INTRODUCTION:

Acyclovir (ACY) is a nucleoside analog with spectrum of antiviral activity against the herpes group of viruses^1-2. ACY (9-[(2-hydroxyethoxy) methyl] guanine, zovirax) is a white, crystalline powder which is slightly soluble in water. ACY is chemically known as 2-amino-1,9-dihydro-9- [(2-hydroxyethoxy) methyl]-6H-purine-6-one, or 9- [(2-hydroxyethoxy) methyl]- guanine. Its molecular formula is C8H11N5O3, and molecular weight 225.21g/mol³.

Intravenous, oral and to some extent topical formulations are an established dosage forms of ACY for therapeutic uses^1-3. The drug offered significant clinical benefit in the treatment and prophylaxis of initials and recurrent episodes of genital and orofacial herpes simplex infection. Moreover, acyclovir is therapeutically effective against the progression of herpes zoster causing varicella (chickenpox) in immunocompetent individuals^1,4.

The mechanism of action of ACY involves competitive inhibition of viral DNA polymerase or inhibition of viral DNA replication by incorporating into the replicating DNA chain⁴

To date, various immunological techniques and high-performance liquid chromatography (HPLC) methods have been established for determination of ACY in biological samples⁵. However, structural similarity of acyclovir with endogenous substances complicates its analysis in human serum.ACY is a polar compound and practically insoluble in most of organic solvents which imposes difficulty in ACY’s solvent extraction⁶. The previously published analytical methods involves use of an ion pairing agent⁷ and column thermostating and fluorometric detection⁸ for ACY analysis. Moreover, reported analytical methods failed to meet the current International Conference on Harmonization guidelines Q1A (R2) (ICH, 2003)⁹and regulatory requirements.

A detailed literature survey did not identify specific, robust and stability-indicating HPLC method development in a QbD environment for ACY. Hence, a systematic approach is desirable for development of an ideal stability indicating chromatographic method using QbD approach^10-12, which helps in the determination of ACY and ensure the quality of drug.

We develop herein for the first time, an accurate, specific and reproducible HPLC method for the determination of ACY that could be applicable in presence of degradation products and stress degradation kinetics. An attempt was made to develop a new specific HPLC method for ACY by using QbD approach.

MATERIALS AND METHODS:

Chemicals, reagents and solutions:

All the reagents as HPLC grade Water and Methanol (HPLC grade), were purchased from Merck Chemicals, India. Reference standard Acyclovir was procured Lupin Limited, Pune (India), as gift samples. Potassium dihydrogen orthophosphate, tetra butyl ammonium hydroxide (TBAH) and o-phosphoric acid, hydrochloric acid, sodium hydroxide, and 30% hydrogen peroxideused were of analytical grade and purchased from S D Fine Chem. Ltd. (Mumbai, India). Buffer was prepared by dissolving 1360mg (10mM) of potassium dihydrogen orthophosphate and 3330mg of TBAH (10mM) in 1 L of HPLC grade water.

HPLC instrumentation and chromatographic procedure:

The HPLC system consisted of two pumps (Analytical Technologies P2230 HPLC pump), a manual injector with 20μL capacity per injection, and a temperature-controlled column oven. The UV–VIS detector (Analytical Technologies UV 2230) was operated at a wavelength of 254nm. The software used waschromatography workstation A-2000, version 1.6. Columns used were Lichrospher C 18, 250 mm 4.6mm, 5.0ml (Merck, Germany), Atlantis C 18, 250mm 4.6 mm, 5.0 lm (Waters Corporation, USA) and Alltima C-8, 250mm 4.6mm, 5.0lm (Grace, USA). Chromatographic separation of RITO was achieved at ambient temperature using a Lichrospher RP C18 (250mm 4.6mm, 5lm) analytical column; the mobile phase consisted of methanol–potassium dihydrogenorthophosphate (pH 3.8; 10mM, tetra butyl ammonium hydroxide; 10mM) (40:60, v/v) at a flow rate of 1.0mL/min. pH of buffer was adjusted with o-phosphoric acid. Before use, the mobile phase was filtered through a 0.22μm nylon membrane filter and sonicated for 15min. Injection volume was 20μL, and the optimum wavelength selected for quantification was 254nm. Photo stability Chamber Model: CS-90 (GMP) internal diameter 50x40x85cm. Design Expert Software Experimental using Statistical (Version 10.0.4.0) Design Wizard- Optimization- factorial/RSM-HTC study type response surface, design type-centric.

