RPLC method development and validation for the quantitative determination of phenytoin and valproic acid in tablet formulations

Abstract

In this study, the reverse phase liquid chromatography (RPLC) method was preferred to determine the chromatographic behavior of phenytoin and valproic acid, the active ingredients of antiepileptic drugs. The optimization of the developed method was based on the capacity factor values of studied compounds in water-organic solvent mixtures varying in constant mobile phase pH value and the organic solvent concentration in the mobile phase where the compounds were analyzed. At constant pH, phenytoin was analyzed in acetonitrile-water binary mixtures containing 30-40% (v/v) acetonitrile, and valproic acid was analyzed in methanol-water binary mixtures containing 10-20% (v/v) methanol at 37^oC. Kinetex EVO C18 Core-Shell (250x4,6mm I.D., 5µm) and Pinnacle DB Cyano (250 x 4,6 mm I.D., 5µm) columns were preferred for the quantitative determination of the compounds. Under all conditions mentioned in the method section, excellent linearity (r>0.99) was obtained in the concentration range of 10-40 μg/mL for phenytoin and 200-600 μg/mL for valproic acid. The recovery value of the method was calculated as 98.48% and 99.40% for phenytoin and valproic acid, respectively. In this study, an analytical procedure suitable for routine use was developed, and the method was validated for the determination of the amount of studied compounds in pharmaceutical dosage forms.

Keywords:
Antiepileptic drugs; Binary mixtures; RPLC; Method validation; Optimization; Quantification.

INTRODUCTION

Epilepsy, a chronic brain condition, is one of the most common neurological disorders in the world, affecting individuals of all ages and with an estimated half a hundred million sufferers (Megiddo et al., 2016). This neurological disease is common among children as well. While antiepileptic drugs (AEDs) are not curative, they effectively manage seizures. Antiepileptic drugs (AEDs) satisfactorily control seizures for the majority of epilepsy patients. These medicines uniquely protect against seizures whilst facilitating the normal functioning of the nervous system. AEDs modify neuronal excitability by selectively acting on multiple molecular targets, thereby blocking seizurerelated firing without affecting non-epileptic activity (Rogawski, Löscher, 2004).

Phenytoin (5,5-diphenylimidazolidine-2,4- dione), chemically named 5,5-diphenylhydantoin and classified as a hydantoin derivative, represents one of the oldest non-sedating antiepileptic/anticonvulsant drugs (Figure 1a). Its principal mechanism of action is characterized by a potent inhibition of Na⁺ conductance, achieved through a selective binding mechanism that prolongs the inactivated state of Na⁺ channels. Despite its association with a relatively elevated incidence of side effects, phenytoin persists as a cornerstone within the realm of antiepileptic therapeutics, owing to its exceptional efficacy and frequent utilization as a firstline treatment option (McNamara et al., 2012). Valproic acid (2-propylpentanoic acid), another compound used in the study, is commonly used as a long-term medication for treating epilepsy acute and chronic partial seizures (Figure 1b). The medication’s wide usage is attributed to its therapeutic effects (Lin Lin Lee et al., 2018; Chateauvieux et al., 2010).

FIGURE 1
Chemical structure of A) Phenytoin and B) Valproic acid.

The detection and monitoring of pharmaceutical compounds in real samples is an essential process for which many methods have been proposed in the scientific literature. Several methods have been reported for the quantitative determinations of phenytoin and valproic acid in pharmaceutical, and biological samples. These methods include HPLC-UV (Patil, Bondkhar, 2005; Samadi et al., 2019; Sakaguchi et al., 2023); HPLC-MS/MS methods (Wen et al., 2018; Sun et al., 2022). UHPLC with mass spectrophotometer detector (Zhao et al., 2017; Jiang et al., 2023; Wachełko et al., 2023); LC-MS/MS method (Patel et al., 2021; Wang et al., 2023; Yang et al., 2023); GC-MS methods (Jin et al., 2023); FT-IR methods (El Orche et al., 2023) have often been preferred.

Among these methods, RPLC (the dominant branch of the HPLC method) is considered the most common technique used and applied in the pharmaceutical field, including the production, development, and analysis of drugs, such as in quality control laboratories, and the analysis of drug active ingredients in biological fluids (Kazakevich, Lobrutto, 2007; Meyer, 2013). In the RPLC method, which is used for the analysis of ionizable and neutral compounds, mobile phase polarity, pH, and column temperature values are used without trial and error (Rosés, Bosch, 2002; Demiralay et al., 2010).The change in organic solvent content also causes a change in the degree of ionization. Achieving a good separation is very important and is often possible at certain pH values. The pH of the mobile phase is an important factor in the separation of ionizable compounds. The effect of pH changes is very important for weak acid/ base analytes because they cause a change in their degree of ionization and thus in their chromatographic retention (Horvath, Melander, 1977; Subirats, Bosch, Rosés, 2006).

