First Acyclovir Determination Procedure via Electrochemically Activated Screen-Printed Carbon Electrode Coupled with Well-Conductive Base Electrolyte

In this work, a new voltammetric procedure for acyclovir (ACY) trace-level determination has been described. For this purpose, an electrochemically activated screen-printed carbon electrode (aSPCE) coupled with well-conductive electrolyte (CH3COONH4, CH3COOH and NH4Cl) was used for the first time. A commercially available SPCE sensor was electrochemically activated by conducting cyclic voltammetry (CV) scans in 0.1 mol L−1 NaOH solution and rinsed with deionized water before a series of measurements were taken. This treatment reduced the charge transfer resistance, increased the electrode active surface area and improved the kinetics of the electron transfer. The activation step and high conductivity of supporting electrolyte significantly improved the sensitivity of the procedure. The newly developed differential-pulse adsorptive stripping voltammetry (DPAdSV) procedure is characterized by having the lowest limit of detection among all voltammetric procedures currently described in the literature (0.12 nmol L−1), a wide linear range of the calibration curve (0.5–50.0 and 50.0–1000.0 nmol L−1) as well as extremely high sensitivity (90.24 nA nmol L−1) and was successfully applied in the determination of acyclovir in commercially available pharmaceuticals.


Introduction
There are many viruses from the Herpesviridae family, known around the world, which infect many living organisms-including humans.These viruses belong to the group of viruses made of double-stranded DNA and are highly specific to the host.There are two types of herpes viruses currently known to attack the human body-Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2).These viruses differ in terms of the place where the symptoms of infection appear.The herpes virus manifests itself as painful red blisters on various areas of the body, usually on the lips or genitals.Viruses from this family have the ability to cause latent infections.Under the influence of certain stimuli, HSV can be activated and, after an infection, go into a hibernation state.In the case of HSV, the infection is lifelong and there is currently no vaccine against Herpes simplex.In order to shorten the duration of the infection, antiviral drug therapy is often involved.The use of such medicaments slows down the process of skin eruption and reduces contagiousness.According to the literature, 70-90% of the human population are serologically positive for HSV type 2. In addition, some sources have revealed some connections between HSV and Alzheimer's disease.This means that the problem is global and, to this day, both vaccines and chemical compounds that help treat HSV are being sought.Some of the well-known antiviral drugs which help in the fight against HSV are ganciclovir (GCV), penciclovir (PCV) and acyclovir (ACY)-the last of which will be further discussed [1][2][3][4][5].
Acyclovir (ACY) (specifically 2-amino-9-[(2-hydroxyethoxy)methyl]-3.9-dihydro-6Hpurin-6-one) is a guanine analogue that is an antiviral drug.It is valued for its high as extremely high sensitivity (90.24 nA nmol L −1 ), and it was successfully used in the determination of ACY in commercially available pharmaceuticals.
Table 1.ACY voltammetric determination procedures with the lowest limit of detection (LOD) value described in the literature.

SPCE Activation Procedure
Each series of measurements (after replacing the basic electrolyte with a new one) were started with SPCE (150) electrochemical activation.This process involved 5 cyclic voltammetry scans between 0 and 2 V (scan rate = 100 mV s −1 ) in a 0.1 mol L −1 solution of NaOH.After this stage, the obtained aSPCE sensor was thoroughly rinsed with deionized water ready for a new analysis.
Electrode activation solution was made by mixing the appropriate amount of NaOH (Merck; Darmstadt, Germany) with deionized water.Tablets containing acyclovir (200 mg per tablet) from two different manufacturers were purchased at a local pharmacy.Sigma-Aldrich (St. Louis, MO, USA) MS-grade nitric acid appropriately diluted with distilled water was used for sample preparation.

