The chromatographic conditions for the analysis were as follows: a YMC C18 column with dimensions of 250 × 4.6 mm and a particle size of 5 µm was used. The column temperature was maintained at 25 °C. A sample volume of 10 µL was injected and the flow rate was set to 1 ml/min. Detection was carried out at a wavelength of 254 nm using a Variance Wavelength Detector. The total run time for the analysis was 30 minutes and water was used as the diluent.

6.0 gm of potassium dihydrogen orthophosphate (KH2PO₄) was dissolved in a 1000 ml volumetric flask with distilled water. The pH of orthophosphoric acid (H3PO₄) was adjusted to 3.0, and the final volume was made up with distilled water to 1000 ml, mixed well, and filtered.

Buffer solution and acetonitrile (95:5) were prepared and filtered.

Acyclovir sodium (25 mg) was weighed into a 25 ml volumetric flask with distilled water which served as the stock solution. Five milliliters were transferred from the stock solution into a 50 ml volumetric flask and made up to the final volume with distilled water to 50 mL which served as the standard.

The lyophilised vial was reconstituted with 10 ml of water for injection. Subsequently, 1 ml of this solution was transferred to 50 ml of water. From this solution, 10 ml was transferred and made up to 100 ml with water (the sample solution).

The related substances were determined by HPLC. The unknown impurities and total impurities were determined and recorded.

Figure 0

The total Impurity content was calculated as follows:

Figure 0

The manufacturing process involved washing and sterilizing the vials, lyo stoppers, and aluminum seals under aseptic conditions. The drug was weighed and fresh water for injection was collected, cooled, and purged with nitrogen. The drug was then added to water, stirred, and filtered. The solution was then filled into 10 ml USP Type I tubular flint glass vials, partially stoppered, and loaded into a lyophilizer. After lyophilization, the rubber plugs were stoppered under nitrogen, sealed with aluminum seals, and stored at a suitable temperature.

Conversion factor=Mol. Weight of Acyclovir SodiumMol. Weight of Acyclovir =247.2 225.2=1. 097

Quantity required for batch=Actual quantity×1.097 Quantity for batch=Strength ×100×100Assay value×(100- Water content)=109.8 × 100 ×10099.5 x (100- 5.18) =117.59 mg/ml

The lyophilized product was evaluated for several formulation characteristics, including description, reconstitution time, pH, water content, identification by IR, particulate matter, colour value, light transmission, and assay.

As a primary step, the drug was subjected to pre-formulation studies to check compliance with the USP specification. The drug was found to be a white crystalline powder, which complies with the standard. Solubility was determined, and it was found that the drug was freely soluble in water, slightly soluble in alcohol, and practically soluble in chloroform and ether. The drug was identified by the IR spectrum, which complied with the standard drug spectrum. The assay was carried out by HPLC, which was found to be 99.50%. The related substances guanine and unknown impurities were found to be 0.14% and 0.02% respectively and the total impurity was found to be 0.16%. The water content of the drug was analyzed by the Karl Fischer titration method and was found to be 4.78%. The pH of the solution was found to be 11.3 and the melting point was 2570C. These results are within the USP limits (Table 2 ).

The lyophilization technique was adopted to formulate the parenteral dosage form of acyclovir sodium, with water for injection as a vehicle. The lyophilization was carried out in five different batches by varying the total cycle time, freezing and holding time, primary drying and secondary drying times Acyclovir sodium strength was maintained at 1gm/vial without any cryoprotectant added.

In batch-I, a cycle duration of 41.33 hours was carried out which resulted in bulging of the cake, which is not desirable for a stable and elegant product. The cycle was altered in batch-II by reducing the primary drying duration and increasing the secondary drying duration of the total cycle to 43.33 hours which resulted in melt back, the condition that was characterized by the extreme shrinkage of material that looked like a sticky mass in the container. This results in incomplete drying, a high moisture content, and decreased solubility. Due to melt back in batch-II, the freezing and primary drying duration increased in batch-III with a total cycle duration of 44.91 hours which resulted in layers of cake.

The cycle was further modified in batch IV with a total duration of 46.25 hours. The cake structure was good, but the moisture content was 6.23%, which was above the limits (4% - 6%) Hence, in batch-V the secondary drying duration was increased 6.5 hours to 14.5 hours, with total duration of 51.75 hours. The cake structure was good, and the moisture content was within the specification range (4% -6%). All other evaluation parameters were found to be within the limits and hence the lyo cycle of batch-V with duration of 51.75 hours was selected as optimized cycle with freezing- 9.25 hours, primary drying- 28.00 hours and secondary drying- 14.50 hours (Table 2 ).

