INTRODUCTION:

The L-valine ester of ACV is valacyclovir, also known as 2- [2-amino-1, 6-dihydro-6-oxo-9Hpurin-9-yl-methoxy] ethyl valinate hydrochloride. The L-valine ester of aciclovir is valacyclovir. It is classified as a DNA polymerase enzyme inhibitor nucleoside analogue.

The metabolite aciclovir, a purine (guanine) nucleoside analogue, significantly boosts the pharmacological effects of valacyclovir. The catalyst valacyclovir hydrolase is likely responsible for the rapid and complete conversion of valacyclovir in humans into aciclovir and valine. Aciclovir is a particular inhibitor of the herpes infections, acting in vitro against human herpesvirus 6, cytomegalovirus (CMV), varicella zoster virus (VZV), and herpes simplex infections (HSV) types 1 and 2. (HHV-6). In conclusion, valacyclovir works to combat viruses in 3 separate ways: 1) Viral DNA polymerase competitive inhibition 2) stop the viral DNA chain formation 3) the viral DNA polymerase is rendered inactive. With increased efficiency, viral thymidine kinase phosphorylates acyclovir, boosting its antiviral efficacy against HSV in comparison to VZV (TK). The purpose of the current work is to establish and validate UV and HPLC techniques for valacyclovir concentration estimate in pharmaceutical products in accordance with ICH Q12R2 standards. Additionally, HPLC is used to examine the force degradation. Certain analytical techniques, such as RP-HPLC, UV, LC-MS, and HPTLC, were described for the simultaneous estimation of other medications with valacyclovir in pharmaceutical formulations, according to a literature review. The force degradation study of valacyclovir was carried out by HPLC method using mobile phase Methanol: 10mM KH2PO4 Buffer (50:50) at pH3, and wavelength 254nm. As an outcome, an exertion has been crafted in the observed study to develop UV and HPLC methods for estimation of in pharmaceutical preparation.

MATERIAL AND METHODS:

Chemicals and Reagents:

Cipla Ltd., Mumbai, India, provided a valacyclovir standard as a gift sample. Thermo Fisher Scientific provided the methanol and water of HPLC grade, as well as valacyclovir tablets were purchased locally.

Instruments:

HPLC:

The HPLC study was carried out using an Analytical Technologies Ltd. HPLC-3000 series (Binary gradient framework) system with UV-Vis detector (UV2012).

UV-Visible spectrophotometer:

The UV-Visible spectrophotometer (UV-3000-M) made by Analytical Technologies Ltd. with a spectral band breadth of 2 centimetres and a quartz cell of 1 millimetre was used to measure the absorbance.

UV-visible Spectrophotometric method optimization^6,7,8,9,10

Selection of mobile phase:

The mobile phase was chosen by testing several solvents in varying concentrations; the solvent that produced the most exact results in the UV spectrophotometer was chosen.

Preparation of standard stock solution:

Produce standard solutions of 1000ug/ml, 10mg of the pure valacyclovir was precisely measured and moved to a 10ml volumetric flask. To this flask, the drug was then dissolved using the proper volume of solvents.

Preparation of working solution:

To produce 100ug/ml solution, 1ml of the standard stock solution was diluted using the mobile phase up to 10ml in a volumetric flask.

Selection of detection wavelength:

For determine the wavelength of maximal absorption (λmax) of valacyclovir, 1mL of experimental solution has been mitigated to 10mL with mobile phase and measured in UV spectrophotometer in spectrum mode over whole UV region, i.e., 200-400nm.

Establishment of calibration curve:

Pipette out 0.9, 1.8, 2.7, 3.6, and 4.5ml of the standard working solution and place them in individual 10ml volumetric flasks. To every flask, mobile phase was added until the volume reached the mark, yielding a solution with concentrations of 9, 18, 27, 36, and 45 g/ml. Figure out whether the concentration range agreed to Beer-Lamberts law, the absorbance of every solution was assessed at the selected wavelength.⁷

Assay:^11,12

The mean mass of the valacyclovir tablets were separately weighing 10 tablets. In a volumetric flask of 10ml, the equivalent weight of 11.96mg of valacyclovir tablet powder (equivalent to 10mg of pure medication) was mixed with 5ml of the mobile phase. Sonication have been used for 10 minutes to dissolve the contents of the powdered tablet, and then the volume was made to the mark using the mobile phase. This solution was placed in falcon tubes and centrifuged for 30 minutes to settle the undissolved contents. A 0.22 membrane filter was used to screen the supernatant after that. 0.3ml of this solution was then diluted up to 10ml with mobile phase to obtain 30ug/ml of solution, which was then scanned in triplicate in a UV spectrophotometer. The % Assay was obtained using the following equation:

%Assay = Concentration of bioactive in μg/ml X Dilution Factor X 100 X Average weight of sample/Label Claim X Weight of the sample taken.

