Rp-Hplc Method for Estimation of Anti-Viral Drug(Valaciclovir) in Pure, Solid Dosage Form and Spiked Human Plasma Using Mbth Reagent as Per Ich Guidelines

Rp-Hplc Method for Estimation of Anti-Viral Drug(Valaciclovir) in Pure, Solid Dosage Form and Spiked Human Plasma Using Mbth Reagent as Per Ich Guidelines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rp-Hplc Method for Estimation of Anti-Viral Drug(Valaciclovir) in Pure, Solid Dosage Form and Spiked Human Plasma Using Mbth Reagent as Per Ich Guidelines K. Bhavya Sri, A. Vaishnavi, J. Pravallika, Mogili Sumakanth This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6287855/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jun, 2025 Read the published version in Accreditation and Quality Assurance → Version 1 posted 19 You are reading this latest preprint version Abstract Background A novel, straightforward, accurate, repeatable, and precise chromogenic UV-visible spectroscopy and bioanalytical technique for measuring valaciclovir in pharmaceutical formulations and bulk. The MBTH reagent and Valaciclovir, in presence of ferric ammonium sulphate, undergo an oxidative condensation/coupling reaction that results in the formation of a green solution with the greatest absorption peak at 618nm. Linearity range of valaciclovir is 10–500µg/ml, and its correlation coefficient is 0.9997. Careful measurement of valaciclovir yielded a value of < 2. Validation of this method is conducted in accordance with ICH recommendationsQ2R1. Enhanced process was designed, verified, and extended to biological material using the protein precipitation extraction method. Results Valaciclovir's detection and quantification limits were 1.5114 µg/mL and 4.58 µg/mL, respectively. Human serum analysis by UV-Visible spectroscopy was created and verified. 1mL serum sample and 1mL of 0.1M was used for protein precipitation, which was vortexed and centrifuged. Standard solutionwhich had standard intensity range of 5–40 µg/mL, required 5.0 minutes to analyze. At 3.177 minutes, valaciclovir was eluted using the described technique. Valaciclovir showed linear behavior in 10–60 µg/ml range. Percentage Accuracy testing revealed that mean recovery was within region of 100%. Limits of quantitation and detection were determined to be 0.070 ng/ml and 0.212 ng/ml, respectively. Conclusion The developed approach was linear, stability indicating, sensitive, rapid, precise, and accurate. As a result, the developed method might be applied to regular quality control of tablets and bulk medications. UV-Spectrophotometer Valaciclovir 3-Methyl-2 Benothiazolinone Hydrazone Human Plasma RP-HPLC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Valaciclovir is a chemically 2-[(2-amino-6-oxo-1h-purin-9-yl)methoxy]ethyl (2s)-2-amino-3-methylbutanoate.One antiviral medication is valaciclovir. Herpes simplex or zoster outbreaks are treated with it. The medication valaciclovir was approved by the FDA in 2001. By way of intestinal and hepatic metabolism, valacyclovir is quickly and almost entirely transformed into acyclovir. Acyclovir is changed into acyclovir monophosphate by virus-specific thymidine kinase, which is then changed into acyclovir a triphosphate by other cellular enzymes. By fighting with deoxyguanosine triphosphate for antiviral DNA polymerase and getting integrated into viral DNA, acyclovir triphosphate prevents DNA synthesis and viral reproduction. Few analytical techniques have been published for determining the concentration of a treatment called by UV Spectral analysis, RP-HPLC, and HPLC, according to a thorough review of the literature. The principle of UV-Visible spectroscopy is the idea that substances absorb visible and ultraviolet light, which causes electronic changes inside molecules [ 1 , 2 ]. When a sample is exposed to visible or ultraviolet light, specific light wavelengths are taken up by the sample, which causes the molecules' electrons to shift from their ground state, which has a lower energy level, to their excited state, which has a higher energy level. The concentration of the absorbing material in the sample determines how much light is absorbed, and the wavelengths at which light is absorbed can reveal details about the composition and structure of the material.The separation principle is based on adsorption in both the normal and reverse phases. Depending on how efficiently a mixture of chemicals binds to the stationary phase28, it runs down an HPLC column. Substance that binds itself to the greater affinity accelerates the adsorbent's movement. It moves more quickly when the component has a lower affinity for the adsorbent. Because each element has a unique affinity for the stationary phase, they are separated. [ 3 – 5 ] Materials and methods Chemicals: Dosage form of valaciclovir tablets that were bought at a nearby pharmacy, the pure API, valaciclovir, which was supplied by the pharmaceutical company, Distilled water, sulphuric acid, ferric ammonium sulphate, plasma, and the MBTH reagent were acquired from a blood bank for UV procedure. The HPLC procedure used HPLC-grade methanol, HPLC-grade distilled water, HPLC-grade acetonitrile, and glacial acetic acid. Analytical conditions and instrumentation UV-Visible spectrophotometer devices: A double-beam Shimadzu Corporation UV-Visible Spectrophotometer 1800, which has two quartz cells that are matched in terms of path length, 1 cm, a 0.1 nm spectral bandwidth, and a wavelength precision of ± 0.1 nm, was used to measure the absorbance of the resultant solution. [6] HPLC devices: The software used was called Spectra Treats. LC Alternatives software was used, and the Shimadzu LC-20AD HPLC kind was used. The UV detection was achieved at 251 nm. Using HPLC grade methanol (60:40) as the mobile phase and HPLC grade water (pH was adjusted with glacial acetic acid up to 5.5), the column C18 column Phenomenix (250 mm×4.6 mm, 5 μm) was pumped at a flow rate of 1.0 ml/min. The mobile element was degassed prior to hoover usage. [7] Creation of an RP-HPLC Technique for Valaciclovir Analysis Preparation of standard solution: Weigh out 10 mg of a medication called medication precisely, then pour it into a clean, dry volumetric flask. Add 10 milliliters of HPLC-grade water to make up the difference. The ultimate concentration is 1000 ppm. By pipetting 1 milliliter of the reference solution into a separate 10 milliliter volumetric vessel and adding water to make it up, a working standard was produced. Its concentration is 100 parts per million. Serial dilutions were prepared using the working standard quantities of 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, and 60 ppm as shown in Table 1. Selection of wavelength UV-Visible spectrometer in scan mode was used to analyse a VLC mixture containing 10 ppm valaciclovir, taking readings between 200 and 400 nm in order to estimate the wavelength. At 251 nm, an ultraviolet absorbance peak was detected. Preparation of solvents HPLC-grade Water Make sure water is free of contaminants that could affect the analysis and is of HPLC-grade quality. Glacial acetic acid can be used to bring the water's pH down to 5.5. Using a calibrated pH meter, add glacial acetic acid drop by drop while monitoring on the pH. To guarantee homogeneity, give the mixture a good stir. Methanol (HPLC-grade) Use methanol of HPLC quality. Make sure it is pure enough to avoid interference or contamination during chromatography. To prevent moisture absorption, methanol should be stored in firmly sealed containers after being filtered using a 0.45 µm filter to eliminate any particles. Mobile Phase Preparation In the appropriate ratio, combine HPLC-grade methanol and water. For the particular procedure described, combine 40% methanol with 60% water (pH calibrated to 5.5). Filtration and Degassing To get rid of any particles, pass the prepared moving phase though a 0.45 µm filter. Degas the solution using vacuum filtration or sonication to get rid of any dissolved gases that can interfere with the chromatographic analysis. [8] Storage To avoid contamination, keep the phase of movement in clean, tightly sealed containers. Put the ingredients and preparation date on the container's label. Optimized method for analysis of valaciclovir To effectively separate and retain valaciclovir, the RP-HPLC method's chromatographic settings were adjusted. The study was conducted using a C18 column (Phenomenix, 250 mm × 4.6 mm, 5 μm). The mobile phase consisted of methanol of HPLC quality. in a 60:40 ratio and HPLC-grade water that had been pH-corrected to 5.5 using glacial acetic acid. With a flow rate of 1.0 mL/min, the procedure was carried out in a isocratic mode. The entire run time was set at 10 minutes, and a injection volume of 20 µL was employed. The chromatogram showed a ‘Gaussian peak’ indicating appropriate retention and peak symmetry, and the retention time for Valaciclovir was 3.1 minutes. [9-15] Validation parameters of HPLC Specificity: By examining the interference that the enhanced method causes, the specificity can be ascertained. When using this technique, the presence of these drugs shouldn't cause interference peaks to appear in the blank samples. Thus, it was asserted that this approach was unique. Specificity was attained by injecting blank solutions. Linearity: A freshly washed volumetric container should be filled with 10 mg of a medicine known as medication that has been carefully weighed out. Add 10 milliliters of water of HPLC quality on top. A final concentration of 1000 ppm is achieved. To create a working standardA different 10 ml weighing container was pipetted with 1 ml of the reference solution, then water was added to equal it. Its concentration is 100 ppm. The Inter-day Reliability %RSD values for Day 1 and Day 2 were 1.8191 and 0.9857, respectively, while the evening value was 1.4158. Using the working benchmark dosages of 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, and 60 ppm, serial dilutions were prepared. The data was shown in Figure 1and 2. Precision: A 10-ml volume flask was pipetted with 0.4 milliliters of 1000 µg/ml of a medication named sol, and HPLC-grade water was used for calibration, yielding a precision of 40 ppm for standard valaciclovir. Accuracy Making a solution that is 50% spiked: One milliliter of solvent was pipetted into a flask with a 10-milliliter capacity, and the solvent was diluted with diluent to obtain 10 parts per million of valaciclovir. Making a solution that is 100% spiked: By pipetting four milliliters of solution into a flask with a capacity of ten milliliters and diluting it with diluent, 40 parts per million of a medication were obtained. Making a solution that is 150% spiked: Six milliliters of solution were pipetted into a 10-milliliter flask, and the solution was diluted with diluent to yield 60 ppm. Data of valaciclovir was shown in Table 2. LOD and LOQ: The lowest possible analyte concentration is known as the detection limit. The quantitation limit is the lowest amount of material that can be detected. calculated using the following formula. [16-24] Robustness: Transmittance at a specific wavelength with a value of one nm is used to measure robustness. At least six measurements were made of the absorbance of a medication known as reference solution at 250, 251, and 252 nm. A percentage was used to calculate the RSD. The data was shown in Table 3. Chromogenic method by using UV-Visible spectrophotometer Standard stock solution: A 25 ml volumetric flask was filled with precisely weighed 25 milligrammes of pure VLC. dissolved in distilled water and adjusted the volume to the appropriate level. The final standard stock solution had a concentration of 1000µg/ml. Preparation of reagents used in chromogenic method Every chemical and reagent used was of analytical quality and was manufactured fresh in distilled water. 2% Preparation of ferric ammonium sulphate Weigh 400 mg of sulphuric acid and 600 mg of ferric ammonium sulphate into a 100 ml volumetric flask, add distilled water to fill up the remaining volume, and then freeze for 10 minutes at 5°. 1% Preparation of MBTH reagent 400 mg of sulphuric acid and 200 mg of MBTH reagent should be weighed into a 100 ml volumetric flask. The volume should then be adjusted with distilled water and allowed to sit at 5°c for 10 minutes. 