudine

Table 9: Method precision data of Stavudine by RP-HPLC method

Table 10: Intermediate precision data of Stavudine RP-HPLC method

Table 11: Accuracy (% Recovery) study of Stavudine by RP-HPLC method

Table 12: Robustness data of Stavudine (4 µg/mL) by RP-HPLC method

Table 13: LOD and LOQ for Stavudine

Figure 5: Chromatogram showing peak of Stavudine at 266 nm in bulk drug

Solution stability:

Table 16: Solution stability data for Stavudine

CONCLUSION:

ACKNOWLEDGEMENT:

REFERENCES:

Figure 1: Chemical structure of Stavudine³

^{Table No.6:}Results of the initial trials for optimization of mobile phase

METHOD VALIDATION²