Delonix Society’s Baramati College of Pharmacy, Barhanpur, Baramati, Dist. Pune, Maharashtra, India 413102
For the quantitative determination of acyclovir (ACV) in bulk medication and tablet dosage form, a straightforward, quick, accurate, and economical UV-visible spectrophotometric approach was created and validated. The technique uses distilled water as a solvent to detect absorbance at 251.5 nm. With a correlation value (R2) of 0.9999, acyclovir demonstrated high linearity in the concentration range of 2–10 ppm, demonstrating outstanding adherence to Beer–Lambert's law. Three estimating techniques were used in the developed method: calibration curve method, standard absorptivity, and single point standardisation. The suggested method was verified according to ICH requirements for factors such as linearity, accuracy, precision, ruggedness, robustness, limit of detection (LOD), and limit of quantification (LOQ). In the range of 98.8% to 101.7%, the accuracy studies demonstrated an acceptable recovery. Precision results showed low %RSD values, indicating moderate precision and acceptable repeatability. The results showed that the LOD and LOQ were 0.098 ppm and 0.297 ppm, respectively. The approach is suitable for routine quality control analysis of acyclovir in pharmaceutical formulations, as evidenced by the 99.01% purity of acyclovir.
The discovery of acyclovir was announced in 1977. Acyclovir, another name for acyclovir (ACV), is an antiviral drug. It is mostly used to treat shingles, chickenpox, and viral infections. Additional applications include prevention of Epstein-Barr virus infections and CMV infections after transplantation. It can be administered intravenously and orally. Since no harm has been reported, it is usually regarded as safe to use during pregnancy. It seems safe to use when nursing. A nucleic acid counterpart derived from guanosine is acyclovir. It functions by reducing the amount of DNA produced by the virus. Acyclovir, also known as acycloguanosine, is an antiviral medication used to treat infections caused by the varicella-zoster virus (VZV), a type of herpes virus that causes shingles and chickenpox, or the herpes simplex virus (HSV). Acyclovir is effective against active, replicating HSV or VZV and was initially identified in the mid-1970s. Acyclovir is a member of a class of synthetic medications known as nucleoside analogues, which are distinguished by their resemblance to naturally occurring nucleosides—the structural components of DNA and RNA—found in viruses and cells. 9-[(2-hydroxyethoxy)-methyl] acyclovir-guanosine, an acyclic derivative of guanosine, has strong therapeutic effects by selectively inhibiting the multiplication of herpes viruses. Antiviral activity against the varicella zoster and herpes simplex viruses.
Fig.No.2: Acyclovir
MATERIAL AND METHOD:
Instrumentation
A Systronics (India) Limited UV-visible spectrophotometer, Model AU-2707, with a 1 cm cuvette and a double beam configuration system was used for the study. Ultrasonicate cleaner was used to degas the solvent. For the weighing, an electronic balance was used.
Chemicals or reagents
Leeford Healthcare Ltd provided an analytically pure sample of ACV, ZOSTER 400 DT a tablet formulation produced by Sun Pharmaceutical Industries Ltd, was purchased from a nearby pharmacy.
METHODOLOGY
Selection of suitable solvent
When selecting the solvent, the drug's solubility, stability, and absorbance maxima in the particular solvent were taken into account. Weighing 100 mg of ACV, its solubility in pure water, methanol, ethanol, acetone, and chloroform was assessed. Among the solvents on the above list, water is the most efficient. As a result, water is used as a solvent.
Preparation of standard stock solution
After being weighed, 100 mg of pure ACV was moved to a 100 ml volumetric flask and dissolved in water. To get a final concentration of 1000 ppm, it was thoroughly dissolved and diluted with diluent. A 10ppm solution was made from the stock solution using water, which served as the working standard.
Determination of wavelength
The wavelength maxima of ACV were found by scanning its UV spectra between 200 and 400 nm after it was synthesised at a concentration of 10 ppm. For the investigation, this wavelength was chosen. The highest absorbance against water was discovered to be 251.5 nm.
Estimation of ACV Tablet
Twenty ACV tablets were weighed and ground into a powder in a glass mortar. The powder equivalent to 10 mg of ACV Tablet was transferred into a 10 ml volumetric flask, dissolved in around 5–6 ml of water, and then subjected to an ultrasonicate for 15 minutes. The
flask was then diluted with water to attain a concentration of 1000 ppm. The mixture was filtered using Whatman filter paper. The sample solution was diluted and analysed, as was previously described.