1% Preparation of FeCl₃ reagent Weigh one gramme of the FeCl₃ reagent into a 100 ml volumetric flask, then add distilled water to fill the remaining volume. Preparation of blank reagent The blank reagent was prepared by adding 1 ml of MBTH Reagent and 1ml of ferric ammonium sulphate into 10ml volumetric flask and make up the volume with distilled water. Preparation of sample: After weighing and coarsely grinding ten 500 mg Valaciclovir pills, the powder was transferred to a 100 mL volumetric flask. After precisely weighing 10 mg of valaciclovir equivalent, it was diluted in a tiny amount of distilled water. The flask's volume was increased to 100 ml by adding distilled water after 15 minutes of sonication. It was discovered that the finished solution included 100 mg of valaciclovir. Method optimization by using UV-Visible spectrophotometer From the standard stock solution, 0.1 ml of the VLC solution was pipetted into a volumetric flask that held 10 ml. After 20 minutes, 1 millilitre of 1% MBTH reagent was added to the pipetted solution, which was then left for 15 minutes. Next, 1 millilitre of 2% ferric ammonium sulphate was added, and the mixture was maintained at 5°C. Distilled water was used to bring the leftover solution up to par. The solution in 400–800 nm range was scanned using UV-visible spectroscopy against a reagent blank.The fourth trial after the first three is the best one. The fourth experiment's wavelength is 618 nm, and its absorbance is 0.2002 nm. The ideal temperature is 5°C for 15 minutes, and the ideal tint is a dark greenish shade. Finally, after four attempts, the strategy is optimised. Colour stability: The drug's colour stability lasts for two hours. After two hours, colour has diminished as a result of prolonged temperature and temporal variations. In house formulation 10mg equivalent of nano valaciclovir solid lipid nano particle by microemulsion technique in house formulation prepared in the college were used for the assay calculation. Validation parameters according to ICH Q2R1 The process of verifying that the specifications for a particular intended use are met through analysis and impartial proof is known as validation. The ICH Q2R1 recommendations were followed in determining the validation parameters. [25-34] Linearity: The developed method follows Beer's law in a concentration ranging from 10-500µg/mL for valaciclovir, with an equation for regression of Y=0.002x + 0.0042 with a correlation coefficient value of 0.9997. A summary of the calibration curves' analytical data is offered together with the standard deviations for the slope, intercept, and system appropriateness characteristics. The calibration graphs' linearity is demonstrated by this dataas shown in Figure 3 and Table 4. Accuracy: Degree to which a single measurement resembles the actual or accepted value is known as accuracy. By computing the recoveries of both medications using the conventional addition approach, the method's correctness was ascertained. Three distinct amounts of standards (i.e., 50%, 100%, and 150%) were triplicately added to pre-quantified samples. It was determined what the mean recovery percentage as shown in Table 5. Precision: Three different concentrations and three duplicates of each concentration were used to evaluate reproducibility (intra-day) and precision at intermediate levels (inter-day). The suggested method's %RSD was found to be very small (<2%), suggesting reasonable reproducibility and intermediate precision. Detection limit: The lowest analyte concentration that produces a detectable response is known as the Limit of Detection (LOD). The following was used to determine LOD. LOD = 3.3 σ /S 1.5114µg/mL Quantification limit: The lowest concentration of the analyte at which a reaction can be precisely measured is known as the Limit of Quantification (LOQ). The following formula was used to determine LOQ. LOQ = 10*SD = 4.58µg/mL Robustness: Robustness is a metric that expresses an analytical procedure's resistance to small, deliberate changes in method parameters as well as its dependability under normal operating conditions. Ruggedness: The degree of consistency of test results achieved by analysing the same samples under various settings, such as different laboratories, different analysts, and different instruments, is known as the ruggedness of an analytical process. Deliberate change in analyst and instrument studies are performed to study their difference in absorbance. The %RSD were found to be very small (<2%). Method extended to Chromogenic bioanalytical method development Using the protein precipitation extraction method, the procedure was devised. The process consists of: 0.1M HCL and 1ml of the standard stock solution (valaciclovir) were added to 1ml of human plasma. Give the vortex solution five minutes or so. For ten minutes, centrifuge the mixture at 4000 rpm. After that, transfer the liquid supernatant to a test tube and add 1 milliliter each of 2% MBTH reagent and 1% ferric ammonium sulphate. Examine the solution at 610 nm using a visible spectrophotometer. Recovery percentage that was obtained was 98.56%.[35-38]. Validation parameters for bioanalytical methods according to M10 guidelines Linearity: Calibration curve illustrates the relationship between an analyte's actual concentration and the testing platform's reaction to it. Calibration curve is made up of the verification criteria, which are created by combining a certain quantity of analyzer into a matrix. Calibration standards also need to be made using same biological material the matrix that was used to make the study samples. Both the LLOQ, the cheapest evaluating standard, and the ULOQ, the most expensive assessing standard, define the calibration range. For every analytical run and every analyte examined during methods validation, a calibration curve ought to exist. Data was shown in Table 5 and Figure 4. Precision and accuracy: Accuracy and precision should be assessed using comparable runs and data. Since the reliability should be between ±15% at every value level and the theoretical level should be within ±20%, do not include the LLOQ. The resolution (%CV) of a concentration must be more than 15% at any level. except for LLOQ, where it should not exceed more than 20%. Three-thirds of all QCs and perhaps 50% of each degree of concentration in order must fall within ±15% of the nominal values for both exact and non-accuracy verification runs. Stability: To ensure that decisions about sample preparation, processing, evaluation, and storage conditions do not impact the analyte concentration, stability studies must be carried out. QCs are used to assess the material's durability in the matrix at both low and large volumes. A portion of both the low and high QCs are examined at zero seconds and following the execution of the storage conditions that need to be assessed. It is necessary to construct a single bulk QC for every concentration level. The bulk sample must be separated into at least three aliquots for each concentration under investigation. These aliquots will then be filtered, stored, and examined. Data was shown in Table 7. Calibration curve and range: In at least one study, the calibration curve was made using freshly spiked calibration standards. Calibration curve was evaluated using six concentration levels of a blank sample, calibration standards (such as the LLOQ and ULOQ), and a zero- concentration specimen (the blank sample spiked with IS). Matrix effect: The matrix effect assesses how elements of biological matrix impact the detection of analytes, which may have an impact on quantitative analysis's precision and accuracy. Reinjection reproducibility: After storage, a run comprising a calibrated standard and at least five re-injections of the middle, inadequate, and high QCs is carried out to assess reinjection repeatability. Injected QCs' accuracy and precision demonstrate the viability of the treated samples. The results ought to be incorporated into the confirmation report or bioanalytical assessment of the study from which the data were derived. Method development of valaciclovir by UV spectrophotometer Valaciclovir Standard solution preparation: Valaciclovir standard solutions were made by diluting 10 mg of the medication with 1000 𝝁g/ml of distilled water in a 10-milliliter flask that was dry and clean. A practical standard is created based on this. To achieve a concentration of 100 parts per million, pipette one mL of the standard solution into a 10-milliliter flask and add the suitable solvent. A 10-ml volume flask should contain 1 ml of this, and the diluent should be added to reach the proper level. The concentration that results is 10 𝝁g /ml. Wavelength was determined by analyzing a reference solution that contained 10 𝝁g/ml of valaciclovir between 200 and 400 nm and accuracy was calculated as shown in Figure 5 and Table 8. Method extending to bioanalytical by using UV spectrophotometer of valaciclovir Preparation of plasma: Procured from blood bank. Preparation of blank plasma: After measuring and transferring 1 ml of plasma to a 10 ml volumetric flask, reagents were added with distilled water. Extraction of plasma with protein precipitation One method for concentrating and purifying proteins from different impurities is protein precipitation. By adding a reagent1, the procedure modifies the solvent's resolution potential, which reduces the solute's solubility1. The water difficulties between protein molecules was broken down by the binding of salt ions with water molecules, enabling contact between the protein's basic regions. As a result, the protein molecules group together and separate from the solvent. The process was developed using the protein precipitation separation method. The procedure includes: One mL of human plasma was mixed with 0.1M Hydrochloric acid and one mL of the usual stock solution (valaciclovir). Let the vortex solution sit for about five minutes. Centrifuged the mixture at 4000 rpm for 10 minutes. The liquid supernatant should then be transferred to cuvettes. Use a visible spectrophotometer to analyse the solution at 251 nm. Obtained recovery percentage was 98.56%. Results and discussion Chromatographic method Linearity Table 1 HPLC Linearity data. Concentration Area Retention Time Theoretical Plate Tailing Factor 10PPM 1072631 3.121 2125.707 0.684 20PPM 2150271 3.135 2842.378 0.713 30PPM 3151734 3.174 2367.700 0.579 40PPM 4222836 3.177 2202.733 0.385 50PPM 5212512 3.317 2916.264 0.973 60PPM 5997982 3.263 2208.314 0.079 Precision Interday and intraday precision %RSD of Repeatability of valaciclovirwas found to be 0.4945877. %RSD of Intra-day Precision ; Morning was found to be 0.49978 and Inter-day Precision %RSD values for Day 1 and Day 2 were 0.856019 and 0.4804377, respectively, while the evening value was 0.66605. According to ICH criteria, all of the results were in limits. Accuracy: Table 2: Percentage of valaciclovir recovered % Percentage Level Sample area Standard area Average Area of three replicas Recovery area % Recovery Mean % Recovery 50% 4222836 1072631 8455602 4232766 100.23% 100.62% 100% 4222836 4222821 8525672 4302836 101.89% 150% 4222836 5997982 10210818 4212836 99.76% Detection and Quantification limits: Using formulas based on the slope of calibration curve and standard deviation of the y-intercept of regression lines, LOD and LOQ were determined and were found to be 0.07022260189 µg/mL and 0.212795763327 µg/mL. Robustness Table 3. Robustness data with respect to HPLC Robustness Flow rate at 0.7ml/min Flow rate at 1.0ml/min Flow rate at 1.2ml/min Concentration Area Area Area 40PPM 4226543 4222836 4222789 40PPM 4228903 4221832 4224567 40PPM 4236547 4222789 4213598 40PPM 4228907 4223132 4224589 40PPM 4228975 4233456 4244672 40PPM 4220987 4221898 4245678 40PPM 4228477 4224324 4228437 40PPM 5011.581307 4505.113 12946.63 40PPM 0.118519772 0.106647 0.30618 Using two wavelengths -1(%RSD 0.118519) and +1(%RSD 0.106647)—the robustness analysis was performed. The findings met ICH standards for outcomes within acceptable limits. Assay: Chromogenic method validation by using UV-Visible spectroscopy parameters Parameters for validation Linearity Table 4 Data on VLC linearity Concentration (µg/ml) Absorbance 10µg/ml 0.03777 50µg/ml 0.1031 100µg/ml 0.2126 150µg/ml 0.3035 200µg/ml 0.4076 250µg/ml 0.5237 300µg/ml 0.6321 350µg/ml 0.7213 400µg/ml 0.8523 450µg/ml 0.9017 500µg/ml 1.0126 Results: As per ICH guidelines r 2 found to be 0.9999 and y= mx +c is 0.002x+0.0042 Precision %RSD of Repeatability of valaciclovirwas found to be 0.0141. %RSD of Intra-day Precision ; Morning was found to be 0.055 and Evening was found to be 0.056 andDay 1's and Day 2's The values of the Inter-day Precision %RSD were found to be 0.0196 and 0.029, respectively. All of the findings were within the limitations, which are less than 2, in accordance with ICH guidelines. Accuracy Limits: ICH Specifies a recovery percentage ranging from 98-102% Results: The accuracy recovery percentage has been found between 98.24-101.83%. Table 5 Valaciclovir formulation recovery study (valaciclovir 500mg) % Percentage Level Sample Absorbance Standard 50ppm, 100ppm 150ppm Absorbances Total Absorbance % Recovery Mean % Recovery 50% (250ppm) + (50ppm) 0.5327 0.1031 0.6170 98.12% 98.16% 0.6175 98.22% 0.6172 98.16% 100% (250ppm) + (100ppm) 0.5327 0.2126 0.7316 99.11% 99.42% 0.7301 99.87% 0.7326 99.30% 150% (250ppm) + (150ppm) 0.5327 0.3035 0.8361 101.69% 101.05% 0.8320 100.91% 0.8301 100.55% *It indicates three replicates Robustness and Ruggedness Using two wavelengths -1(%RSD 0.0292) and +1(%RSD 0.181)—the robustness analysis was performed. Two separate analysts performed the ruggedness analysis; their findings were %RSD 0.042 for Analyst 1 and %RSD 0.028 for Analyst 2. The findings met ICH standards for outcomes within acceptable limits. Detection limit and Quantitation limit Using formulas based on the slope of the calibration curve and the standard deviation of the y-intercept of regression lines, LOD and LOQ were determined and were found to be 1.5114µg/mL and4.58µg/mL. Assay Calculation: Chromogenic Bio Analytical Method Validation Parameters Validation parameters Linearity Table 6 Chromogenic bioanalytical linearity Concentration (µg/ml) Absorbance 50µg/ml 0.1001 100µg/ml 0.2016 150µg/ml 0.3011 200µg/ml 0.4003 250µg/ml 0.5103 300µg/ml 0.6132 350µg/ml 0.7053 Results: the correlation coefficient was found to be 0.9998 Accuracy and precision All concentration levels should have accuracy within ±15% of the nominal focused attention, with the exception of the LLOQ, when it should be within ±20%. Apart at the LLOQ, where the value shouldn't be more than 20%, the accuracy (%CV) of the quantities measured at each level shouldn't be more than 15%.Accuracy and precision were found to be within limits. Stability Bench top stability studies Table 7 Stability of chromogenic bioanalytical method of LQC and HQC Percentage Level(LQC) 0hour 2hour 4hour 6hour 8hour Mean 0.143833 0.1469 0.114767 0.134766667 0.1168 SD 0.028471 0.02816 0.015581 0.04434144 0.015655989 %CV 19.79414 19.16968 13.57612 13.90237969 12.4041004 Percentage Level (HQC) 0hour 2hour 4hour 6hour 8hour Mean 0.7136 0.7541 0.6373333 0.622366667 0.487366667 SD 0.0090006 0.0443819 0.0404829 0.064030019 0.077951352 %CV 11.261288 13.8854085 12.351915 10.28815045 15.99439553 Percentage Recovery Calculation % Recovery = Sample absorbance/Standard absorbance*100 = 0.5103/0.5205*100 =99.8% UV Spectrophotometer Validation Parameters of Valaciclovir Linearity Results: As per ICH guidelines r 2 found to be 0.9999 and y= mx+c is 0.02159x+0.9993 Precision %RSD of Repeatability of valaciclovirwas found to be 0.5627. %RSD of Intra-day Precision ; Morning was found to be 1.7755 and The Inter-day Reliability %RSD values for Day 1 and Day 2 were 1.8191 and 0.9857, respectively, while the evening value was 1.4158. All of the findings were within the limitations, which are less than 2, in accordance with ICH guidelines. Accuracy Limits: ICH Specifies a recovery percentage ranging from 98-102% Results: The accuracy recovery percentage has been found between 98.24-101.83%. Table 8 Recovery Study of valaciclovir formulation (valaciclovir 500mg) % Percentage Level Sample Absorbance Standard 50ppm, 100ppm 150ppm Absorbances Total Absorbance % Recovery Mean % Recovery 50% (35ppm) + (50ppm) 1.5275 1.0323 2.0193 98.12% 98.25% 2.0540 98.32% 2.0870 98.26% 100% (35 ppm) + (100ppm) 1.5275 1.2776 2.5051 99.15% 99.62% 2.5076 99.90% 2.5043 99.30% 150% (35ppm) + (150ppm) 1.5275 1.6274 3.0013 101.69% 101.10% 3.0065 100.91% 3.0023 100.55% *It indicates three replicates Robustness and ruggedness: Using two wavelengths -1(%RSD 1.0852) and +1(%RSD 1.0269)—the robustness analysis was performed. Two separate analysts performed the ruggedness analysis; their findings were %RSD 1.3430 for Analyst 1 and %RSD 1.5522 for Analyst 2. The findings met ICH standards for outcomes within acceptable limits. Detection and Quantitation limits: DL: 0.1391µg/mL DQ: 0.4271µg/mL Discussion RP-HPLC technique developed for valaciclovir analysis demonstrated effective chromatographic separation with a well-defined Gaussian peak at a retention time of 3.1 minutes. The method utilized an isocratic mobile phase of methanol and water (60:40), with a flow rate of 1.0 mL/min, ensuring precision and reproducibility. UV-Visible spectrophotometry confirmed an optimal detection wavelength at 251 nm, facilitating further analytical studies. Validation parametersadhered to ICH Q2R1 guidelines, confirming method's reliability. The method exhibited high linearity (R² = 0.9997) within a concentration range of 10–500 µg/mL, demonstrating its suitability for quantitative analysis. Accuracy assessments using spiked solutions at different concentrations yielded satisfactory recovery rates, supporting method validity. Bioanalytical method development using protein precipitation techniques successfully extracted valaciclovir from plasma with a high recovery of 98.56%. The method showed stability under various conditions, ensuring its robustness and reproducibility in different laboratory settings. Reinjection reproducibility and matrix effect evaluations confirmed that biological matrix components did not interfere significantly with the analysis. Overall, the developed RP-HPLC and UV-based techniques proved to be reliable, sensitive, and applicable for routine quality control and bioanalytical studies of valaciclovir. Conclusion It is concluded that suggested approach for measuring valaciclovir in pharmaceutical dose forms was found to be straightforward, sensitive and accurate. Assay results showed a strong correlation with corresponding label claim. Because of its low solvent content, lack of extraction steps, specificity, and sensitivity, this spectrophotometric approach has been proven to be superior. The regular application of the recommended technique in control of quality for valaciclovir analyses in medication dosage forms is supported by these advantages. High precision and dependability are demonstrated by the devised UV spectroscopy method for measuring valaciclovir in human serum. Efficacy of the approach is demonstrated by excellent correlation and quantification limits and conformity to Beer's law within designated concentration range. The method's correctness is confirmed by its 100% recovery rate, and its application is validated by the findings' conformity with labels of commercial products. In compliance with ICH bioanalytical criteria, the validation of the standard pharmaceutical solution analysis showed strong performance. For the intensity range of 5–40 µg/mL, the 5.0-minute analysis time was effective. High dependability is indicated by the method's accuracy, which ranges from 101.26–101.96%, and yield, which surpasses 89.48%. The approach exhibits exceptional precision, with relative standard deviation (RSD) values for both incurred plasma and quality control specimens below 10%. Standard had intensity range of 5–40 µg/mL, required 5.0 minutes to analyze. At 3.177 minutes, valaciclovir was eluted using described technique. Valaciclovir showed linear behaviour in the 10–60 µg/ml range. Relative variance of 0.85% serves as an example of the accuracy. Percentage Accuracy testing revealed. Abbreviations MBTH: 3-Methyl-2 Benothiazolinone Hydrazone; LOD: Limit of detection; LOQ: Limit ofquantitation; % RSD: Relative standard deviation; ICH: International Conference on Harmonization; Rs: Resolution; SD: Standarddeviation; μg: Microgram; mL: Milliliters; mg: Milligrams; %: Percentage; RP: Reverse phase; HPLC: High-performance liquid chromatography. LLOQ: Lower limit of Quantification; ULOQ: Upper limit of Quantification. Declarations Acknowledgment I would like to thank our beloved principle, prof. M. Sumakanth madam and faculty of the department of pharmaceutical analysis for giving me the opportunity. Authors’ contributions The authors have read and approved the manuscript. K. Bhavyasri designedthe study. Remaining authors performed the experiment and analyzed and reviewed thedata. K. Bhavyasri and M. Sumakanth supervised the experiment, reviewed the data, and supported forwriting the manuscript. Funding Not Applicable Availability of data and materials All data and materials are available upon request. Ethics approval and consent to participate Plasma was collected from blood banks. Consent for publication Not Applicable Competing interests The authors declare that they have no competing interests References Choudhary A (2017) The principle of ultraviolet (UV) spectrophotometer. Medium. Sri Varsha M, Raghavendra Babu N, et al. (2015) Development of new spectrophotometric method for estimation of tenofovir disoproxil fumarate using MBTH reagent. Int Curr Pharm J 4(4):378-381. Khagga Bhavyasri, Murali Balaram V, Nageswarao R, Rambabu D, Sasikiran Goud E, Ajitha M (2013) Development and validation of forced degradation studies of Atazanavir using RP-HPLC and characterization of degradants by LC-MS/MS. Int J Pharm Sci Rev Res 33(2):11-15. Khagga Bhavyasri, Murali Balaram V, Nageswarao R, Rambabu D, Ajitha M (2013) RP-UPLC method development and validation for the simultaneous estimation of Proguanil and Atovaquone in pharmaceutical dosage form. J Chem Pharm Res 5(12):1222-1229. Khagga Bhavyasri, Murali Balaram V, Nageswarao R, Rambabu D, Ajitha M (2013) Rapid simultaneous determination of Naproxen and Esomeprazole magnesium in combined tablets by validated Ultra Performance Liquid Chromatographic method. J Chem Pharm Res 5(12):1230-1236. Rahul Reddy MB, Gurupadayya BM, et al. (2011) Spectrophotometric determination of Lamuvidine using acidic dye and coupling reagent. Indian J Chem Technol 18:431-438. Jadhav AS, Pathare DB, Shingare MS (2007) Development and validation of high-performance liquid chromatographic method for valacyclovir, an antiviral drug. J Pharm Biomed Anal 43(4):1568-1572. Bhavyasri K, Manisha KV, Swethasri R, Sumakanth M (2019) Method development and validation for the estimation of esmolol hydrochloride in bulk and ophthalmic formulation by UV-Visible spectrophotometer. Paripex - Indian J Res 8(6):143-145. Mounika G, Bhavyasri K, Swethasri R, Sumakanth M (2019) Development and validation of RP-HPLC method for estimation of Canagliflozin. Samriddhi S-JPSET 11(2):85-92. Bhavyasri K, Bhairavi B, Swethasri R, Sumakanth M (2020) Three-dimensional nuclear magnetic resonance spectroscopy – a review. Int J Anal Exp Modal Anal 12(3):1456-1463. Padmalatha H, Anupama V, et al. (2014) Spectrophotometric estimation of chlorthalidone in pharmaceutical formulation using MBTH & FC reagent. World J Pharm Pharm Sci 3(2):1443-1440. Kutty SV, Eapen SC, et al. (2012) Validated UV-Visible spectrophotometric method for the estimation of fenofibrate in pure and pharmaceutical formulation using MBTH reagent. Int J Pharm Sci Drug Res 4(1):74-76. Borman P, Elder D (2017) Q2 (R1) validation of analytical procedures: text and methodology. ICH quality guidelines: an implementation guide. 127-166. Shravya A, Chandan RS, et al. (2011) Spectrophotometric determination of Ezetimibe using MBTH reagent in pharmaceutical dosage form. Int J Res Ayurveda Pharm 2(2):521-525. Guideline IH (2022) Validation of analytical procedures Q2 (R1). ICH: Geneva, Switzerland. Guideline IH (2005) Validation of analytical procedures: text and methodology. Q2 (R1) 1(20):05. Srinivasa Rao K, Sunil M (2009) Stability-indicating liquid chromatographic method for valacyclovir. Int J ChemTech Res 1(3):702-708. UV spectrophotometric method for the estimation of valacyclovir HCl in tablet dosage form. E-J Chem 6(3):814-818. Anand BS, Katragadda S, Mitra AK (2004) ICH harmonized tripartite guidelines, validation of analytical procedures: text and methodology, Q2A. J Pharmacol Exp Ther 311:659. Ishpreet K, Sharad W, Harsharan PS (2015) Development and validation of UV spectroscopic method for determination of pioglitazone in bulk and pharmaceutical dosage form. Pharm Methods 6(2):1-1. Shah RS, Shah RR, Pawar RB, Gayakar PP (2015) UV-Visible spectrophotometry - a review. Int J Inst Pharm Life Sci 5(5):490-505. Abdel-Rehim M, Dahlgren M, Blomberg L (2006) Quantification of ropivacaine and its major metabolites in human urine samples utilizing microextraction in a packed syringe automated with LC-MS/MS. J Sep Sci 29(11):1658-1661. Mercier-Letondal P, Marton C, Godet Y, Galaine J (2021) Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1). J Transl Med 19:1-5. Almeida C, Nogueira JM (2006) Determination of steroid sex hormones in water and urine matrices by stir bar sorptive extraction and liquid chromatography with diode array detection. J Pharm Biomed Anal 41(4):1303-1311. Anderson NL, Polanski M, Pieper R, et al. (2004) The human plasma proteome: A nonredundant list developed by combination of four separate sources. Mol Cell Proteomics 3(4):311-326. Sawicki E, Hauser TR, Stanley TW, Elbert W (1961) The 3-methyl-2-benzothiazolone hydrazone test. Anal Chem 33:93-96. Anthon GE, Barrett DM (2002) Determination of reducing sugars with 3-methyl-2-benzothiazolinone hydrazone. Anal Biochem 305:287-289. Shah RS, Shah RR, Pawar RB, Gayakar PP (2015) UV-Visible spectrophotometry - a review. Int J Inst Pharm Life Sci 5(5):490-505. ICH (1994) Q2A, Text on Validation of Analytical Procedures. Int Conf Harmonization, Geneva, October:1‐5. Sylvie LR, Francoise SL, Agnes PL, et al. (2003) Ribavirin quantification in combination treatment of chronic hepatitis C. AAC 47(1):124-129. Alvarez D, Dieterich DT, Brau N, et al. (2013) Zidovudine use but not weight-based ribavirin dosing impacts anemia during HCV treatment in HIV. 12(5):498-513. Bani SF, Carrat F, Pol S, et al. (2006) Risk factors for symptomatic mitochondrial toxicity in HIV-infected persons. J Viral Hepat 13(10):683-. Granero GE, Amidon GL (2006) Stability of valacyclovir: implications for its oral bioavailability. Int J Pharm 317(1):14-18. Venkata Reddy P, Sudha Rani B (2006) Estimation of some antiviral drugs using TP as analytical reagent. E-J Chem 3(3):154-158. Bhavyasri K, Fatima A, Rambabu D (2019) Spectrophotometric estimation of metformin in human plasma. J Biomed Pharm Res 8(3):61-64. Bhavyasri K, Benazir SB, Rambabu D (2019) Method development and validation of UV spectrophotometric method for estimation of nevirapine in human plasma. Panama Res J 9(6):115-124. Bhavyasri K, Bhairavi B, Rambabu D (2019) Method development and validation for estimation of paracetamol in human plasma by UV spectrophotometry. J Emerg Technol Innov Res 6(6):572-577. Sugumaran M, Jothieswari D (2010) Development and validation of spectroscopic method for estimation of valacyclovir in tablet dosage form. Oriental J Chem 26(1):163-165. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 16 Jun, 2025 Read the published version in Accreditation and Quality Assurance → Version 1 posted Editorial decision: Revision requested 06 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviews received at journal 04 Apr, 2025 Reviews received at journal 03 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviewers invited by journal 25 Mar, 2025 Editor assigned by journal 25 Mar, 2025 Submission checks completed at journal 24 Mar, 2025 First submitted to journal 23 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6287855","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":438156562,"identity":"bbb9a262-53c1-489c-9e18-7bc7de1cccd8","order_by":0,"name":"K. Bhavya Sri","email":"data:image/png;base64,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","orcid":"","institution":"RBVRR women’s college of pharmacy","correspondingAuthor":true,"prefix":"","firstName":"K.","middleName":"Bhavya","lastName":"Sri","suffix":""},{"id":438156563,"identity":"be4c20c0-4f28-4705-91df-41f6396265cd","order_by":1,"name":"A. Vaishnavi","email":"","orcid":"","institution":"RBVRR women’s college of pharmacy","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"","lastName":"Vaishnavi","suffix":""},{"id":438156564,"identity":"88fa482c-9880-4a22-827b-a89ee5916bea","order_by":2,"name":"J. Pravallika","email":"","orcid":"","institution":"RBVRR women’s college of pharmacy","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"","lastName":"Pravallika","suffix":""},{"id":438156565,"identity":"08569862-b000-4a24-9448-cd208b252b67","order_by":3,"name":"Mogili Sumakanth","email":"","orcid":"","institution":"RBVRR women’s college of pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Mogili","middleName":"","lastName":"Sumakanth","suffix":""}],"badges":[],"createdAt":"2025-03-23 11:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6287855/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6287855/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00769-025-01638-w","type":"published","date":"2025-06-16T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79899941,"identity":"f8ab40a1-1bf2-4ed7-b0a4-8310e0068391","added_by":"auto","created_at":"2025-04-04 09:50:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22133,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curve of valaciclovir\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6287855/v1/f238cbf0032ff156c96991a7.jpg"},{"id":79899902,"identity":"07cefa59-d5a9-4c42-971a-8466b36690e0","added_by":"auto","created_at":"2025-04-04 09:50:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23877,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of standard VLC of 10 ppm\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6287855/v1/1ae3756ca36ad3c5cc607259.jpg"},{"id":79899942,"identity":"752c701c-99e9-4a8d-91d8-bc3d8ac9f99c","added_by":"auto","created_at":"2025-04-04 09:50:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":23369,"visible":true,"origin":"","legend":"\u003cp\u003eVLC Calibration Curve using MBTH\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6287855/v1/67e37f11bd4eb4cac22847ff.jpg"},{"id":79899906,"identity":"c99a935e-6d27-4a81-b1cf-0b10e89130df","added_by":"auto","created_at":"2025-04-04 09:50:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25547,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration Curve of VLC\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6287855/v1/623a511fceb62ebf12e61fc0.jpg"},{"id":79899908,"identity":"bc0cff91-fe95-4be1-94ad-b51a5e2c0cd1","added_by":"auto","created_at":"2025-04-04 09:50:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22604,"visible":true,"origin":"","legend":"\u003cp\u003eValaciclovir Calibration Curve with Diluent\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6287855/v1/6d041b07519dcd0cc75b57c0.jpg"},{"id":85231439,"identity":"d0a3325d-9602-4387-8cb4-f35b2610ba52","added_by":"auto","created_at":"2025-06-23 16:08:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1862120,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6287855/v1/27540261-b94a-4cbd-967c-489ab0dccb9e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eRp-Hplc Method for Estimation of Anti-Viral Drug(Valaciclovir) in Pure, Solid Dosage Form and Spiked Human Plasma Using Mbth Reagent as Per Ich Guidelines\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eValaciclovir is a chemically 2-[(2-amino-6-oxo-1h-purin-9-yl)methoxy]ethyl (2s)-2-amino-3-methylbutanoate.One antiviral medication is valaciclovir. Herpes simplex or zoster outbreaks are treated with it. The medication valaciclovir was approved by the FDA in 2001.\u003c/p\u003e \u003cp\u003eBy way of intestinal and hepatic metabolism, valacyclovir is quickly and almost entirely transformed into acyclovir. Acyclovir is changed into acyclovir monophosphate by virus-specific thymidine kinase, which is then changed into acyclovir a triphosphate by other cellular enzymes. By fighting with deoxyguanosine triphosphate for antiviral DNA polymerase and getting integrated into viral DNA, acyclovir triphosphate prevents DNA synthesis and viral reproduction. Few analytical techniques have been published for determining the concentration of a treatment called by UV Spectral analysis, RP-HPLC, and HPLC, according to a thorough review of the literature.\u003c/p\u003e \u003cp\u003eThe principle of UV-Visible spectroscopy is the idea that substances absorb visible and ultraviolet light, which causes electronic changes inside molecules [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. When a sample is exposed to visible or ultraviolet light, specific light wavelengths are taken up by the sample, which causes the molecules' electrons to shift from their ground state, which has a lower energy level, to their excited state, which has a higher energy level. The concentration of the absorbing material in the sample determines how much light is absorbed, and the wavelengths at which light is absorbed can reveal details about the composition and structure of the material.The separation principle is based on adsorption in both the normal and reverse phases. Depending on how efficiently a mixture of chemicals binds to the stationary phase28, it runs down an HPLC column. Substance that binds itself to the greater affinity accelerates the adsorbent's movement. It moves more quickly when the component has a lower affinity for the adsorbent. Because each element has a unique affinity for the stationary phase, they are separated. [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eChemicals:\u003c/strong\u003eDosage form of valaciclovir tablets that were bought at a nearby pharmacy, the pure API, valaciclovir, which was supplied by the pharmaceutical company, Distilled water, sulphuric acid, ferric ammonium sulphate, plasma, and the MBTH reagent were acquired from a blood bank for \u0026nbsp;UV procedure. The HPLC procedure used HPLC-grade methanol, HPLC-grade distilled water, HPLC-grade acetonitrile, and glacial acetic acid.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalytical conditions and instrumentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUV-Visible spectrophotometer devices:\u003c/strong\u003e A double-beam Shimadzu Corporation UV-Visible Spectrophotometer 1800, which has two quartz cells that are matched in terms of path length, 1 cm, a 0.1 nm spectral bandwidth, and a wavelength precision of \u0026plusmn; 0.1 nm, was used to measure the absorbance of the resultant solution. [6]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC devices:\u003c/strong\u003e The software used was called Spectra Treats. LC Alternatives software was used, and the Shimadzu LC-20AD HPLC kind was used. The UV detection was achieved at 251 nm. Using HPLC grade methanol (60:40) as the mobile phase and HPLC grade water (pH was adjusted with glacial acetic acid up to 5.5), the column C18 column Phenomenix (250 mm\u0026times;4.6 mm, 5 \u0026mu;m) was pumped at a flow rate of 1.0 ml/min. The mobile element was degassed prior to hoover usage. [7]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCreation of an RP-HPLC Technique for Valaciclovir Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of standard solution:\u003c/strong\u003eWeigh out 10 mg of a medication called medication precisely, then pour it into a clean, dry volumetric flask. Add 10 milliliters of HPLC-grade water to make up the difference. The ultimate concentration is 1000 ppm. By pipetting 1 milliliter of the reference solution into a separate 10 milliliter volumetric vessel and adding water to make it up, a working standard was produced. Its concentration is 100 parts per million. Serial dilutions were prepared using the working standard quantities of 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, and 60 ppm as shown in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of wavelength\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUV-Visible spectrometer in scan mode was used to analyse a VLC mixture containing 10 ppm valaciclovir, taking readings between 200 and 400 nm in order to estimate the wavelength. At 251 nm, an ultraviolet absorbance peak was detected.\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003cstrong\u003ePreparation of solvents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC-grade Water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMake sure water is free of contaminants that could affect the analysis and is of HPLC-grade quality. Glacial acetic acid can be used to bring the water\u0026apos;s pH down to 5.5. Using a calibrated pH meter, add glacial acetic acid drop by drop while monitoring on the pH. To guarantee homogeneity, give the mixture a good stir.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cstrong\u003eMethanol (HPLC-grade)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUse methanol of HPLC quality. Make sure it is pure enough to avoid interference or contamination during chromatography. To prevent moisture absorption, methanol should be stored in firmly sealed containers after being filtered using a 0.45 \u0026micro;m filter to eliminate any particles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Mobile Phase Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the appropriate ratio, combine HPLC-grade methanol and water. For the particular procedure described, combine 40% methanol with 60% water (pH calibrated to 5.5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Filtration and Degassing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo get rid of any particles, pass the prepared moving phase though a 0.45 \u0026micro;m filter.\u0026nbsp;\u003cbr\u003e\u0026nbsp;Degas the solution using vacuum filtration or sonication to get rid of any dissolved gases that can interfere with the chromatographic analysis. [8]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStorage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo avoid contamination, keep the phase of movement in clean, tightly sealed containers. Put the ingredients and preparation date on the container\u0026apos;s label.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimized method for analysis of valaciclovir\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo effectively separate and retain valaciclovir, the RP-HPLC method\u0026apos;s chromatographic settings were adjusted. The study was conducted using a C18 column (Phenomenix, 250 mm \u0026times; 4.6 mm, 5 \u0026mu;m). The mobile phase consisted of methanol of HPLC quality. in a 60:40 ratio and HPLC-grade water that had been pH-corrected to 5.5 using glacial acetic acid. With a flow rate of 1.0 mL/min, the procedure was carried out in a isocratic mode. The entire run time was set at 10 minutes, and a injection volume of 20 \u0026micro;L was employed. The chromatogram showed a \u0026lsquo;Gaussian peak\u0026rsquo; indicating appropriate retention and peak symmetry, and the retention time for Valaciclovir was 3.1 minutes. [9-15]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation parameters of HPLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity:\u003c/strong\u003e By examining the interference that the enhanced method causes, the specificity can be ascertained. When using this technique, the presence of these drugs shouldn\u0026apos;t cause interference peaks to appear in the blank samples. Thus, it was asserted that this approach was unique. Specificity was attained by injecting blank solutions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity:\u003c/strong\u003eA freshly washed volumetric container should be filled with 10 mg of a medicine known as medication that has been carefully weighed out. Add 10 milliliters of water of HPLC quality on top. A final concentration of 1000 ppm is achieved. To create a working standardA different 10 ml weighing container was pipetted with 1 ml of the reference solution, then water was added to equal it. Its concentration is 100 ppm. The Inter-day Reliability %RSD values for Day 1 and Day 2 were 1.8191 and 0.9857, respectively, while the evening value was 1.4158.\u003c/p\u003e\n\u003cp\u003eUsing the working benchmark dosages of 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, and 60 ppm, serial dilutions were prepared. The data was shown in Figure 1and 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision:\u003c/strong\u003eA 10-ml volume flask was pipetted with 0.4 milliliters of 1000 \u0026micro;g/ml of a medication named sol, and HPLC-grade water was used for calibration, yielding a precision of 40 ppm for standard valaciclovir.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaking a solution that is 50% spiked:\u0026nbsp;\u003c/strong\u003eOne milliliter of solvent was pipetted into a flask with a 10-milliliter capacity, and the solvent was diluted with diluent to obtain 10 parts per million of valaciclovir.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaking a solution that is 100% spiked:\u003c/strong\u003e By pipetting four milliliters of solution into a flask with a capacity of ten milliliters and diluting it with diluent, 40 parts per million of a medication were obtained.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaking a solution that is 150% spiked:\u003c/strong\u003eSix milliliters of solution were pipetted into a 10-milliliter flask, and the solution was diluted with diluent to yield 60 ppm. Data of valaciclovir was shown in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLOD and LOQ:\u003c/strong\u003eThe lowest possible analyte concentration is known as the detection limit. The quantitation limit is the lowest amount of material that can be detected. calculated using the following formula. [16-24]\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"123\" height=\"45\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness:\u003c/strong\u003eTransmittance at a specific wavelength with a value of one nm is used to measure robustness. At least six measurements were made of the absorbance of a medication known as reference solution at 250, 251, and 252 nm. A percentage was used to calculate the RSD. The data was shown in Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromogenic method by using UV-Visible spectrophotometer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStandard stock solution:\u003c/strong\u003eA 25 ml volumetric flask was filled with precisely weighed 25 milligrammes of pure VLC. dissolved in distilled water and adjusted the volume to the appropriate level. The final standard stock solution had a concentration of 1000\u0026micro;g/ml.