According to the solvophobic theory, the retention will be a function of the chromatographic behavior of the analyte determined in the mobile phase (Horvath, Melander, 1977). Using the equations for estimation of retention in chromatography ensures the performance of the experiment in a shorter time and the selectivity of the compounds (Secilmis-Canbay et al., 2012). The relationship between the percentage of the organic solvent in the mobile phase and the capacity factor (k) value is given in Equation (1).

(1) $$\log k={\log }_{\mathrm{kw}}-S\phi$$

φ is the volume fraction of the organic modifier in the binary mixture. The S value is a factor associated with the solvent strength of the organic modifier. As seen in Equation 1, the k value of the compounds shows a linear relationship depending on the volume percentage of the organic modifier in the mobile phase. With the slope value of this linear function, S can be calculated, and with the intercept value, the capacity factor (k_w) value of the compounds in the pure water can be calculated. logk_w is a measure of hydrophobicity in the RPLC method. Although the S value depends on the solvent power of the pure organic modifier, it is not constant for analytes with different chemical structures. S values can range from -3.0 to -6.0. This value indicates that a given increase in organic modifier concentration causes large differences in retention (Poole, Poole, 2012). Equation 1, also referred to as the linear solvent strength (LSS) model, is used to develop methods in RPLC (Snyder, Dolan, 2002).

Since phenytoin and valproic acid are poorly soluble compounds in water, water-organic solvent binary mixtures were used in their analysis with the RPLC method. For the analysis of phenytoin, acetonitrile was preferred as the organic solvent in the mobile phase. Valproic acid could not be analyzed at high acetonitrile concentration on the Kinetex EVO C18 (250x4.6mm,5µm) column chosen for the analysis of phenytoin. Therefore, in the analysis of valproic acid, the Pinnacle DB cyano column (250x4.6mm, 5µm) was preferred because the compound retained less on the HPLC column. In analyses carried out on cyano-containing columns, methanol is the more environmentally friendly solvent preferred than acetonitrile. For this purpose, low-concentration methanol was used in the analysis of valproic acid.

This study aims to explain the effect of changing the amount of organic solvent in the pH value of the stationary mobile phase on the retention behavior of phenytoin and valproic acid. The pH value of the mobile phase to be studied was chosen within the pK_a±1.5 range of the compound. To distinguish the compounds with the selected internal standards in the quantitative determination of the compounds, k value, selectivity factor (α), and separation factor (R_s) values were calculated. Additionally, the method was validated according to the International Conference on Harmonization (ICH) parameter and then the quantitative determination was carried out in tablet formulations.

MATERIAL AND METHODS

Reagents and Apparatus

In this study, phenytoin (5,5-diphenylhydantoin) and valproic acid (2-propylpentanoic acid) were purchased from Sigma-Aldrich (USA). Rivastigmine tartrate and oxacillin sodium were used as internal standards, and acetonitrile and methanol were used as organic solvents in the preparation of the mobile phase and were obtained from Sigma-Aldrich. Sodium hydroxide, ammonia, and ammonium chloride used as buffers to adjust the mobile phase pH was obtained from Merck KGaA (Darmstadt, Germany) and o-phosphoric acid was obtained from Riedel-de Haën. The Shimadzu HPLC system was used in the study and consisted of an LC-20AD pump, SPD-20A UV detector, CTO- 20A column oven, and DGU-20A3 degasser unit. pH measurements were made using Mettler Toledo MA 235 pH/Ion analyzer Schwerzenbach, Switzerland) and InLab 413 glass electrode. According to the International Union of Fundamental and Applied Chemistry (IUPAC), potassium hydrogen phthalate (obtained from Sigma-Aldrich) was chosen as the reference standard for the calibration of the electrode in the acetonitrilewater and methanol-water binary mixtures (Mussini et al., 1985). The ultrapure water used in the preparation of the mobile phase was obtained from the Direct Q3 (Millipore, Bedford, MA, USA) device.

Chromatographic Study

Analysis of phenytoin, valproic acid, and selected internal standards was performed on the Kinetex EVO C18 (Phenomenex, 250x4.6mm, 5µm) and Pinnacle DB cyano (Restek 250x4.6mm, 5µm) columns. The column temperature is 37 ^oC and the flow rate is 1.0 mL/min. Compounds injected into the manual injection system in a volume of 20 µL were analyzed in triplicate and the relative standard deviation of the analysis was calculated below 1%. The maximum absorbance wavelengths for phenytoin and valproic acid were 215 nm. The wavelengths of rivastigmine tartrate and oxacillin sodium, the compounds used as internal standards, and uracil were 210 nm.