Table No. 1: Drug estimation
|
Concentration (API) |
Abs (API) |
Concentration (Tablet) |
Abs at 251.5 nm |
Specifications |
|
2 ppm |
0.086 |
10 ppm |
0.504 |
Brand name: ZOSTER 400 DT |
|
4 ppm |
0.195 |
10 ppm |
0.506 |
Label claim:400 mg |
|
6 ppm |
0.301 |
10 ppm |
0.501 |
Mfg. by: Leeford Healthcare Ltd |
|
8 ppm |
0.407 |
Mean |
0.503 |
Manufacturing date:03/2025 |
|
10 ppm |
0.509 |
SD |
0.167901 |
Expiry date:02/2027 |
|
|
|
RSD |
1.000075 |
Batch no:ZDT502C |
|
|
|
%RSD |
100.0075 |
% Purity of drug: 99% |
Estimation of ACV by using Standard Absorptivity Method
Prepare standard solutions of ACV in water at concentrations ranging from 2 ppm to 10 ppm. Measure the absorbance of each solution at 251.5 nm using a UV-visible spectrophotometer. Calculate the absorptivity (a) using Beer-Lambert’s law
A = abc
Where: A = Absorbance, a = Molar Absorptivity, b = Path length (1 cm), c = Cocn in ppm
The standard absorptivity method is based on the principle of Beer-Lambert’s law, which states that absorbance is directly proportional to concentration by determining the absorptivity coefficient (a), unknown sample concentrations can be estimated accurately. This method is widely used due to its simplicity and ability to provide reliable quantitative results. By using formula, it was noted that the percentage purity of drug by using this method was found 99.01%. (By using Table No. 1)
Estimation of ESC by using single point method [6,30]
Make a standard ACV solution in water at a pre-determined concentration, such as 10 ppm.
Make the water solution for the test sample. Using a UV-visible spectrophotometer, determine the absorbance of the test sample and the standard at 251.5 nm. Determine the concentration of the test sample using the following formula:
Ctest = (Atest X Cstd) / Astd
Where, Ctest = concentrations of sample, Cstd = concentrations of standard, Atest = absorbance of the sample, Astd = absorbance of the standard By comparing the absorbance of an unknown sample with that of a standard solution with a known concentration, the Single Point Standardisation Method provides a straightforward and straightforward method for determining the concentration of an unknown sample. It is very helpful for routine, fast analysis where great precision is not necessary. Using the procedure, it was discovered that the drug's purity percentage was 99.01% (Table No. 1).
Calibration curve method [6,30]
Make standard ACV solutions in water with concentrations between 2 and 10 parts per million. Using a UV-visible spectrophotometer, determine each solution's absorbance at 251.5 nm. Plot absorbance against concentration to create a calibration curve. Use linear regression analysis to find the calibration curve's equation. By interpolating the absorbance values of unknown samples, you can use the calibration curve equation to determine their concentration.
Fig. No. 2: Calibration Curve ACV
Beer-Lambert's law, which states that absorbance is directly proportional to concentration, is the foundation of the Calibration Curve Method. The concentration of unknown samples can be precisely ascertained by building a standard calibration curve. This technique guarantees excellent accuracy and dependability in numerical analysis. The percentage purity of the substance was discovered to be 99.01% by utilising the equation of line after the formula was calculated.
Method of validation [6,31-44]
Linearity [6,31-33]
The absorbance of each concentration was measured at 251.5 nm using water as a blank.
To make new aliquots, standard stock-2 solution with concentrations ranging from 2 to 10 ppm was utilised. The regression coefficient (R2) value for the linearity curve was found to be
0.9999. Table 1 displays the linearity results.
Table No. 02: Linearity
|
Concentration |
Absorbance |
Calculation |
|
|
2 |
0.086 |
MEAN |
0.299 |
|
4 |
0.195 |
MODE |
N/A |
|
6 |
0.301 |
MEDIAN |
0.301 |
|
8 |
0.407 |
SD |
0.00191 |
|
10 |
0.509 |
RSD |
0.00874 |
|
Tablet -10 PPM |
0.504 |
%RSD |
0.87% |
|
|
C.C |
0.9999 |
|
|
INTERSECT |
(-0.0178) |
||
|
SLOPE |
0.1058 |
||
Precision [6,34-35]
An analytical method's degree of repeatability under typical operating circumstances. It is separated into:
Intraday and Interday precision
The precision of the approach was demonstrated by the results, which showed little change in absorbance levels over various time intervals and days.
Table No. 03: Intra day precision
|
Time |
Concentration |
Absorbance |
Calculation |
|
0 Hrs. |
10PPM |
0.509 |
MEAN - 0.506 |
|
2 Hrs. |
10PPM |
0.499 |
SD - 0.006 |
|
4 Hrs. |
10PPM |
0.501 |
RSD - 0.0119 |
|
8 Hrs. |
10PPM |
0.505 |
%RSD – 1.19% |
|
12 Hrs. |
10PPM |
0.510 |
|
|
24 Hrs. |
10PPM |
0.515 |
|
Table No. 04: Inter day precision
|
Time |
Concentration |
Day 1 |
Day 2 |
Day 3 |
|
0 Hrs. |
10PPM |
0.509 |
0.507 |
0.503 |
|
2 Hrs. |
10PPM |
0.499 |
0.500 |
0.502 |
|
4 Hrs. |
10PPM |
0.501 |
0.505 |
0.501 |
|
8 Hrs. |
10PPM |
0.505 |
0.511 |
0.505 |
|
12 Hrs. |
10PPM |
0.510 |
0.506 |
0.507 |
|
24 Hrs. |
10PPM |
0.515 |
0.509 |
0.511 |
|
|
MEAN |
0.506 |
0.506 |
0.504 |
|
SD |
0.00597 |
0.00377 |
0.00371 |
|
|
RSD |
0.0118 |
0.00745 |
0.00735 |
|
|
%RSD |
1.18 % |
0.75% |
0.74% |
Accuracy [6,36-37]
The accuracy of the proposed approach was tested using recovery experiments at different replicate levels in triplets for 80%, 100%, and 120%. The pre-analysed formulation was mixed with a known amount of pure medication to create the sample solutions, and the mean percent recovery was computed and presented in ACV.