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of reagents used in chromogenic method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvery chemical and reagent used was of analytical quality and was manufactured fresh in distilled water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;2% Preparation of ferric ammonium sulphate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeigh 400 mg of sulphuric acid and 600 mg of ferric ammonium sulphate into a 100 ml volumetric flask, add distilled water to fill up the remaining volume, and then freeze for 10 minutes at 5\u0026deg;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1% Preparation of MBTH reagent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e400 mg of sulphuric acid and 200 mg of MBTH reagent should be weighed into a 100 ml volumetric flask. The volume should then be adjusted with distilled water and allowed to sit at 5\u0026deg;c for 10 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1% Preparation of FeCl₃ reagent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeigh one gramme of the FeCl₃ reagent into a 100 ml volumetric flask, then add distilled water to fill the remaining volume.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of blank reagent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blank reagent was prepared by adding 1 ml of MBTH Reagent and 1ml of ferric ammonium sulphate into 10ml volumetric flask and make up the volume with distilled water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of sample:\u0026nbsp;\u003c/strong\u003eAfter weighing and coarsely grinding ten 500 mg Valaciclovir pills, the powder was transferred to a 100 mL volumetric flask. After precisely weighing 10 mg of valaciclovir equivalent, it was diluted in a tiny amount of distilled water. The flask\u0026apos;s volume was increased to 100 ml by adding distilled water after 15 minutes of sonication. It was discovered that the finished solution included 100 mg of valaciclovir.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod optimization by using UV-Visible spectrophotometer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom the standard stock solution, 0.1 ml of the VLC solution was pipetted into a volumetric flask that held 10 ml. After 20 minutes, 1 millilitre of 1% MBTH reagent was added to the pipetted solution, which was then left for 15 minutes. Next, 1 millilitre of 2% ferric ammonium sulphate was added, and the mixture was maintained at 5\u0026deg;C. Distilled water was used to bring the leftover solution up to par. The solution in 400\u0026ndash;800 nm range was scanned using UV-visible spectroscopy against a reagent blank.The fourth trial after the first three is the best one. The fourth experiment\u0026apos;s wavelength is 618 nm, and its absorbance is 0.2002 nm. The ideal temperature is 5\u0026deg;C for 15 minutes, and the ideal tint is a dark greenish shade. Finally, after four attempts, the strategy is optimised.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColour stability:\u003c/strong\u003eThe drug\u0026apos;s colour stability lasts for two hours. After two hours, colour has diminished as a result of prolonged temperature and temporal variations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn house formulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e10mg equivalent of nano valaciclovir solid lipid nano particle by microemulsion technique in house formulation prepared in the college were used for the assay calculation.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"427\" height=\"147\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation parameters according to\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eICH Q2R1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe process of verifying that the specifications for a particular intended use are met through analysis and impartial proof is known as validation. The ICH Q2R1 recommendations were followed in determining the validation parameters. [25-34]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity:\u003c/strong\u003eThe developed method follows Beer\u0026apos;s law in a concentration ranging from 10-500\u0026micro;g/mL for valaciclovir, with an equation for regression of Y=0.002x + 0.0042 with a correlation coefficient value of 0.9997. A summary of the calibration curves\u0026apos; analytical data is offered together with the standard deviations for the slope, intercept, and system appropriateness characteristics. The calibration graphs\u0026apos; linearity is demonstrated by this dataas shown in Figure 3 and Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy:\u0026nbsp;\u003c/strong\u003eDegree to which a single measurement resembles the actual or accepted value is known as accuracy. By computing the recoveries of both medications using the conventional addition approach, the method\u0026apos;s correctness was ascertained. Three distinct amounts of standards (i.e., 50%, 100%, and 150%) were triplicately added to pre-quantified samples. It was determined what the mean recovery percentage\u0026nbsp;as shown in Table 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision:\u0026nbsp;\u003c/strong\u003eThree different concentrations and three duplicates of each concentration were used to evaluate reproducibility (intra-day) and precision at intermediate levels (inter-day). The suggested method\u0026apos;s %RSD was found to be very small (\u0026lt;2%), suggesting reasonable reproducibility and intermediate precision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection limit:\u003c/strong\u003e The lowest analyte concentration that produces a detectable response is known as the Limit of Detection (LOD). The following was used to determine LOD.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;LOD = 3.3 \u0026sigma; /S\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 1.5114\u0026micro;g/mL\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification limit:\u0026nbsp;\u003c/strong\u003eThe lowest concentration of the analyte at which a reaction can be precisely measured is known as the Limit of Quantification (LOQ). The following formula was used to determine LOQ.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;LOQ = 10*SD\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; = 4.58\u0026micro;g/mL\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness:\u003c/strong\u003e Robustness is a metric that expresses an analytical procedure\u0026apos;s resistance to small, deliberate changes in method parameters as well as its dependability under normal operating conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRuggedness:\u003c/strong\u003e The degree of consistency of test results achieved by analysing the same samples under various settings, such as different laboratories, different analysts, and different instruments, is known as the ruggedness of an analytical process. Deliberate change in analyst and instrument studies are performed to study their difference in absorbance. The %RSD were found to be very small (\u0026lt;2%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod extended to Chromogenic bioanalytical method development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing the protein precipitation extraction method, the procedure was devised. The process consists of: 0.1M HCL and 1ml of the standard stock solution (valaciclovir) were added to 1ml of human plasma. Give the vortex solution five minutes or so. For ten minutes, centrifuge the mixture at 4000 rpm. After that, transfer the liquid supernatant to a test tube and add 1 milliliter each of 2% MBTH reagent and 1% ferric ammonium sulphate. Examine the solution at 610 nm using a visible spectrophotometer. Recovery percentage that was obtained was 98.56%.[35-38].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation parameters for bioanalytical methods according to M10 guidelines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity:\u003c/strong\u003eCalibration curve illustrates the relationship between an analyte\u0026apos;s actual concentration and the testing platform\u0026apos;s reaction to it. Calibration curve is made up of the verification criteria, which are created by combining a certain quantity of analyzer into a matrix. Calibration standards also need to be made using same biological material the matrix that was used to make the study samples. Both the LLOQ, the cheapest evaluating standard, and the ULOQ, the most expensive assessing standard, define the calibration range. For every analytical run and every analyte examined during methods validation, a calibration curve ought to exist. Data was shown in Table 5 and Figure 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision and accuracy:\u0026nbsp;\u003c/strong\u003eAccuracy and precision should be assessed using comparable runs and data. Since the reliability should be between \u0026plusmn;15% at every value level and the theoretical level should be within \u0026plusmn;20%, do not include the LLOQ. The resolution (%CV) of a concentration must be more than 15% at any level. except for LLOQ, where it should not exceed more than 20%. Three-thirds of all QCs and perhaps 50% of each degree of concentration in order must fall within \u0026plusmn;15% of the nominal values for both exact and non-accuracy verification runs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability:\u003c/strong\u003eTo ensure that decisions about sample preparation, processing, evaluation, and storage conditions do not impact the analyte concentration, stability studies must be carried out. QCs are used to assess the material\u0026apos;s durability in the matrix at both low and large volumes. A portion of both the low and high QCs are examined at zero seconds and following the execution of the storage conditions that need to be assessed. It is necessary to construct a single bulk QC for every concentration level. The bulk sample must be separated into at least three aliquots for each concentration under investigation. These aliquots will then be filtered, stored, and examined. Data was shown in Table 7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalibration curve and range:\u003c/strong\u003eIn at least one study, the calibration curve was made using freshly spiked calibration standards. Calibration curve was evaluated using six concentration levels of a blank sample, calibration standards (such as the LLOQ and ULOQ), and a zero- concentration specimen (the blank sample spiked with IS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMatrix effect:\u003c/strong\u003eThe matrix effect assesses how elements of biological matrix impact the detection of analytes, which may have an impact on quantitative analysis\u0026apos;s precision and accuracy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReinjection reproducibility:\u003c/strong\u003eAfter storage, a run comprising a calibrated standard and at least five re-injections of the middle, inadequate, and high QCs is carried out to assess reinjection repeatability. Injected QCs\u0026apos; accuracy and precision demonstrate the viability of the treated samples. The results ought to be incorporated into the confirmation report or bioanalytical assessment of the study from which the data were derived.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod development of valaciclovir by UV spectrophotometer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValaciclovir Standard solution preparation:\u0026nbsp;\u003c/strong\u003eValaciclovir standard solutions were made by diluting 10 mg of the medication with 1000\u0026nbsp;𝝁g/ml of distilled water in a 10-milliliter flask that was dry and clean. A practical standard is created based on this. To achieve a concentration of 100 parts per million, pipette one mL of the standard solution into a 10-milliliter flask and add the suitable solvent. A 10-ml volume flask should contain 1 ml of this, and the diluent should be added to reach the proper level. The concentration that results is 10\u0026nbsp;𝝁g /ml. Wavelength was determined by analyzing a reference solution that contained 10\u0026nbsp;𝝁g/ml of valaciclovir between 200 and 400 nm and accuracy was calculated as shown in Figure 5 and Table 8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod extending to bioanalytical by using UV spectrophotometer of valaciclovir\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of plasma:\u0026nbsp;\u003c/strong\u003eProcured from blood bank.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of blank plasma:\u0026nbsp;\u003c/strong\u003eAfter measuring and transferring 1 ml of plasma to a 10 ml volumetric flask, reagents were added with distilled water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of plasma with protein precipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne method for concentrating and purifying proteins from different impurities is protein precipitation. By adding a reagent1, the procedure modifies the solvent\u0026apos;s resolution potential, which reduces the solute\u0026apos;s solubility1. The water difficulties between protein molecules was broken down by the binding of salt ions with water molecules, enabling contact between the protein\u0026apos;s basic regions. As a result, the protein molecules group together and separate from the solvent.\u0026nbsp;\u003cbr\u003e\u0026nbsp;The process was developed using the protein precipitation separation method. The procedure includes: One mL of human plasma was mixed with 0.1M Hydrochloric acid and one mL of the usual stock solution (valaciclovir). Let the vortex solution sit for about five minutes. Centrifuged the mixture at 4000 rpm for 10 minutes. The liquid supernatant should then be transferred to cuvettes. Use a visible spectrophotometer to analyse the solution at 251 nm. Obtained recovery percentage was 98.56%.