Acyclovir and Interferent Standards Preparation
All acyclovir standard solutions (1 mmol L −1 , 0.1 mmol L −1 as well as 0.01 mmol L −1 ) were prepared by dissolving ACY white powder (Sigma-Aldrich; St. Louis, MO, USA) in deionized water stored in a refrigerator and sonicated in an ultrasonic bath daily for a couple of minutes before use.The 1 mmol L −1 solution was prepared once every 3 weeks, in contrast to the 0.1 and 0.01 mol L −1 standards which were unstable in the long term.To avoid further complications, these solutions were prepared daily.

Pharmaceuticals Preparation
Tablets containing ACY from two different manufacturers were prepared in the same way.Three tablets were weighed and mortared homogeneously.Next, the mass corresponding to the average mass of the three previously weighed tablets was quantitatively transferred into 200 mL volumetric flask and filled with 0.1 mol L −1 HNO 3 solution to the mark.Then, a volumetric flask was placed in an ultrasonic bath for one hour.Finally, the obtained extract was filtered with a 0.22 µm Millipore filter.

Acyclovir DPAdSV Analysis
Differential-pulse adsorptive stripping voltammetry (DPAdSV), aSPCE and the CH 3 COONH 4 , CH 3 COOH and NH 4 Cl solution were used for ACY determination under optimized conditions.Firstly, an electrochemical cleaning step was involved by applying a potential of 1.4 V for 5 s onto the working electrode, while stirring the solution.Next, ACY was accumulated on the aSPCE by stirring the base electrolyte for 60 s (t acc. ) and applying a potential of −0.1 V (E acc.).Finally, the DPAdSV voltammogram was registered from 0.5 to 1.4 V with the following technique parameters: amplitude (∆E A ) of 150 mV, a scan rate (ν) of 250 mV s −1 , modulation time (t m ) of 6 ms and 5 ms equilibrium time.The baseline was corrected for each voltammogram and the background was subtracted as well.

Selection of the Sensor and Influence of Electrochemical Activation on ACY Signal
At the first stage of our research, an optimal sensor was established.For this purpose, differential-pulse voltammograms (DPV) were recorded in the presence of increasing concentrations of ACY (1, 3 and 5 µmol L −1 ) under initial conditions (0.075 mol L −1 PBS pH = 6.8,DPV parameters: ∆E A of 125 mV, ν of 175 mV s −1 and t m of 10 ms) at the following electrodes: SPCE (110), SPCE (150), (SPCE/CNFs), (SPCE/GPH), (SPCE/MWCNTs) and (SPCE/SWCNTs).Figure 1A shows a comparison of the signals registered at these electrodes (1 µmol L −1 of ACY).Measurements taken at SPCE/MWCNTs indicate no signal increments corresponding with standard additions.The current signals of ACY obtained with SPCE/CNFs and SPCE/GPH are at the noise level.However, in the case of the other sensors, a 1 µmol L −1 acyclovir signal was visible at the following potentials and peak current intensity: 0.94 V, 1.1 nA SPCE (110); 0.85 V, 2.9 nA SPCE (150) and 0.90 V, 1.38 nA (SPCE/SWCNTs).The highest signal (2.9 nA) was obtained at the SPCE (150); therefore, this sensor was chosen for further experiments.
a potential of 1.4 V for 5 s onto the working electrode, while stirring the solution.Nex ACY was accumulated on the aSPCE by stirring the base electrolyte for 60 s (tacc.)an applying a potential of −0.1 V (Eacc.).Finally, the DPAdSV voltammogram was registere from 0.5 to 1.4 V with the following technique parameters: amplitude (∆EA) of 150 mV, scan rate (ν) of 250 mV s −1 , modulation time (tm) of 6 ms and 5 ms equilibrium time.Th baseline was corrected for each voltammogram and the background was subtracted well.