Table No. 5: Accuracy
|
Spiked Level (%) |
Added Concentration |
Measured Concentration |
% Recovery |
|
80 |
8 ppm |
7.9 |
98.8 |
|
100 |
10 ppm |
10.1 |
101 |
|
120 |
12 ppm |
12.2 |
101.7 |
*Result shows mean of 3 readings
Ruggedness [6,38-40]
The analysis was conducted by two independent analysts, and the absorbance of each was noted to determine the percentage RSD. Table No. 06 presents the results.
Table No. 6: Ruggedness
|
Analyst – 1 |
Analyst – 2 |
||
|
Concentration ( PPM ) |
Absorbance |
Concentration |
Absorbance |
|
10 PPM |
0.509 |
10PPM |
0.512 |
|
10 PPM |
0.511 |
10PPM |
0.519 |
|
10 PPM |
0.506 |
10PPM |
0.506 |
|
10 PPM |
0.515 |
10PPM |
0.501 |
|
10 PPM |
0.499 |
10PPM |
0.495 |
|
10 PPM |
0.501 |
10PPM |
0.509 |
|
Average |
0.506 |
Average |
0.507 |
|
SD |
0.00608 |
SD |
0.00841 |
|
RSD |
0.0120 |
RSD |
0.0166 |
|
%RSD |
1.20% |
%RSD |
1.66 |
Robustness [6,41,42]
The robustness of the approach was assessed using three different wavelengths of analysis. The results were displayed in Table No. 07 after the relative absorbance was recorded.
Table no. 07: Robustness
|
Concentration (PPM) |
Wavelength (nm) |
||
|
10 |
249.5 |
251.5 |
253.5 |
|
10 |
0.498 |
0.503 |
0.500 |
|
10 |
0.500 |
0.505 |
0.502 |
|
10 |
0.499 |
0.504 |
0.501 |
|
10 |
0.501 |
0.506 |
0.503 |
|
10 |
0.497 |
0.502 |
0.499 |
|
AVERAGE |
0.499 |
0.5040 |
0.5010 |
|
SD |
0.00158 |
0.00158 |
0.00158 |
|
%RSD |
0.32% |
0.31% |
0.32% |
Limit of Detection (LOD) and Limit of Quantification (LOQ)[6,43,44]
LOQ is the lowest amount of analyte that can be quantitatively determined with reasonable precision and accuracy, while LOD is the lowest amount of analyte that can be detected but not necessarily quantified under specified conditions.
LOD = 3.3×
σ /S LOQ = 10×σ/S
Where, σ = Standard deviation of the response S = Slope of the calibration curve
LOD and LOQ were calculated as 0.098 PPM and 0.297 PPM, respectively.
DISCUSSION
The UV-spectrophotometric approach was developed by scanning ACV oxalate in the UV region between 200 and 400 nm using methanol as the solvent. 243 nm was found to be the maximum wavelength. This approach has been validated in compliance with ICH standards. A number of properties, including as linearity, accuracy, precision, robustness, ruggedness, LOD, and LOQ, are used to assess and determine ACV oxalate. For linearity investigations, several doses between 4 and 20 µg/ml were generated from the same solution, and the linearity curve's R2 value was 0.9997. was observed that the mean % recovery was within the region that supported the accuracy of the method's development. At 80%, 100%, It, and 120%, the accuracy metric was assessed. The LOD and LOQ values were computed using the linearity curve.
RESULT AND CONCLUSION
In compliance with ICH recommendations, a simple UV-spectrophotometric method for ESC has been developed and confirmed. The findings of our study showed that the recommended UV-spectrophotometric method was very sensitive, accurate, and affordable.
in contrast to the methods that have already been published. ACV oxalate in bulk could be determined using the recommended UV spectrophotometric approach.
Conflict of interest
Regarding this article, the authors disclose no relevant conflicts of interest.
REFERENCES
Suyash Gaikwad, Shrikrishna Baokar, Dhananjay Ghodke, Rajendra Patil, Comparative UV–Visible Spectrophotometric Quantification of Acyclovir Using Single-Point Standardization, Absorptivity and Calibration Curve Approaches, , Int. J. of Pharm. Sci., 2026, Vol 4, Issue 3, 3752-3761, https://doi.org/10.5281/zenodo.19281235
10.5281/zenodo.19281235
