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%.