Selection of the Sensor and Influence of Electrochemical Activation on ACY Signal
At the first stage of our research, an optimal sensor was established.For this purpos differential-pulse voltammograms (DPV) were recorded in the presence of increasing co centrations of ACY (1, 3 and 5 μmol L −1 ) under initial conditions (0.075 mol L −1 PBS pH 6.8, DPV parameters: ∆EA of 125 mV, ν of 175 mV s −1 and tm of 10 ms) at the followin electrodes: SPCE (110), SPCE (150), (SPCE/CNFs), (SPCE/GPH), (SPCE/MWCNTs) an (SPCE/SWCNTs).Figure 1A shows a comparison of the signals registered at these ele trodes (1 μmol L −1 of ACY).Measurements taken at SPCE/MWCNTs indicate no sign increments corresponding with standard additions.The current signals of ACY obtaine with SPCE/CNFs and SPCE/GPH are at the noise level.However, in the case of the oth sensors, a 1 μmol L −1 acyclovir signal was visible at the following potentials and peak cu rent intensity: 0.94 V, 1.1 nA SPCE (110); 0.85 V, 2.9 nA SPCE (150) and 0.90 V, 1.38 n (SPCE/SWCNTs).The highest signal (2.9 nA) was obtained at the SPCE (150); therefor this sensor was chosen for further experiments.In order to improve the sensitivity, SPCE (150) was electrochemically activated in strongly alkaline medium (0.1 mol L −1 NaOH) in accordance with the procedure alread described in our previous studies [12,13].In the article [13], the changes in the surfa morphology and electrochemical properties of the SPCE (150) before and after electr chemical activation (pre-anodization) were examined using scanning electron microscop In order to improve the sensitivity, SPCE (150) was electrochemically activated in a strongly alkaline medium (0.1 mol L −1 NaOH) in accordance with the procedure already described in our previous studies [12,13].In the article [13], the changes in the surface morphology and electrochemical properties of the SPCE (150) before and after electrochemical activation (pre-anodization) were examined using scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV).The obtained results showed the numerous advantages of subjecting the SPCE to the activation process, including a significant increase in the active surface of the working electrode, a decrease in the charge-transfer resistance and improvement of the kinetics of electron transfer.In addition, SEM imaging showed that the electrode gained a highly porous structure as a result of electrochemical activation in a strongly alkaline medium.These factors have a direct influence on the acyclovir signal, which is shown in Figure 1B.The acyclovir signal obtained with the activated sensors (blue line) is wider, better shaped and 2.8 times higher in comparison with the signal obtained at the non-activated electrode (black line).

Supporting Electrolyte Composition-Impact of Concentration and pH Value
In order to optimize the composition of the basic electrolyte, the measurements were performed in the presence of three ACY additions ( 2A).This is associated with the improvement of the conductivity of the supporting electrolyte, which was confirmed in our previous work [15].For this reason, the next step relied on examination of the influence of pH (Figure 2B) as well as the concentration of the buffer solution (Figure 2C) on the ACY signal.Finally, a 0.075 mol L −1 solution of CH 3 COONH 4 , CH 3 COOH and NH 4 Cl with pH = 4.2 was established as the best choice and used for further studies.
2.8 times higher in comparison with the signal obtained at the non-activated electrode (black line).

Supporting Electrolyte Composition-Impact of Concentration and pH Value
In order to optimize the composition of the basic electrolyte, the measurements were performed in the presence of three ACY additions (1, 3 and 5 μmol L −1 ) in 0.075 mol L −1 (pH = 4.2) PBS, acetate buffer (CH3COOH/CH3COONa) and (CH3COONH4, CH3COOH and NH4Cl) solution.The obtained results indicate that the highest peak current intensity (Ip) of ACY for every acyclovir standard addition is with the new electrolyte composition (CH3COONH4, CH3COOH and NH4Cl) (Figure 2A).This is associated with the improvement of the conductivity of the supporting electrolyte, which was confirmed in our previous work [15].For this reason, the next step relied on examination of the influence of pH (Figure 2B) as well as the concentration of the buffer solution (Figure 2C) on the ACY signal.Finally, a 0.075 mol L −1 solution of CH3COONH4, CH3COOH and NH4Cl with pH = 4.2 was established as the best choice and used for further studies.