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eChromatographic method\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003eHPLC Linearity data.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eArea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eRetention Time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eTheoretical Plate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eTailing Factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e10PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e1072631\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3.121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2125.707\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.684\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e20PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2150271\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3.135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2842.378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.713\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e30PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3151734\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3.174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2367.700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.579\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e4222836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3.177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2202.733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.385\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e50PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e5212512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2916.264\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.973\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e60PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e5997982\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e3.263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2208.314\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.079\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterday and intraday precision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e%RSD of Repeatability of valaciclovirwas found to be 0.4945877. %RSD of Intra-day Precision\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003eMorning was found to be 0.49978 and Inter-day Precision %RSD values for Day 1 and Day 2 were 0.856019 and 0.4804377, respectively, while the evening value was 0.66605. According to ICH criteria, all of the results were in limits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Percentage of valaciclovir recovered\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e% Percentage\u003c/p\u003e\n \u003cp\u003eLevel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003cp\u003earea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003eStandard area\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003eAverage Area of three replicas\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eRecovery area\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eRecovery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003eMean % Recovery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e50%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e4222836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e1072631\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e8455602\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e4232766\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e100.23%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e100.62%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e100%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e4222836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e4222821\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e8525672\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e4302836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e101.89%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e150%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e4222836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e5997982\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e10210818\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e4212836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e99.76%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eDetection and Quantification limits:\u0026nbsp;\u003c/strong\u003eUsing formulas based on the slope of \u0026nbsp;calibration curve and \u0026nbsp;standard deviation of the y-intercept of regression lines, LOD and LOQ were determined and were found to be 0.07022260189 \u0026micro;g/mL and 0.212795763327 \u0026micro;g/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eRobustness data with respect to HPLC\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eRobustness\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eFlow rate at 0.7ml/min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eFlow rate at 1.0ml/min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eFlow rate at 1.2ml/min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eConcentration\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eArea\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eArea\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eArea\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4226543\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4222836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4222789\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4228903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4221832\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4224567\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4236547\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4222789\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4213598\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4228907\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4223132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4224589\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4228975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4233456\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4244672\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4220987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4221898\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4245678\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4228477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4224324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4228437\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e5011.581307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4505.113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e12946.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40PPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e0.118519772\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e0.106647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e0.30618\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eUsing two wavelengths -1(%RSD 0.118519) and +1(%RSD 0.106647)\u0026mdash;the robustness analysis was performed. The findings met ICH standards for outcomes within acceptable limits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssay:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"435\" height=\"184\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromogenic method validation by using UV-Visible spectroscopy parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Parameters for validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003eData on VLC linearity\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003eConcentration (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAbsorbance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e10\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.03777\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e50\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.1031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e100\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.2126\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e150\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.3035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e200\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.4076\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e250\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.5237\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e300\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.6321\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e350\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.7213\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e400\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.8523\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e450\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.9017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 153px;\"\u003e\n \u003cp\u003e500\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1.0126\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u0026nbsp;\u003c/strong\u003eAs per ICH guidelines r\u003csup\u003e2\u003c/sup\u003e found to be 0.9999 and y= mx +c is 0.002x+0.0042\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e%RSD of Repeatability of valaciclovirwas found to be 0.0141. %RSD of Intra-day Precision\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003eMorning was found to be 0.055 and Evening was found to be 0.056 andDay 1\u0026apos;s and Day 2\u0026apos;s\u0026nbsp;The values of the Inter-day Precision %RSD were found to be 0.0196 and 0.029, respectively.\u0026nbsp;All of the findings were within the limitations, which are less than 2, in accordance with ICH guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimits:\u0026nbsp;\u003c/strong\u003eICH Specifies a recovery percentage ranging from 98-102%\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u0026nbsp;\u003c/strong\u003eThe accuracy recovery percentage has been found between 98.24-101.83%.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u0026nbsp;\u003c/strong\u003eValaciclovir formulation recovery study (valaciclovir 500mg)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e% Percentage\u003c/p\u003e\n \u003cp\u003eLevel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003cp\u003eAbsorbance\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eStandard 50ppm, 100ppm 150ppm Absorbances\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eTotal Absorbance\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eRecovery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eMean % Recovery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e50%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(250ppm) + (50ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.5327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.1031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.6170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e98.12%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e98.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.6175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e98.22%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.6172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e98.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e100%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(250ppm) + (100ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.5327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.2126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.7316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e99.11%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e99.42%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.7301\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e99.87%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.7326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e99.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e150%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(250ppm) + (150ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.5327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.3035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.8361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e101.