Preparation of Solutions and Mobile Phase

Stock solutions of rivastigmine tartrate and phenytoin were prepared as 50 μg/mL, and stock solutions of oxacillin sodium and valproic acid were prepared as 10 μg/mL and 700 μg/mL, respectively. Compounds were stored in the refrigerator at +4 °C, away from sunlight, in the dark. To determine the capacity factors of the compounds, uracil solution was prepared by dissolving 100 μg/mL in the working mobile phase medium. The primary standard was prepared using the reference potassium hydrogen phthalate compound in the mobile phase medium, where the electrode calibration solution was 0.05 mol/kg.

For phenytoin analysis, acetonitrile-water binary mixtures containing 33%, 35%, and 40% (v/v) acetonitrile with pH adjusted to 10 were prepared as mobile phase. For the valproic acid, the methanol-water mixture containing 10%, 15%, and 20% (v/v) methanol with pH adjusted to 4.0 were prepared in the same way.

Analysis of Pharmaceutical Samples

In the existing study, quantitative analyses were performed on Epdantoin^® 100 mg tablets for phenytoin and Convulex CR 300 mg tablets for valproic acid. For the quantification of phenytoin and valproic acid in tablet formulations, 10 tablets were crushed and tablet powder equivalent to 1 tablet was taken. The weighted amounts were dissolved in the mobile phase and the total volume was completed with the mobile phase to 100 mL. The prepared solutions were filtered and prepared at different dilution ratios to concentrations within the calibration range.

RESULTS AND DISCUSSION

In this study, the retention behavior of phenytoin and valproic acid was investigated using the combined effect of pH of the mobile phase and organic solvent concentration. Analysis of compounds containing these acidic functional groups by the RPLC method largely depends on the degree of ionization of these compounds and therefore the pH of the mobile phase. Since the analyzed phenytoin and valproic acid have slightly soluble solubility, water-organic solvent binary mixtures are preferred in liquid chromatographic analysis of these compounds. In the study, water-soluble polar aprotic acetonitrile was chosen for the analysis of phenytoin. Pinnacle DB and polar solvent methanol were preferred for valproic acid, which could not be analyzed on the Kinetex EVO C18 (250x4.6mm, 5µm) column even though it was studied at a high concentration. Phenytoin, which has a symmetrical peak shape, was analyzed with the Kinetex EVO C18 column, which has a wide pH working range, instead of classical alkyl chain columns such as C18 and C8. For valproic acid analysis, a Pinnacle DB cyano (250x4.6mm, 5µm) column, which has a cyanopropyl silane ligand type with a stable bond structure and provides less interaction of the analytes with the column, was preferred.

The t_R values of phenytoin in mobile phases containing acetonitrile-water mixtures containing 30%, 45%, and 40% (v/v) acetonitrile at a constant column temperature of 37 °C and a flow rate of 1.0 mL/min were determined by averaging the results of three replications. In addition, dead time (t_o) values were determined by using the type of uracil that was not retained in the column under each condition. The capacity factor values of the compound were calculated for each condition using the data obtained as a result of the qualitative analysis. The pH value of the mobile phase to be studied was chosen in the pK_a±1.5 range of phenytoin. Since there is no experimental data on the pK_a value of the compound, the Chemicalize program, which makes estimation, was used to determine this physicochemical parameter. The pK_a value of the acidic nitrogen (Number 3 nitrogen) atom in the hydantoin ring in the chemical structure of the compound is around 8.49 (Chemicalize, 2023). For this, the pH values to be analyzed were chosen as pH 10.0. Since the peak symmetry and reproducibility of phenytoin will not be good at pH 8, no study has been carried out at pH where the pK_a value of the compound. Under the same chromatographic conditions, valproic acid was analyzed in methanol-water binary mixtures containing 10%, 15%, and 20% (v/v) methanol. The pH value of the mobile phase was chosen within the pK_a±1.5 range of the compound. The pK_a value of the carboxylic acid functional group in the structure of the compound was calculated as 5.14 from the Chemicalize program (Chemicalize, 2023). According to this pH value, the pH value at which the compound is in molecular form was selected for analysis.

According to the LSS model, the linear relationship obtained when the logarithmic capacity factor (logk) values of phenytoin and valproic acid are plotted with the volume percentage of acetonitrile and methanol (φ) in the mobile phase is given in Figure 2 (Snyder, Kirkland, Dolan, 2011).

FIGURE 2
Graphs showing the φ-logk relationship A) phenytoin, B) valproic acid.