Validation of method as per guidelines of ICH ^13,14,15,16,

Accuracy:

The drug recovery investigations were done using a conventional addition approach with known amounts of standard drug in three levels, specifically 50%, 100%, and 150%, which were mixed with pre-analysed samples. At each level, three determinations were prepared, and the outcomes observed were assessed to the potential results.

Precision:

Intermediate precision was executed by utilizing the optimized method for 27µg/ml of solution in triplicates for 2 times a day and for 2 consequent days (Interday). Estimation of absorbance was used to calculate % relative standard deviation (%RSD).

Linearity:

The sample solution 9 to 45ug/ml were transferred to 10 ml volumetric flask individually and mobile phase used to get desired solution. The calibration curve’s linearity was determined through regression coefficient by plotting a graph of absorbance versus concentration.

Robustness:

Calculate the optimized method robustness, two parameters were deliberately altered. The parameters changed in this study are flow rate (±1) and the wavelength of determination (±1). The procedure was performed by using 27μg/ml concentration and the factor changed was detection wavelength to measure its impact on the optimized method.

Ruggedness:

The many analysts concluded their investigations into drugs, and the quantitative influence of the components was determined in order to verify the robustness of the approach. The approach determination was evaluated at various drug conc. levels. Insignificant differences in drug absorbance were observed.

Selection and validation of HPLC method:^17,18,19

Specificity:

The specificity of the technique was controlled by taking chromatogram of mobile phase, standard solution and test sample solution for decide the obstruction of excipients in the test sample.

Accuracy (Recovery):

A standard addition system was followed to additionally assess the exactness of the technique

Precision:

When a procedure is repeatedly used on various samplings of a homogeneous sample, precision is the degree of agreement among individual test results. Standard deviation or relative standard deviation is usually applied to indicate the accuracy of an analytical technique.

Precision is the ratio of an analytical technique's level of repeatability under normal activity.

Repeatability:

By evaluating the sample solution six times and calculating the relative standard deviation, the repeatability of the framework is established.

Intermediate Precision:

Intraday Precision:

The sample solution is examined three times, each at roughly the same time, using alternate hours like 0, 4, and 8hrs., and the relative standard deviation is calculated.

Inter day Precision:

The sample solution is evaluated at repeatedly on different days, such as 1, 2, and 3, and the relative standard deviation is calculated to control the framework's interday accuracy.

Linearity:

The linearity of an analytical strategy was controlled by its capacity to demonstrate that test outcomes are straightforwardly relative to the strength of an analyte inside a particular limit.

Limit of Detection (LOD) and limit of Quantitation (LOQ):

In accordance with ICH guidelines, the LOD and LOQ were established using the following equations.

LOD= 3.3 × σ/S

LOQ= 10 × σ/S

Robustness:

It measures the technique's ability to remain unaffected by small but deliberate variations in the strategy parameter and indicates its dependability when used as intended.

Ruggedness:

It is the level of reproducibility of the test outcome acquired by investigation of tests, under an assortment of condition, for example, various lab, scientist, instrument, loads of reagents, passed time, diverse time, temp, days and so on.

Forced degradation studies:^20,21,22

Constrained decomposition is an exploitation of novel drug constituents and medicine items at conditions more than quickened conditions. These analyses show the chemical conduct of the particles which thus helps the improvement of formulation and packaging. Henceforth, in the present investigation constrained decomposition examines were built up by oppressing the samples of Valacyclovir standard solution for decomposition in oxidative, hydrolytic, photolytic and thermal.

RESULT AND DISCUSSION:

Development of UV Visible spectrophotometric method for Valacyclovir.

Figure 1: UV spectrum of Valacyclovir at 254 nm

Assay:

As a result of experimental results, the amount of drug in powder that was reported as a % of the label claim could not be said to be in acceptable agreement with the label claim, therefore implying that there is no impediment from any excipients.