Table 2

Preformulation studies data of Acyclovir sodium
Sr. No.	Tests		Standards				Results
1	Description		A White or almost white crystalline powder				White crystalline powder
2	Solubility		Freely soluble in water Slightly soluble in methanol Practically insoluble in Chloroform and Acetone				Passes
3	Melting Point		257°C				Passes
4	Identification by I R		Sample spectrum must be Concordant with the Standard spectrum				Passes
5	Foreign matter		Sample should be free from black particles and foreign matter				Passes
6	pH		Not less than 11				11.3
7	Water content (by KFT)		Between 4 and 6%				4.78%
8	Assay		Between 98.0 and 101.0%				99.50%
9	Related compounds by HPLC Guanine Unknown impurity Total impurity		Not more than 0.5% Not more than 0.1% Not more than 1.0 %				0.14% 0.02% 0.16%
Analysis of Acyclovir sodium formulation
Evaluation parameters		Batch-I		Batch-II	Batch-III	Batch-IV		Batch-V
Appearance		White in colour		White in colour	White in colour	White in colour		White in colour
Moisture content %		6.10%		5.56%	5.86%	6.23%		4.75%
Cake formation		Bulging of cake		Melt back	Cracking of cake	Good cake formation		Good cake Formation
Reconstitution time(sec)		24 sec		21 sec	23 sec	19 sec		16 sec
pH(initial)		11.03		11.12	10.99	11.30		11.31
pH (After 24 hours)		11.01		11.09	10.97	11.29		11.34
Assay		97.50%		98.32%	98.12%	99.90%		100.50%
Color value (At 430nm)		0.0213		0.0298	0.0193	0.0198		0.0195
Light transmittance (At 630nm)		98.21%		98.61%	96.96%	98.95%		99.56%
Particulate matter ≥10µm: Not more than 6000/vial ≥25µm: Not   more   than 600/vial		3665		4021	4121	4900		4201
		348		398	438	468		235

Evaluation of the lyophilized product across the five batches revealed that the lyophilized cakes in all batches were white, as shown in Table 2 . The moisture content, a critical factor, was found to be within the acceptable range of 4%-6% for batches I (6.10%), II (5.56%), III (5.86%), IV (6.23%), and V (4.75%), indicating that all products had moisture content within the limits. The cake formation varied: batch I exhibited bulging, batch II showed melt back, and batch III had layers of cake, whereas batches IV and V showed complete cake formation without issues such as sticking or melting back. The reconstitution times for batches I, II, and III were 21-24 seconds, whereas those for batches IV and V were shorter at 19 seconds. The pH of the reconstituted products in all batches was within the USP limits of 11-11.5, indicating no stability problems, such as hydrolysis and esterification. The assay results, determined by HPLC, were within the USP specification range of 97.50%-100.50%. Additionally, the colour value and % light transmittance, assessed using the UV method, were within the USP limits for all batches. Based on these results, batches IV and V were identified as good formulations. Given that cake formation plays a crucial role in the final selection of the formulation, complete and stable cakes in batches IV and V were significant. Furthermore, moisture content is important for preventing chemical reactions and microbial growth, with batch V having the lowest moisture content compared to batch IV. The reconstitution time was also shorter in batch V. Therefore, batch V was selected as the optimized formulation and subjected to further studies including particulate matter determination and differential scanning calorimetry.

Particulate matter analysis was performed for the batch-V formulation, and the particulate matter was found to be 4201 (10 µm) and 235 (25µm). The results showed that the values were within the limits of the USP.

Differential scanning calorimetry (DSC) was performed on the lyophilized drug product at a ramp rate of 5ºC/min from -60ºC to 60 °C. The DSC thermogram of the acyclovir sodium liquid formulation before lyophilization exhibited an endothermic peak at -32.03ºC, indicating its eutectic point. The optimized lyophilized product, reconstituted with 10 mL of WFI, showed an endothermic peak at -33.37 ºC, demonstrating no change in the eutectic point. This result confirmed that the crystalline nature of the drug was preserved after lyophilization.

Stability studies were performed in accordance with ICH guidelines at 40 °C ± 2 °C and 75% RH ± 5% RH. The optimised lyophilised formulation was analysis at one-month intervals over a period of three months. The evaluated parameters included the colour of the product, reconstitution time, pH, assay, particulate matter, colour value, and percentage light transmittance, as summarised in Table 2. After the first month, the colour of the product remained consistent with the initial formulation, with no observable colour changes. The reconstitution time was 17 seconds, and the pH was 11.35, showing no significant deviations from the initial values. HPLC assay results indicated that the formulation remained within the USP limits. The colour value and percentage light transmittance, determined via UV methods, were 0.0196 and 99.58%, respectively, and were also within the USP limits. Particulate matter counts were recorded as 4224 for particles ≥10 µm and 239 for particles ≥25 µm, indicating that the product was stable after one month. During the second and third months, the appearance of the product remained unchanged and no colour variations were observed. All the evaluation parameters continued to meet the specified limits, as shown in Table 2 .

The results confirm that the product maintains stability under accelerated conditions, supporting an expected shelf-life of two years for batch-V. Based on these findings, it is clear that batch-V was effectively optimised as the target lyophilised product of acyclovir sodium IV injection (Table 3 ).

We observed superior stability compared to existing marketed formulations, thereby achieving the goal of developing a stable and long-lasting lyophilised acyclovir sodium IV injection.

Batch-V, with a duration of 51.75 hours, was chosen as the best formulation due to its good cake formation, pH, particulate matter, water content, colour value, and light transmission being within USP limits. Stability studies confirmed the stability of the formulation, indicating that lyophilisation is advantageous for developing a stable injectable dosage form of acyclovir sodium.