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e101.05%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.8320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e100.91%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e0.8301\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e100.55%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e*It indicates three replicates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness and Ruggedness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing two wavelengths -1(%RSD 0.0292) and +1(%RSD 0.181)\u0026mdash;the robustness analysis was performed. Two separate analysts performed the ruggedness analysis; their findings were %RSD 0.042 for Analyst 1 and %RSD 0.028 for Analyst 2. The findings met ICH standards for outcomes within acceptable limits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection limit and Quantitation limit\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing formulas based on the slope of the calibration curve and the standard deviation of the y-intercept of regression lines, LOD and LOQ were determined\u0026nbsp;and were found to be 1.5114\u0026micro;g/mL and4.58\u0026micro;g/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssay Calculation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"258\" height=\"267\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromogenic Bio Analytical Method Validation Parameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6\u0026nbsp;\u003c/strong\u003eChromogenic bioanalytical linearity\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003eConcentration (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003eAbsorbance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 105px;\"\u003e\n \u003cp\u003e50\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e0.1001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 105px;\"\u003e\n \u003cp\u003e100\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e0.2016\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 105px;\"\u003e\n \u003cp\u003e150\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e0.3011\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 105px;\"\u003e\n \u003cp\u003e200\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e0.4003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 105px;\"\u003e\n \u003cp\u003e250\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e0.5103\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 105px;\"\u003e\n \u003cp\u003e300\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e0.6132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 105px;\"\u003e\n \u003cp\u003e350\u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e0.7053\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u0026nbsp;\u003c/strong\u003ethe correlation coefficient was found to be 0.9998\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy and precision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll concentration levels should have accuracy within \u0026plusmn;15% of the nominal focused attention, with the exception of the LLOQ, when it should be within \u0026plusmn;20%. Apart at the LLOQ, where the value shouldn\u0026apos;t be more than 20%, the accuracy (%CV) of the quantities measured at each level shouldn\u0026apos;t be more than 15%.Accuracy and precision were found to be within limits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBench top stability studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7\u0026nbsp;\u003c/strong\u003eStability of chromogenic bioanalytical method of LQC and HQC\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003ePercentage Level(LQC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0hour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e2hour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e4hour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e6hour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e8hour\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.143833\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.1469\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.114767\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.134766667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.1168\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.028471\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.02816\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.015581\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.04434144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.015655989\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e%CV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e19.79414\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e19.16968\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e13.57612\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e13.90237969\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e12.4041004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003ePercentage Level (HQC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0hour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e2hour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e4hour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e6hour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e8hour\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.7136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.7541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.6373333\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.622366667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.487366667\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.0090006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e0.0443819\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.0404829\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.064030019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.077951352\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e%CV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e11.261288\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e13.8854085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e12.351915\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e10.28815045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e15.99439553\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003ePercentage Recovery Calculation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e% Recovery = Sample absorbance/Standard absorbance*100\u003c/p\u003e\n\u003cp\u003e= 0.5103/0.5205*100\u003c/p\u003e\n\u003cp\u003e=99.8%\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUV Spectrophotometer Validation Parameters of Valaciclovir\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u0026nbsp;\u003c/strong\u003eAs per ICH guidelines r\u003csup\u003e2\u003c/sup\u003e found to be 0.9999 and y= mx+c is 0.02159x+0.9993\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision\u003c/strong\u003e\u003cbr\u003e%RSD of Repeatability of valaciclovirwas found to be 0.5627. %RSD of Intra-day Precision\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003eMorning was found to be 1.7755 and\u0026nbsp;The Inter-day Reliability %RSD values for Day 1 and Day 2 were 1.8191 and 0.9857, respectively, while the evening value was 1.4158.\u0026nbsp;All of the findings were within the limitations, which are less than 2, in accordance with ICH guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimits:\u0026nbsp;\u003c/strong\u003eICH Specifies a recovery percentage ranging from 98-102%\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u0026nbsp;\u003c/strong\u003eThe accuracy recovery percentage has been found between 98.24-101.83%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 8\u0026nbsp;\u003c/strong\u003eRecovery Study of valaciclovir formulation (valaciclovir 500mg)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"603\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e% Percentage\u003c/p\u003e\n \u003cp\u003eLevel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003cp\u003eAbsorbance\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eStandard 50ppm, 100ppm 150ppm Absorbances\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eTotal Absorbance\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eRecovery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003eMean % Recovery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e50%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(35ppm) + (50ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e1.5275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e1.0323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e2.0193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e98.12%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e98.25%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e2.0540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e98.32%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e2.0870\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e98.26%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e100%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(35\u0026nbsp;ppm) + (100ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e1.5275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e1.2776\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e2.5051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e99.15%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e99.62%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e2.5076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e99.90%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e2.5043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e99.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e150%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(35ppm) + (150ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e1.5275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e1.6274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e3.0013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e101.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e101.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e3.0065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e100.91%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e3.0023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 89px;\"\u003e\n \u003cp\u003e100.55%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e*It indicates three replicates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness and ruggedness:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing two wavelengths -1(%RSD 1.0852) and +1(%RSD 1.0269)\u0026mdash;the robustness analysis was performed. Two separate analysts performed the ruggedness analysis; their findings were %RSD 1.3430 for Analyst 1 and %RSD 1.5522 for Analyst 2. The findings met ICH standards for outcomes within acceptable limits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection and Quantitation limits:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDL:\u003c/strong\u003e 0.1391\u0026micro;g/mL\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;DQ:\u003c/strong\u003e 0.4271\u0026micro;g/mL\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRP-HPLC technique developed for valaciclovir analysis demonstrated effective chromatographic separation with a well-defined Gaussian peak at a retention time of 3.1 minutes. The method utilized an isocratic mobile phase of methanol and water (60:40), with a flow rate of 1.0 mL/min, ensuring precision and reproducibility. UV-Visible spectrophotometry confirmed an optimal detection wavelength at 251 nm, facilitating further analytical studies. Validation parametersadhered to ICH Q2R1 guidelines, confirming method's reliability. The method exhibited high linearity (R\u0026sup2; = 0.9997) within a concentration range of 10\u0026ndash;500 \u0026micro;g/mL, demonstrating its suitability for quantitative analysis. Accuracy assessments using spiked solutions at different concentrations yielded satisfactory recovery rates, supporting method validity. Bioanalytical method development using protein precipitation techniques successfully extracted valaciclovir from plasma with a high recovery of 98.56%. The method showed stability under various conditions, ensuring its robustness and reproducibility in different laboratory settings. Reinjection reproducibility and matrix effect evaluations confirmed that biological matrix components did not interfere significantly with the analysis. Overall, the developed RP-HPLC and UV-based techniques proved to be reliable, sensitive, and applicable for routine quality control and bioanalytical studies of valaciclovir.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIt is concluded that suggested approach for measuring valaciclovir in pharmaceutical dose forms was found to be straightforward, sensitive and accurate. Assay results showed a strong correlation with corresponding label claim. Because of its low solvent content, lack of extraction steps, specificity, and sensitivity, this spectrophotometric approach has been proven to be superior. The regular application of the recommended technique in control of quality for valaciclovir analyses in medication dosage forms is supported by these advantages. High precision and dependability are demonstrated by the devised UV spectroscopy method for measuring valaciclovir in human serum. Efficacy of the approach is demonstrated by excellent correlation and quantification limits and conformity to Beer's law within designated concentration range. The method's correctness is confirmed by its 100% recovery rate, and its application is validated by the findings' conformity with labels of commercial products. In compliance with ICH bioanalytical criteria, the validation of the standard pharmaceutical solution analysis showed strong performance. For the intensity range of 5\u0026ndash;40 \u0026micro;g/mL, the 5.0-minute analysis time was effective. High dependability is indicated by the method's accuracy, which ranges from 101.26\u0026ndash;101.96%, and yield, which surpasses 89.48%. The approach exhibits exceptional precision, with relative standard deviation (RSD) values for both incurred plasma and quality control specimens below 10%. Standard had intensity range of 5\u0026ndash;40 \u0026micro;g/mL, required 5.0 minutes to analyze. At 3.177 minutes, valaciclovir was eluted using described technique. Valaciclovir showed linear behaviour in the 10\u0026ndash;60 \u0026micro;g/ml range. Relative variance of 0.85% serves as an example of the accuracy. Percentage Accuracy testing revealed.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMBTH: 3-Methyl-2 Benothiazolinone Hydrazone; LOD: Limit of detection; LOQ: Limit ofquantitation; % RSD: Relative standard deviation; ICH: International Conference on Harmonization; Rs: Resolution; SD: Standarddeviation; \u0026mu;g: Microgram; mL: Milliliters; mg: Milligrams; %: Percentage; RP: Reverse phase; HPLC: High-performance liquid chromatography. LLOQ: Lower limit of Quantification; ULOQ: Upper limit of Quantification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI would like to thank our beloved principle, prof. M. Sumakanth madam and faculty of the department of pharmaceutical analysis for giving me the opportunity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have read and approved the manuscript. K. Bhavyasri designedthe study. Remaining authors performed the experiment and analyzed and reviewed thedata. K. Bhavyasri and M. Sumakanth supervised the experiment, reviewed the data, and supported forwriting the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and materials are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma was collected from blood banks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col start=\"1\" type=\"1\"\u003e\n\u003cli\u003eChoudhary A (2017) The principle of ultraviolet (UV) spectrophotometer. Medium.