Table 1: Assay of Valacyclovir

Sr. No	Conc. (μg/ml)	Abs. of sample	% Assay	Mean % Assay	S.D. (±)	% RSD
1	27	0.7039	99.45	99.4566	0.220075	0.221278
2	27	0.7041	99.68
3	27	0.7043	99.24

Validation of UV-Visible spectrophotometric method

Accuracy (Recovery studies)

Table 2: Accuracy determination of Valacyclovir by UV

Sr. No	Conc. (µg/ml)	Area	Mean	%SD	% RSD
1	9	0.3517	0.35183333	0.00015275	0.009709157
2	9	0.352
3	9	0.3518
4	27	0.7043	0.7043	0.0002
5	27	0.7041
6	27	0.7045
7	45	1.0591	1.05933333	0.00049329
8	45	1.059
9	45	1.0599

Table 3: Recovery determination of Valacyclovir by UV

Sr. No.	Concentration	% Added	Absorbance	% Recovery
1	18+9	50%	0.704	99.75
2	18+18	100%	0.875	98.65
3	18+27	150%	1.0585	98.91
			Mean	99.1033

Once the suggested method was used to estimate the amount of valacyclovir in bulk and pharmaceutical products after they had been spiked with additional drug, the recovery rate was seen to be 99.103% valacyclovir with a %RSD of < 2%.

Precision:

The %RSD of Valacyclovir was seen as 0.0578 for intraday precision and 0.089 for interday precision. As a result, the suggested UV approach was considered precise.

Linearity:

By selecting correlation coefficient and S.D., it was determined that the calibration graph was linear, and that the framework adhered to Beer's law. As much as feasible, there are ideals that were visible inside as well.

Table 4: Study of linearity for Valacyclovir

Sr. No.	Conc. (μg/ml)	Absorbance
1	9	0.3517
2	18	0.5367
3	27	0.7043
4	36	0.8756
5	45	1.0591

Robustness:

Robustness of the method was assessed at a 27 μg/ml concentration (n=3).

Table 5: Result at different wavelength

Sr. No.	Concentration	Wavelength	Absorbance
1	27	254 nm	0.7045
2	27	255 nm	0.7044
3	27	256 nm	0.7048
		Mean	0.70457
		SD	0.00021

Ruggedness:

Table 6: Ruggedness study for Valacyclovir

Sr. No.	Concentration	Absorbance
1	9	0.3519
2	18	0.5369
3	27	0.7045
4	36	0.8759
5	45	1.059

Development of HPLC method for Valacyclovir:

Preparation of Calibration curve by HPLC method:

Calibration curve of Valacyclovir got by plotting diagram Concentration versus Area. Regression coefficient acquired inside utmost for Valacyclovir 0.998.

Table 7: Calibration by HPLC method

Sr. No.	Conc. (µg/ml)	Avg. Area
1	10	578693
2	20	968475
3	30	1325973
4	40	1678272
5	50	2108373

System suitability studies:

Number of theoretical plates ought to be more prominent than 2000. It demonstrates the proficiency of column. Evaluation of asymmetry factor or subsequent variable ought to be < 2. For Valacyclovir, R.T, Theoretical plate and Tailing factor was seen as 5.024 min, 8451, 1.14 individually

Assay of Valacyclovir:

Valacyclovir contain at the very least 90.00% and not over 105.0% of Labelled claim.

Valacyclovir was seen as 99.5605%.

HPLC method validation:

Accuracy (Recovery studies)

Mean recovery ought to be in the range of 90-102%. The Relative Standard Deviation ought not be over 2.0%.

Table 8: Accuracy of Valacyclovir by HPLC

Sr. No	Conc. (µg/ml)	Area	Mean	%SD	% RSD
1	10	578693	578772.6667	0.242271	0.068809349
2	10	580213
3	10	577412
4	30	1325973	1325388	0.2255745
5	30	1328042
6	30	1322149
7	50	2108373	2108010.333	0.1156219
8	50	2110246
9	50	2105412

Table 9: Recovery study of Valacyclovir by HPLC

Sr. No.	% Recovery level	Area of standard	Area of sample	% Recovery
1	50%	1325973	1321548	99.6662828
2	100%	1678272	1674258	99.76082542
3	150%	2108373	2099704	99.58882987
			Mean	99.67197936

Precision:

A) Intermediate precision (Intra-day and Inter-day)

The intraday and interday precision reads for Valacyclovir was done by assessing the comparing reactions multiple times around the same time and on three unique days for the three degree of linearity concentrations.

The %RSD was seen as 0.21 for Valacyclovir for intra-day precision and 0.19 inter day precision. Hence the proposed HPLC strategy was seen as exact.

Linearity:

The linearity of calibration graph and observance of the framework to Beer's law was approved by assurance correlation coefficient and R.S.D. There are values which were seen as well inside as far as possible.

Table 10: Linear regression data for calibration curve for Valacyclovir (n=3)

Parameters	Result
Linearity range	10-50μg/ml
R²	0.998
Slope	37692x
Intercept	20121
Y=mX+C	37692x + 20121

Limit of detection and Limit of quantitation (LOD & LOQ):

LOD and LOQ were resolved dependent on standard deviation of the reaction of calibration curve.