Acyclovir Electrochemical Behavior
For the next step, a series of cyclic voltammograms at different scan rates (from 5 to 150 mV s −1 ) were recorded in the presence of 0.1 mmol L −1 ACY under optimized supporting electrolyte conditions.Increasing the scan rate affects onto the ACY peak potential, which shifts slightly towards more positive values (a  c), as well as peak current intensity (Ip) values, which increase slightly (Figure 3A).There is no visible reduction peak, which is evidence of the irreversible nature of the examined process.On the basis of relationships between peak current intensity (Ip) and square root of scanning rate (ν 1/2 ) (there is a linear course of that dependency  r = 0.9888) (Figure 3B), as well as log Ip and log ν (the slope of the curve is higher than theoretical value = 0.5) (Figure 3C), it was found that the investigated process is mixed (adsorption-diffusion controlled) [25].In turn, the value of the slope

Acyclovir Electrochemical Behavior
For the next step, a series of cyclic voltammograms at different scan rates (from 5 to 150 mV s −1 ) were recorded in the presence of 0.1 mmol L −1 ACY under optimized supporting electrolyte conditions.Increasing the scan rate affects onto the ACY peak potential, which shifts slightly towards more positive values (a → c), as well as peak current intensity (I p ) values, which increase slightly (Figure 3A).There is no visible reduction peak, which is evidence of the irreversible nature of the examined process.On the basis of relationships between peak current intensity (I p ) and square root of scanning rate (ν 1/2 ) (there is a linear course of that dependency → r = 0.9888) (Figure 3B), as well as log I p and log ν (the slope of the curve is higher than theoretical value = 0.5) (Figure 3C), it was found that the investigated process is mixed (adsorption-diffusion controlled) [25].In turn, the value of the slope of dependency between peak potential (E p ) and log υ equal 0.058 was used in the Laviron equation [26] and allows us to conclude that two electrons are involved in the process of electrooxidation of acyclovir at the aSPCE under optimized supporting electrolyte conditions. of dependency between peak potential (Ep) and log υ equal 0.058 was used in the Laviron equation [26] and allows us to conclude that two electrons are involved in the process of electrooxidation of acyclovir at the aSPCE under optimized supporting electrolyte conditions.

Optimization of Signal Registration Technique Parameters
According to our previous conclusions, the process of electrooxidation of acyclovir on aSPCE is mixed.Therefore, the influence of the potential (Eacc.)applied to aSPCE as well as various mixing times (tacc.)on the ACY analytical signal was examined with the optimized electrolyte composition.Changes in the applied potentials from 0 to −0.2 V with a constant tacc.= 60 s value resulted in significant changes in the peak current intensity of

Optimization of Signal Registration Technique Parameters
According to our previous conclusions, the process of electrooxidation of acyclovir on aSPCE is mixed.Therefore, the influence of the potential (E acc. ) applied to aSPCE as well as various mixing times (t acc. ) on the ACY analytical signal was examined with the optimized electrolyte composition.Changes in the applied potentials from 0 to −0.2 V with a constant t acc.= 60 s value resulted in significant changes in the peak current intensity of the 50 nmol L −1 ACY signals (Figure 5A).The best results were obtained for E acc.= −0.1 V.For the next step, t acc .optimization was performed for constant value E acc. and the most effective time was chosen (60 s) (Figure 5B).