Preparation of standard solutions:

A standard ACY stock solution was prepared in methanol at a concentration of 10mg mL^-1. Working standard solutions were prepared freshly before use with a suitable dilution of mobile phase of 100μg mL^-1 for achieving appropriate concentration levels.

Construction of the calibration curve:

The working standard stock solution was diluted with the mobile phase to prepare calibration samples in the concentration range of 0.5–100 lg mL^-1. Triplicate injections of 20 lL were made for each calibration sample and chromatographed under the specified HPLC conditions decayed previously. Peak areas were plotted against the corresponding concentration to obtain the calibration curve.

Forced degradation of ACY:

Forced degradation studies (FDS) were employed to ensure the specificity of and suitability of proposed analytical method. The current stress testing includes evaluation of the effect of oxidizing and reducing agents, temperature and light on chemical behaviour of ACY.These samples were further subjected to the stress conditions as mentioned in (Table 1).

Sample collection:

Before collecting samples, the volume was made up to the mark with respective solvent. Sample (200 lL) was collected at specified sampling points as indicated in Table 1. The samples from acid and base induced degradation were neutralized by adding 200 lL of appropriate strength of NaOH and HCl. All samples were stored at 2–8⁰C in the refrigerator. On the day of analysis samples were diluted with the mobile phase up to 10mL, filtered with a 0.22 lm membrane syringe filter and injected three times for each sample into HPLC.

Table 1: Hydrolytic, oxidative, thermal and photolytic stress testing conditions for drugs

Stress condition	Solvents	Temperature 0 C	Time (days)	Sampling time (days)
Hydrolytic	H₂O	60	25	1,2,4,8,10,15,20
Neutral	2 N HCL	60	30	1,2,4,8,10,15,20
Acidic	5 N HCL	60	30	1,2,4,8,10,15,20
Basic	2N NaOH 5N NaOH	60	30	1,2,4,8,10,15,20
Oxidizing	3% H₂O₂	60	30	1,2,4,8,10,15,20
thermal	10% H₂O₂	Room temp.	7	1,4,8,20
Moist heat	Methanol	Room temp.	7	1,4,8,20
Dry heat	Methanol	60	14	1,2,4,8,10,15,20
Photolytic	Methanol	60	14	1,2,4,8,10,15,20
Direct sunlight	Methanol	-	25	1,2,4,8,10,15,20

Table 2. Factors and level used in the experimental design

Factor	Level (-1)	Level (0)	Level (+1)
TBAH (mM)	2.5	4.5	6.5
pH	4.4	4.5	4.6
Organic phase	15	20	25

Mean+- SD. RSD (%), n =

System suitability and Method validation parameter:

Retention time (Rt), capacity factor (k), number of theoretical plates (N) and tailing factor (T), were calculated for six replicate injections of the drug at a concentration of 60μg mL^-1. The values presented in (Table 4) are within the acceptable limits and demonstrated adequate performance of the applied chromatographic system.

Linearity of the proposed method was verified over the concentration range of 10-80μg mL^-1, with coefficient of determination was r² = 0.999. The regression equation was found to be Y= 12.50X+1.785, where Y is peak area and X is concentration of ACY (μg mL^-1).

The limit of detection (LOD) was defined as the lowest concentration of ACY resulting in a signal-to-noise ratio of 3:1 and limit of quantification (LOQ) was expressed as a signal-to noise ratio of 10:1. Due to the difference in detector response, different concentrations ranging from 0.01 to 2 lg mL_1 were prepared and analyzed. The LOD and LOQ obtained were 0.3 and 0.9 lg mL_1, respectively.

Accuracy of the method was determined in triplicate at three known different concentration levels (80%, 100% and 120%) and the percentage recovery was calculated. The mixture of degraded sample was fortified with known concentration of drug at 80%, 100% and 120% levels. Peak area of the standard was measured as a difference of peak area between fortified and unfortified samples. Three replicate samples of each concentration level were prepared and the percentage recovery at each level (n = 3) was determined (Table 4). For ACY, the recovery standard is 100 % at all three levels indicating the accuracy of proposed method.

The analysis of within- and between-day variance was determined by injecting four different concentrations (10, 40, 60, and 80μg mL^-1) of ACY. For the evaluation of intra-day precision, sets of six replicates of the four concentrations were analysed on the same day. The inter-day variation was calculated from the data generated from six replicates analysed on six different days. The intra-day and inter-day coefficient of variation and percentage error was found to be less than 2% (Table 5), suggesting that the method was precise.