Table 11: LOD and LOQ of Valacyclovir.

Parameters	Result
LOD (μg/ml)	0.2617
LOQ (μg/ml)	0.7932

Robustness:

The parameter like variety in flow rate and wavelength. Concentration of the method was assessed at a 20 μg/ml. (n=3).

Table 12: Valacyclovir at different flowrate

Sr. No.	Concentration	Flowrate	Area
1	20	0.9 ml/min	970105
2	20	1.0 ml/min	968475
3	20	1.1 ml/min	966485
		Mean	968355
		SD	1812.98
		%RSD	0.187

Table 13: Valacyclovir at different wavelength

Sr. No.	Concentration	Wavelength	Area
1	20	254 nm	968475
2	20	252 nm	967429
3	20	256 nm	966520
		Mean	967475
		SD	978.3
		%RSD	0.101

Ruggedness:

Table 14: Result of ruggedness study for Valacyclovir

Sr. No.	Concentration	Area
1	10	580412
2	20	974102
3	30	1327820
4	40	1680102
5	50	2111044

Forced degradation studies:^19,20,21,22

Figure 2: Hydrolytic degradation of Valacyclovir

Figure 3: Acid degradation of Valacyclovir

Figure 4: Alkaline degradation of Valacyclovir

Figure 5: Photolytic degradation of Valacyclovir

Figure 6: Thermal degradation of Valacyclovir

Constrained decomposition studies done on Valacyclovir show that 12.41%, 26.57%, 31.09%, 0.61% and 0.47% when exposed to hydrolytic, acid, basic, photolytic and heat decomposition individually. The delegate chromatograms of the constrained decomposition studies uncover that all the decomposition items were completely settled, this shows the explicitness of the method. Along these lines the technique can be utilized for observing the stability of valacyclovir in bulk medication.

CONCLUSIONS:

The investigation was attempted to develop analytical strategy and validation of valacyclovir by utilizing spectrophotometric and chromatographic procedure. RP-HPLC separation of the drug Valacyclovir were accomplished on Cosmosil C18 (250mm×4.6ID, Particle size: 5µ) utilizing UV identification at 254nm. The advanced mobile stages for Valacyclovir comprised of Methanol:10mM KH₂PO₄ Buffer (50:50) and stream rate 1 ml/min., Valacyclovir were acceptably settled with retention time, 5.03 min. The strategy was approved for linearity, exactness, precision, explicitness, LOD, LOQ, robustness and ruggedness according to ICH and USP rules. The technique was seen as direct for Valacyclovir over fixation scopes of 10-50μg/ml (R2 = 0.998). The % relative standard deviation esteems for precision examines were seen as inside a farthest point. The investigations of robustness assessment of the strategy uncovered unimportant contrasts in peak zones and less changeability in retention time esteems shows that the developed technique was robust.

Valacyclovir with the Beer's-Lambert's law over the range of 9-45μg/ml. The absorption maxima (λmax) of Valacyclovir 254nm. The developed technique was approved by methods for precision, accuracy, explicitness, linearity and range, robustness and ruggedness according to ICH and USP rules.

The developed RP-HPLC and UV-Vis Spectrophotometric techniques were exact and explicit. The developed strategy offers a few focal points as far as effortlessness in solvents, simple sample preparation steps and efficient which makes the technique explicit and solid for its expected use in the assurance of Valacyclovir in pharmaceutical products, with no obstruction from the excipients. The chromatographic technique was approved according to ICH and USP rules. statistical examination shows that the proposed UV and HPLC strategy decrease the span of investigation and seem, by all accounts, to be similarly appropriate for routine assurance of Valacyclovir in pharmaceutical product in quality control research facilities, where economy and time are fundamental.

The after effect of stress testing embraced by the ICH rules uncover that the HPLC technique is explicit and stability demonstrating. The proposed technique can isolate these medications from their decomposed items in pharmaceutical products and subsequently can be applied to the examination of routine quality control samples got from stability contemplates.

CONFLICT OF INTEREST:

There is no Conflict of Interest of author for this research work.

ACKNOWLEDGEMENT:

The authors extend their gratitude to the Department of Quality Assurance of R.C. Patel Institute of Pharmaceutical education research for providing essential facilities and supportive encouragement for research work.

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Received on 10.03.2023 Modified on 08.06.2023

Asian J. Pharm. Ana. 2024; 14(2):53-59.

DOI: 10.52711/2231-5675.2024.00010