Optimization of Signal Registration Technique Parameters
According to our previous conclusions, the process of electrooxidation of acyclovir on aSPCE is mixed.Therefore, the influence of the potential (Eacc.)applied to aSPCE as well as various mixing times (tacc.)on the ACY analytical signal was examined with the optimized electrolyte composition.Changes in the applied potentials from 0 to −0.2 V with a constant tacc.= 60 s value resulted in significant changes in the peak current intensity of the 50 nmol L −1 ACY signals (Figure 5A).The best results were obtained for Eacc.= −0.1 V.For the next step, tacc.optimization was performed for constant value Eacc.and the most effective time was chosen (60 s) (Figure 5B).Additionally, the influence of registration technique choice on the 50 nmol L −1 ACY signal was examined.The SWV (square wave voltammetry) voltammograms, which we performed with the parameters corresponding to previously used DPV parameters, were very jagged and lacked in the presence of an ACY peak.For this reason, during further optimization, we decided to stick to the DPV technique and optimize the following parameters: scan rate (ν), amplitude (∆EA) and modulation time (tm).Firstly, the value of ν was changed in the range of 150-300 mV s −1 (Figure 6A).At the same time, the values of the potential step and modulation time remained unchanged and were the same as under initial conditions.The ACY peak current increased with the increasing scan rate at a value Additionally, the influence of registration technique choice on the 50 nmol L −1 ACY signal was examined.The SWV (square wave voltammetry) voltammograms, which we performed with the parameters corresponding to previously used DPV parameters, were very jagged and lacked in the presence of an ACY peak.For this reason, during further optimization, we decided to stick to the DPV technique and optimize the following parameters: scan rate (ν), amplitude (∆E A ) and modulation time (t m ).Firstly, the value of ν was changed in the range of 150-300 mV s −1 (Figure 6A).At the same time, the values of the potential step and modulation time remained unchanged and were the same as under initial conditions.The ACY peak current increased with the increasing scan rate at a value equal to 250 mV s −1 and then started to diminish.Hence, this value was considered optimal.Next, the influence of the ∆E A on the ACY peaks was tested, with the optimized value of ν and constant value of t m (Figure 6B).The best result was obtained with the amplitude value of 150 mV.Finally, the modulation time parameter was optimized in a similar way and t m = 6 ms was selected as optimal (Figure 6C).
For each of the measurements provided, voltammograms were recorded in the range of 0.1 to 1.4 V.However, it was decided to reduce this range, and in subsequent stages of this research, DPAdSVs were recorded from 0.4 to 1.4 V.This change did not affect the peak current intensity but shortened the duration of the analysis and also improved the visibility of the signals.
equal to 250 mV s −1 and then started to diminish.Hence, this value was considered optimal.Next, the influence of the ∆EA on the ACY peaks was tested, with the optimized value of ν and constant value of tm (Figure 6B).The best result was obtained with the amplitude value of 150 mV.Finally, the modulation time parameter was optimized in a similar way and tm = 6 ms was selected as optimal (Figure 6C).For each of the measurements provided, voltammograms were recorded in the range of 0.1 to 1.4 V.However, it was decided to reduce this range, and in subsequent stages of this research, DPAdSVs were recorded from 0.4 to 1.4 V.This change did not affect the peak current intensity but shortened the duration of the analysis and also improved the visibility of the signals.

Analytical Parameters and Robustness Studies
In the next part of the research, analytical parameters were determined under optimized conditions, such as the calibration curve linear range, the limit of detection (LOD), the limit of quantification (LOQ) and the sensitivity of the developed procedure.DPAdSV voltammograms were registered in the presence of increasing ACY additions a  k (0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0 and 1000.0 nmol L −1 ) (Figure 7A).According to voltammograms, a calibration curve consisting of two linear ranges (0.5-50.0 and 50.0-1000.0nmol L −1 ) was presented (Figure 7B) and LOD = 0.12 nmol L −1 and LOQ = 0.41 nmol L −1 values were calculated from the following equations: LOD = 3SDa/b and LOQ = 10SDa/b, where SDa is the intercept of the standard deviation for n = 3 and b is the slope of the calibration plot [29].The proposed procedure is characterized by very high repeatability and sensitivity.The RSD values calculated for each of the standard additions were within the range of 0.23-4.90%.Additionally, for the lower range of the linear calibration curve (0.5-50.0 nmol L −1 ), the sensitivity was 90.24 nA/nmol L −1 , which is one of the best values according to the literature reports presented in Table 1.Moreover, the reproducibility was calculated for the determination of 50 nmol L −1 ACY for three sensors.The RSD value was 5.9%, which confirmed the acceptable reproducibility of the aSPCE.