To investigate the robustness of present method, small changes were purposely done including variation of C18 columns from different manufacturers, pH of the buffer, flow rate and percentage of methanol in the mobile phase. Robustness of method was verified by using two analytical columns, namely Lichrospher C 18 column (Germany) and Atlantis C 18 column (USA). Each of the three examined factors (pH, flow rate, and methanol percentage) selected was changed one at a time to estimate the effect. Replicate injections (n = 6) of standard solution (60 lg mL^-1) were performed under small changes of chromatographic parameters(factors). Flow rate was varied by 1±0.1mL min^-1; level of methanol in the mobile phase was varied by 25±2% (v/v), while pH was varied by 2.8±0.1. The observed results are presented in (Table 6), indicating that the results remained unaffectedby small variations of these parameters. The results from the two columns indicated that there is no significant difference between the results from the two columns.

Table 3 System suitability data

Property	Mean ± SD, n=6	RSD (%)	Required limits
Retention time (Rt)	8.31 ± 0.03	0.43	RSD≤ 2
Capacity factor (k)	2.04 ± 0.002	0.18	-
Theoretical plates (N)	30126 ±184	0.53	N ≥ 2000
Tailing factor (T)	1.36 ± 0.01	1.52	T ≤ 2

Table 6. Results for the analysis of robustness

Factors	level	Retention Mean ± SD (n=6)	Time (min) Mean ± SD (n=6)	Peak area Mean ± SD (n=6)
A Flow rate (ml/min)
0.9	-1	8.33 ± 0.02	1.41 ± 0.03	5441.28 ±,1.27
1.0	0	8.31 ± 0.02	1.30 ± 0.01	5452.21 ±1.27
1.1	+1	8.32 ± 0.03	1.22 ± 0.01	5447.27 ±1.27
Mean		8.32 ± 0.02	1.34 ± 0.02	5449.39 ±1.27
B percentage of methanol in the mobile phase (v/v)
23	-1	8.32 ± 0.01	1.32 ± 0.03	5381.21 ±1.24
25	0	8.31 ± 0.02	1.31 ± 0.01	5498.79 ±,1.19
27	+1	8.33 ± 0.03	1.31 ± 0.01	5471.29 ±,1.57
Mean		8.32 ± 0.02	1.31 ± 0.02	5414.79 ±1.39
C pH of buffer
2.7	-1	8.30 ± 0.02	1.27 ± 0.03	5461.29 ±1.27
2.8	0	8.33 ± 0.02	1.24 ± 0.02	5461.29 ±15.31,1.27
2.9	+1	8.32 ± 0.03	1.26 ± 0.01	5461.29 ±15.31,1.27
Mean		8.32 ± 0.02	1.25 ± 0.02	5460.39 ±,1.27
D: columns from different manufactures
I Lichrospher C 18 column		8.33 ± 0.02	1.22 ± 0.03	5421.45 ±1.17
II Atlantis C18 column		8.30 ± 0.02	1.28 ± 0.02	5434.21 ±15.31,1.32
Mean		8.32 ± 0.03	1.24 ± 0.01	5477.23 ±1.37

Table 7. Summary of drug degradation kinetics

Stress condition	K (day ^-1)^a	t _½(days)^b	t₉₀(days)^c	Degraded (%)
2 N NaOH	3.43 ×10 ^-3	195.77	31.89	1.45
5 N NaOH	13.03 ×10 ^-3	45.33	5.89	17.30
2N HCL	5.85 ×10 ^-3	101.21	18.54	07.04
5N HCL	14.73 ×10 ^-3	34.71	4.87	21.90
3% H₂O₂	18.13 ×10 ^-3	69.32	7.64	1.09
10% H₂O₂	63.45 ×10 ^-3	11.09	1.98	23.91
Dry heat	23.69 ×10 ^-3	21.75	2.89	23.08
Moist heat	28.40 ×10 ^-3	25.98	3.87	25.12
Hydrolysis	17.43 ×10 ^-3	32.09	6.02	29.08
photolysis	19.83 ×10 ^-3	31.89	5.34	19.91

a Rate constant per day, _b Half –life ,_cTime left for 90% potency

DISCUSSION:

Method development and optimization:

The current QbD work was conducted to evaluate the method performance and effect of various variables on method response. In order to reduce the base line noise at absorption maximum (208nm) of ACY, optimum wavelength of 220nm was selected. Considering the solubility of ACY, methanol was chosen as organic phase. To start with, reversed-phase analytical columns (C18 and C8) were tested with mobile phase composed of variable composition of methanol (80–20% v/v) and water. Subsequently, water was replaced with buffer (10 mM potassium dihydrogen orthophosphate) at different pH levels in between 2.5 to 6.0 with a flow rate of 1 mL min^-1. In the applied conditions, ACY failed to get any capacity factor (k). ACY was eluted along with the mobile phase, i.e. the retention volume was equal to void volume. Based on this elution behavior of ACY, we applied ion pair methodology, using TBAH as an ion pair agent at the concentration of 10mM. The pH of the buffer (10mM potassium dihydrogen orthophosphate) was adjusted to 3.2 with o-phosphoric acid, which is more than two units below the pKa (6.2) to ionize ACY by 100%. Keeping above conditions in consideration, the mobile phase was selected which is composed of methanol: buffer at the 30:70 ratio eluted ACY through the C18 stationary phase (Lichrospher, RP C18, 250mm · 4.6mm, 5 l) having a k of 1.44. The pH of buffer was varied between 2.6 and 3.2, level of methanol was varied between 20% and 30% v/v and TBAH concentration was varied between 5 and 10mM. In total, 27 experiments were performed using the full factorial design (3 factors, 3 levels, 27 runs). This help to rationally examine the effects of TBAH concentration, buffer pH and organic phase concentration on the capacity factor of ACY. Experimental factors and levels used in the experimental design are presented in Table 2. The factors and ranges selected for consideration were based on previous conducted univariate studies and chromatographic intuition. The statistical analysis of the data generated was performed with Statistical (Version 6.0) software.

(Figures.3) showed the normal probability plot of the residuals and the plot of the residuals versus the predicted response for capacity factor. (Figure.4) reveals that the residuals generally fall on a straight line and this suggest that the errors are normally distributed. This supports the fact that the model fits the data adequately. These plots are usually required to check the normality assumption in a fitted model, ensuring that the model provides an adequate approximation to the optimization process. This clearly indicated that there is no obvious pattern followed in the residual versus predicted response. The plot showed an almost equal scatter above and below the X-axis, and suggests the proposed model is adequate.

The optimized chromatographic conditions obtained from the design were mixture of 10 mM potassium dihydrogen orthophosphate (pH 2.8) containing 10 mM TBAH and methanol (25:75, v/v), at a flow rate of 1.0 mL min_1. These chromatographic conditions achieved reasonable retention (k= 2.06) and symmetric peak shape for ACY with a retention time of 7.05 min (Figure. 2). No interference from the blank and cream formulation excipients was observed at the retention time of ACY (Figure. 2). Percentage of recovery (n= 6) obtained from the formulation was 100.5 ±1.8.

Kinetic investigation:

When subjected to the stress studies, ACY exhibited gradual decomposition in all specified stress conditions. The degradation of ACY was performed using a large amount of solvent (9 mL) compared to drug solution (1 mL). The degradation of ACY followed pseudo-first-order kinetics as shown by a linear relationship between log percentage of ACY remaining and time was established with good correlation coefficients (Figure.5–7). Pseudo-first-order kinetic reveals the involvement of two reactants in the reaction with one of them present in a large amount. Any change in its concentration is negligible in comparison to the change in concentration of the other reactant (drug).

The observed kinetic parameters are depicted in (Table 7).

Rate constant (K), time left for 50% potency (t1/2) and time left for 90% potency (t90) for each stress condition were calculated using Eqs. (1)– (3), respectively:

where K is the rate constant, [C0] is the concentration of ACY at time t= 0 and [Ct] is its concentration at time t.

The K values per day were found to be 3.48 · 10_3,

19.18 · 10_3, 6.78 · 10_3, 19.84 · 10_3, 12.14 · 10_3,

63.79 · 10_3, 30.69 · 10_3, 23.53 · 10_3, 20.20 · 10_3 and

21.72 · 10_3 for 2 N NaOH, 5 N NaOH, 2 N HCl, 5 N HCl,

3% H2O2, 10% H2O2, dry heat, moist heat, water hydrolysis and photolytic conditions, respectively.