\u003c/li\u003e\n\u003cli\u003eSri Varsha M, Raghavendra Babu N, et al. (2015) Development of new spectrophotometric method for estimation of tenofovir disoproxil fumarate using MBTH reagent. Int Curr Pharm J 4(4):378-381.\u003c/li\u003e\n\u003cli\u003eKhagga Bhavyasri, Murali Balaram V, Nageswarao R, Rambabu D, Sasikiran Goud E, Ajitha M (2013) Development and validation of forced degradation studies of Atazanavir using RP-HPLC and characterization of degradants by LC-MS/MS. Int J Pharm Sci Rev Res 33(2):11-15.\u003c/li\u003e\n\u003cli\u003eKhagga Bhavyasri, Murali Balaram V, Nageswarao R, Rambabu D, Ajitha M (2013) RP-UPLC method development and validation for the simultaneous estimation of Proguanil and Atovaquone in pharmaceutical dosage form. J Chem Pharm Res 5(12):1222-1229.\u003c/li\u003e\n\u003cli\u003eKhagga Bhavyasri, Murali Balaram V, Nageswarao R, Rambabu D, Ajitha M (2013) Rapid simultaneous determination of Naproxen and Esomeprazole magnesium in combined tablets by validated Ultra Performance Liquid Chromatographic method. J Chem Pharm Res 5(12):1230-1236.\u003c/li\u003e\n\u003cli\u003eRahul Reddy MB, Gurupadayya BM, et al. (2011) Spectrophotometric determination of Lamuvidine using acidic dye and coupling reagent. Indian J Chem Technol 18:431-438.\u003c/li\u003e\n\u003cli\u003eJadhav AS, Pathare DB, Shingare MS (2007) Development and validation of high-performance liquid chromatographic method for valacyclovir, an antiviral drug. J Pharm Biomed Anal 43(4):1568-1572.\u003c/li\u003e\n\u003cli\u003eBhavyasri K, Manisha KV, Swethasri R, Sumakanth M (2019) Method development and validation for the estimation of esmolol hydrochloride in bulk and ophthalmic formulation by UV-Visible spectrophotometer. Paripex - Indian J Res 8(6):143-145.\u003c/li\u003e\n\u003cli\u003eMounika G, Bhavyasri K, Swethasri R, Sumakanth M (2019) Development and validation of RP-HPLC method for estimation of Canagliflozin. Samriddhi S-JPSET 11(2):85-92.\u003c/li\u003e\n\u003cli\u003eBhavyasri K, Bhairavi B, Swethasri R, Sumakanth M (2020) Three-dimensional nuclear magnetic resonance spectroscopy \u0026ndash; a review. Int J Anal Exp Modal Anal 12(3):1456-1463.\u003c/li\u003e\n\u003cli\u003ePadmalatha H, Anupama V, et al. (2014) Spectrophotometric estimation of chlorthalidone in pharmaceutical formulation using MBTH \u0026amp; FC reagent. World J Pharm Pharm Sci 3(2):1443-1440.\u003c/li\u003e\n\u003cli\u003eKutty SV, Eapen SC, et al. (2012) Validated UV-Visible spectrophotometric method for the estimation of fenofibrate in pure and pharmaceutical formulation using MBTH reagent. Int J Pharm Sci Drug Res 4(1):74-76.\u003c/li\u003e\n\u003cli\u003eBorman P, Elder D (2017) Q2 (R1) validation of analytical procedures: text and methodology. ICH quality guidelines: an implementation guide. 127-166.\u003c/li\u003e\n\u003cli\u003eShravya A, Chandan RS, et al. (2011) Spectrophotometric determination of Ezetimibe using MBTH reagent in pharmaceutical dosage form. Int J Res Ayurveda Pharm 2(2):521-525.\u003c/li\u003e\n\u003cli\u003eGuideline IH (2022) Validation of analytical procedures Q2 (R1). ICH: Geneva, Switzerland.\u003c/li\u003e\n\u003cli\u003eGuideline IH (2005) Validation of analytical procedures: text and methodology. Q2 (R1) 1(20):05.\u003c/li\u003e\n\u003cli\u003eSrinivasa Rao K, Sunil M (2009) Stability-indicating liquid chromatographic method for valacyclovir. Int J ChemTech Res 1(3):702-708.\u003c/li\u003e\n\u003cli\u003eUV spectrophotometric method for the estimation of valacyclovir HCl in tablet dosage form. E-J Chem 6(3):814-818.\u003c/li\u003e\n\u003cli\u003eAnand BS, Katragadda S, Mitra AK (2004) ICH harmonized tripartite guidelines, validation of analytical procedures: text and methodology, Q2A. J Pharmacol Exp Ther 311:659.\u003c/li\u003e\n\u003cli\u003eIshpreet K, Sharad W, Harsharan PS (2015) Development and validation of UV spectroscopic method for determination of pioglitazone in bulk and pharmaceutical dosage form. Pharm Methods 6(2):1-1.\u003c/li\u003e\n\u003cli\u003eShah RS, Shah RR, Pawar RB, Gayakar PP (2015) UV-Visible spectrophotometry - a review. Int J Inst Pharm Life Sci 5(5):490-505.\u003c/li\u003e\n\u003cli\u003eAbdel-Rehim M, Dahlgren M, Blomberg L (2006) Quantification of ropivacaine and its major metabolites in human urine samples utilizing microextraction in a packed syringe automated with LC-MS/MS. J Sep Sci 29(11):1658-1661.\u003c/li\u003e\n\u003cli\u003eMercier-Letondal P, Marton C, Godet Y, Galaine J (2021) Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1). J Transl Med 19:1-5.\u003c/li\u003e\n\u003cli\u003eAlmeida C, Nogueira JM (2006) Determination of steroid sex hormones in water and urine matrices by stir bar sorptive extraction and liquid chromatography with diode array detection. J Pharm Biomed Anal 41(4):1303-1311.\u003c/li\u003e\n\u003cli\u003eAnderson NL, Polanski M, Pieper R, et al. (2004) The human plasma proteome: A nonredundant list developed by combination of four separate sources. Mol Cell Proteomics 3(4):311-326.\u003c/li\u003e\n\u003cli\u003eSawicki E, Hauser TR, Stanley TW, Elbert W (1961) The 3-methyl-2-benzothiazolone hydrazone test. Anal Chem 33:93-96.\u003c/li\u003e\n\u003cli\u003eAnthon GE, Barrett DM (2002) Determination of reducing sugars with 3-methyl-2-benzothiazolinone hydrazone. Anal Biochem 305:287-289.\u003c/li\u003e\n\u003cli\u003eShah RS, Shah RR, Pawar RB, Gayakar PP (2015) UV-Visible spectrophotometry - a review. Int J Inst Pharm Life Sci 5(5):490-505.\u003c/li\u003e\n\u003cli\u003eICH (1994) Q2A, Text on Validation of Analytical Procedures. Int Conf Harmonization, Geneva, October:1‐5.\u003c/li\u003e\n\u003cli\u003eSylvie LR, Francoise SL, Agnes PL, et al. (2003) Ribavirin quantification in combination treatment of chronic hepatitis C. AAC 47(1):124-129.\u003c/li\u003e\n\u003cli\u003eAlvarez D, Dieterich DT, Brau N, et al. (2013) Zidovudine use but not weight-based ribavirin dosing impacts anemia during HCV treatment in HIV. 12(5):498-513.\u003c/li\u003e\n\u003cli\u003eBani SF, Carrat F, Pol S, et al. (2006) Risk factors for symptomatic mitochondrial toxicity in HIV-infected persons. J Viral Hepat 13(10):683-.\u003c/li\u003e\n\u003cli\u003eGranero GE, Amidon GL (2006) Stability of valacyclovir: implications for its oral bioavailability. Int J Pharm 317(1):14-18.\u003c/li\u003e\n\u003cli\u003eVenkata Reddy P, Sudha Rani B (2006) Estimation of some antiviral drugs using TP as analytical reagent. E-J Chem 3(3):154-158.\u003c/li\u003e\n\u003cli\u003eBhavyasri K, Fatima A, Rambabu D (2019) Spectrophotometric estimation of metformin in human plasma. J Biomed Pharm Res 8(3):61-64.\u003c/li\u003e\n\u003cli\u003eBhavyasri K, Benazir SB, Rambabu D (2019) Method development and validation of UV spectrophotometric method for estimation of nevirapine in human plasma. Panama Res J 9(6):115-124.\u003c/li\u003e\n\u003cli\u003eBhavyasri K, Bhairavi B, Rambabu D (2019) Method development and validation for estimation of paracetamol in human plasma by UV spectrophotometry. J Emerg Technol Innov Res 6(6):572-577.\u003c/li\u003e\n\u003cli\u003eSugumaran M, Jothieswari D (2010) Development and validation of spectroscopic method for estimation of valacyclovir in tablet dosage form. Oriental J Chem 26(1):163-165.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"accreditation-and-quality-assurance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acqa","sideBox":"Learn more about [Accreditation and Quality Assurance](http://link.springer.com/journal/769)","snPcode":"769","submissionUrl":"https://submission.nature.com/new-submission/769/3","title":"Accreditation and Quality Assurance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"UV-Spectrophotometer, Valaciclovir, 3-Methyl-2 Benothiazolinone Hydrazone, Human Plasma, RP-HPLC","lastPublishedDoi":"10.21203/rs.3.rs-6287855/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6287855/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eA novel, straightforward, accurate, repeatable, and precise chromogenic UV-visible spectroscopy and bioanalytical technique for measuring valaciclovir in pharmaceutical formulations and bulk. The MBTH reagent and Valaciclovir, in presence of ferric ammonium sulphate, undergo an oxidative condensation/coupling reaction that results in the formation of a green solution with the greatest absorption peak at 618nm. Linearity range of valaciclovir is 10\u0026ndash;500\u0026micro;g/ml, and its correlation coefficient is 0.9997. Careful measurement of valaciclovir yielded a value of \u0026lt;\u0026thinsp;2. Validation of this method is conducted in accordance with ICH recommendationsQ2R1. Enhanced process was designed, verified, and extended to biological material using the protein precipitation extraction method.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eValaciclovir's detection and quantification limits were 1.5114 \u0026micro;g/mL and 4.58 \u0026micro;g/mL, respectively. Human serum analysis by UV-Visible spectroscopy was created and verified. 1mL serum sample and 1mL of 0.1M was used for protein precipitation, which was vortexed and centrifuged. Standard solutionwhich had standard intensity range of 5\u0026ndash;40 \u0026micro;g/mL, required 5.0 minutes to analyze. At 3.177 minutes, valaciclovir was eluted using the described technique. Valaciclovir showed linear behavior in 10\u0026ndash;60 \u0026micro;g/ml range. Percentage Accuracy testing revealed that mean recovery was within region of 100%. Limits of quantitation and detection were determined to be 0.070 ng/ml and 0.212 ng/ml, respectively.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe developed approach was linear, stability indicating, sensitive, rapid, precise, and accurate. As a result, the developed method might be applied to regular quality control of tablets and bulk medications.\u003c/p\u003e","manuscriptTitle":"Rp-Hplc Method for Estimation of Anti-Viral Drug(Valaciclovir) in Pure, Solid Dosage Form and Spiked Human Plasma Using Mbth Reagent as Per Ich Guidelines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-04 09:50:07","doi":"10.21203/rs.3.rs-6287855/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-06T12:04:29+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"189089159998017509348889030263097873257","date":"2025-04-05T14:16:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-04T18:26:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-03T21:50:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114079176473698800005633621529458186151","date":"2025-04-02T06:56:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139272646486765979558153913504482724722","date":"2025-04-01T11:28:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270552244533372217849836866403879677819","date":"2025-04-01T08:05:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156743045130312915149003877943877834572","date":"2025-04-01T07:39:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160844743020900340827934149472722897143","date":"2025-04-01T03:58:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224847671532548803990353396305344671328","date":"2025-03-31T15:45:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214791767679167975513182679903117821993","date":"2025-03-31T14:28:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285597588404528328536288765915684707436","date":"2025-03-31T14:25:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11059120156143166834074255318554929195","date":"2025-03-31T13:18:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200823706405343328665737294210191817503","date":"2025-03-31T13:13:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308699950388619467098670741683262693639","date":"2025-03-28T09:52:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-25T13:34:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-25T13:30:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-24T06:47:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Accreditation and Quality Assurance","date":"2025-03-23T10:55:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"accreditation-and-quality-assurance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acqa","sideBox":"Learn more about [Accreditation and Quality Assurance](http://link.springer.com/journal/769)","snPcode":"769","submissionUrl":"https://submission.nature.com/new-submission/769/3","title":"Accreditation and Quality Assurance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a11b4203-700f-43b6-8203-ee3a271faf43","owner":[],"postedDate":"April 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-23T16:04:21+00:00","versionOfRecord":{"articleIdentity":"rs-6287855","link":"https://doi.org/10.1007/s00769-025-01638-w","journal":{"identity":"accreditation-and-quality-assurance","isVorOnly":false,"title":"Accreditation and Quality Assurance"},"publishedOn":"2025-06-16 15:57:37","publishedOnDateReadable":"June 16th, 2025"},"versionCreatedAt":"2025-04-04 09:50:07","video":"","vorDoi":"10.1007/s00769-025-01638-w","vorDoiUrl":"https://doi.org/10.1007/s00769-025-01638-w","workflowStages":[]},"version":"v1","identity":"rs-6287855","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6287855","identity":"rs-6287855","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}