Analytical Parameters and Robustness Studies
In the next part of the research, analytical parameters were determined under optimized conditions, such as the calibration curve linear range, the limit of detection (LOD), the limit of quantification (LOQ) and the sensitivity of the developed procedure.DPAdSV voltammograms were registered in the presence of increasing ACY additions a → k (0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0 and 1000.0 nmol L −1 ) (Figure 7A).According to voltammograms, a calibration curve consisting of two linear ranges (0.5-50.0 and 50.0-1000.0nmol L −1 ) was presented (Figure 7B) and LOD = 0.12 nmol L −1 and LOQ = 0.41 nmol L −1 values were calculated from the following equations: LOD = 3SD a /b and LOQ = 10SD a /b, where SD a is the intercept of the standard deviation for n = 3 and b is the slope of the calibration plot [29].The proposed procedure is characterized by very high repeatability and sensitivity.The RSD values calculated for each of the standard additions were within the range of 0.23-4.90%.Additionally, for the lower range of the linear calibration curve (0.5-50.0 nmol L −1 ), the sensitivity was 90.24 nA/nmol L −1 , which is one of the best values according to the literature reports presented in Table 1.Moreover, the reproducibility was calculated for the determination of 50 nmol L −1 ACY for three sensors.The RSD value was 5.9%, which confirmed the acceptable reproducibility of the aSPCE.

Figure 3 .
Figure 3. Cyclic voltammetry measurements registered on aSPCE in the presence of 0.1 mmol L −1 ACY in optimized electrolyte formulation with a scan rate between 5 and 150 mV s −1 .(A) Selected CV voltammograms; (B) dependency between Ip and ν 1/2 ; (C) relationship between log Ip and log ν; (D) dependency between Ep and log υ.According to the data in the literature and the results obtained, the process of acyclovir oxidation relies on deprotonation and cleavage of the double bond in the imidazole ring between the N(7) = C(8) atoms.The described process of ACY electrooxidation into an oxoguanine analogue is shown in Figure4[4,27,28].

Figure 3 .
Figure 3. Cyclic voltammetry measurements registered on aSPCE in the presence of 0.1 mmol L −1 ACY in optimized electrolyte formulation with a scan rate between 5 and 150 mV s −1 .(A) Selected CV voltammograms; (B) dependency between I p and ν 1/2 ; (C) relationship between log I p and log ν; (D) dependency between E p and log υ.According to the data in the literature and the results obtained, the process of acyclovir oxidation relies on deprotonation and cleavage of the double bond in the imidazole ring between the N (7) = C (8) atoms.The described process of ACY electrooxidation into an oxoguanine analogue is shown in Figure4[4,27,28].Sensors 2024, 24, x FOR PEER REVIEW 8 of 12

Figure 4 .
Figure 4. Acyclovir behavior at aSPCE under an optimized basic electrolyte formulation.

Figure 4 .
Figure 4. Acyclovir behavior at aSPCE under an optimized basic electrolyte formulation.

Figure 4 .
Figure 4. Acyclovir behavior at aSPCE under an optimized basic electrolyte formulation.

Figure 5 .
Figure 5. Dependency between (A) Ip and Eacc.with a constant value of tacc.; (B) Ip and tacc.with a constant and optimized value of Eacc..The standard deviation was calculated for n = 3.

Figure 5 .
Figure 5. Dependency between (A) I p and E acc. with a constant value of t acc.; (B) I p and t acc.with a constant and optimized value of E acc. .The standard deviation was calculated for n = 3.

Figure 6 .
Figure 6.Relationship between Ip and (A) ν; (B) ∆EA as well as (C) tm. for 50 nmol L −1 ACY addition.Experiments were performed in optimized base electrolyte formulation.The standard deviation was calculated for n = 3.

Figure 6 .
Figure 6.Relationship between I p and (A) ν; (B) ∆E A as well as (C) t m .for 50 nmol L −1 ACY addition.Experiments were performed in optimized base electrolyte formulation.The standard deviation was calculated for n = 3.