The kinetic behaviour of the system can be explained by using equations:

The rate constant values were increased with increase in the strength of NaOH, HCl and H2O2. The rise in K value was approximately six times when the strength of NaOH was increased from 2 N to 5 N. At the same time, the rate of degradation was increased up to approximately three times under the same conditions of acid (HCl) treatment, suggesting more susceptibility of ACY under basic media compared to acidic. The K value for water degradation was almost similar to the degradation induced by 5 N HCl and 5 N NaOH, indicating the importance of water in ACY degradation. In thermal treatments, K value for dry heat was reported to be higher compared to moist heat. Extensive degradation taken place in oxidative conditions, where K value was higher among all the tested conditions. This indicates the effect of oxygen during topical formulation of ACY. K value obtained for photolytic degradation was similar to water hydrolysis, 5 N NaOH and 5 N HCl, suggesting the significant impact of light toward the stability of ACY. Table 7 showed t _1/2 and t ₉₀ values for all the tested stress conditions and were found to be lowest. (Figure 7,8), first order plots for the degradation of ACY under hydrolytic and photolytic stress conditions (each point represents the mean ± SD, n=3). Quality by Design (QbD) approach to develop HPLC method for Acyclovir: Application S321 and highest (198.99 and 30.24 days) for alkaline hydrolysis with 2 N NaOH.

Figure 1: Typical chromatogram of (RT 8.31 min)

Figure 2: Perturbation (a) the effect of each of the independent factors on Rt of ACY, while keeping other factors at their respective mid-point levels (A; pH = 4.5; B: flow rate =1.0ml/min C % MeOH = 50 % v/v)

Figure 3: Graphic representation of the overall desirability function D Flow Rate (B) is plotted against pH (A) with MeOH % v/v (C) held constant at 60 % v/v.

Figure 4: contour plot for capacity factor as a function of TBAH concentration and organic phase for ACY

Figure 5: first order plots for the degradation of ACY under acidic and basic stress condition (each point represents the mean±SD, n=3)

Figure 6: First order plots for the deradation of ACY under thermal stress conditions (each point represents the mean ± SD, n=3)

Figure 7: First order plots for the degradation of ACY under oxidative stress condition (each point represents the mean±SD, n=3)

Figure 8. First order plots for the degradation of ACY under hydrolysis and photolytic stress conditions (each point represents the mean±SD, n=3)

CONCLUSION:

In summary, the proposed HPLC method for ACV is simple and accurate. The developed method exhibited acceptable system suitability, linearity, accuracy, precision and specificity.

It offers distinguishable benefits over other established analytical methods in terms of providing reproducible and accurate results in the presence of ACY’s degradants. ACY was noted to rapidly degraded under oxidative, hydrolytic (acid and alkali) and photolytic conditions. The degradation of ACY was found to be of pseudo-first-order kinetics in analyte’s concentration. The reaction rate increases with increase in strength of the acid/base/H₂O₂solution. The proposed method can be used for ACY analysis in quality control unit to advance pharmaceutical development.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 02.03.2022 Modified on 07.08.2022

Research J. Pharm. and Tech 2023; 16(8):3691-3696.

DOI: 10.52711/0974-360X.2023.00607

Level of standard added (%)	Amount of sample std added (µg)	Sample	Mean peak area ± SD, RSD (%) n=3		Amount of standard found (µg)	Recovery for standard (%)
Level of standard added (%)	Amount of sample std added (µg)	Sample	Standard + Sample	Standard	Amount of standard found (µg)	Recovery for standard (%)
80	40	5222.29 ±15.31,1.07	6202.29 ±23.31,1.83	610.09 ±23.31,1.03	40.05	100.02
100	50	5461.29 ±15.31,1.27	6431.29 ±25.31,1.23	629.91 ±25.31,1.16	50.02	100.3
120	60	5672.29 ±15.31,1.10	6852.29 ±27.31,1.08	638.35 ±27.31,1.24	60.04	100.03

Concentration µg/ml	Intra- day precision	RSD (%)	Inter-day precision	RSD (%)
	Peak area		Peak area
	Mean ± SD (n=6)		Mean ± SD (n=6)
10	5461.29 ±12.21,1.62	1.89	5001.19 ±12.34,1.29	1.29
20	6628.23 ±12.43,1.22	1.56	6208.10 ±12.97,1.10	1.77
40	7629.12±15.29,1.67	1.64	7602.11±15.29,1.43	1.19
60	9738.09 ±12.33,1.29	1.32	9298.09 ±12.63,1.32	1.28