The intercept value of the linear functions obtained from these graphs gives the logk_w value according to Equation 1. According to this data, the k_w value of phenytoin at the studied pH values was calculated without any experiment. Accordingly, the k_w value for phenytoin at pH 10.0 was 23.50, while the k_w value for valproic acid was calculated as 3.24 at pH 4. The desired k value in chromatographic analyses is between 1-5. According to these calculated data, the calculated k values in the aqueous medium for phenytoin are very large. According to this value, it was concluded that the t_R value would also be very high, and under this condition, this compound could not be analyzed in a 100% water medium. Since the k_w value for valproic acid is in the range of 1-5, this compound can also be analyzed in water. However, since this compound has a slight solubility in water, analysis of the compound in 100% water is not possible.

In the linear functions of the graphs given in Figure 2 (Equation 1), k values for phenytoin and valproic acid can be estimated when any percentage by volume value of the studied organic solvent outside the experimental study is substituted for the φ value. For acetonitrilewater binary mixtures containing 10%, 50%, and 70% (v/v) acetonitrile outside the experimental working range, the k values of the compound could also be calculated without any experiment (Table I). Likewise, using the linear function, k values could be calculated for methanol-water binary mixtures containing 30%, 50%, and 70% (v/v) methanol (Table I).

TABLE I
k values calculated for phenytoin and valproic acid

Compounds	Percentage of organic solvent (v/v)
Phenytoin	10% (v/v)	50% (v/v)	70% (v/v)
	8.991	0.193	0.028
Valproic acid	30% (v/v)	50% (v/v)	70% (v/v)
	0.180	0.026	0.004

According to the data in Table I, since the k value must be between 1-5, it is not possible to analyze valproic acid in methanol-water binary mixtures containing 30%, 50%, and 70% (v/v) methanol. According to the equation, it is possible to determine phenytoin in acetonitrile-water binary mixtures containing 32% (v/v) acetonitrile and below atpH 10.0. Additionally, as the amount of organic solventin the mobile phases increases, the interaction of the compound with the HPLC column will decrease and the k value will decrease. The data in Table I supports this situation.

For the qualitative and quantitative determination of studied compounds, which will be determined by the RPLC method, the capacity factor (k) value should be 1 and above. these compounds, which have low solubility in an aqueous medium, were carried out in binary mixtures. For this purpose, mobile phases containing organic solvents at three different concentrations (v/v%) by volume were prepared. Since phenytoin and valproic acid are an ionizable compound, it is affected by changing mobile phase pH values. In determining the pH value to be studied, the pH value of the compounds in the range of pK_a±1.5 is selected. In a chromatographic analysis, it is desiredto analyze a compound as quickly as possible. For this reason, it is important to determine the k value.In RPLC analyses performed for this, k=1.407 at pH 7 and k=1.330 at pH 8.5 were calculated for phenytoin. According to these data, the optimum mobile phase pH value was determined as pH 10, which is 1.5 units above the pK_a value of the compound for which the shortest analysis time was obtained (k=1.277). In the analyses performed for valproic acid, the pK_a value and the k value beyond 1.5 units were calculated below 1 (pH 4.5 k= 0.986; pH 5 k=0.913; pH 6.5 k=0.827). According to these data, the optimum pH value was 4.0 (k=1.249). In the acetonitrile-water binary mixture containing 40% (v/v) acetonitrile adjusted to pH 10.0, a symmetrical peak with a k value above 1 was obtained at 37 ^oC for phenytoin. Under the same conditions,the methanol-water binary mixture containing 20% (v/v) methanol at pH 4.0 was chosen as the analysis condition for valproic acid.

In this study, the internal standard method was used for the quantitative determination of phenytoinin commercial tablet formulation. For this, different standards with UV properties and chromatographic separation from phenytoin and valproic acid were tried. In the separation performed for the quantitative determination, the k value should be 1 and above 1, the selectivity factor (α) value should be 1.15 and above, and the resolution factor (R_s) value should be 2 and above to be accepted as separation of the two compounds. As a result, rivastigmine tartrate, a cholinesterase inhibitor drug that meets these conditions, was chosen as the internal standard (IS). Rivastigmine tartrate is better than hydrophobic (logP 2.29) than phenytoin (logP 0.90) (Swis ADME, 2023). Likewise, penicillinase-resistant oxacillin sodium was selected as the appropriate IS for the analysis of valproic acid. Oxacillin sodium is better than hydrophobic (logP 2.38) than valproic acid (logP 1.60) (Swis ADME, 2023).

The tests performed to determine the suitability and effectiveness of the chromatographic system before the quantitative determination in a chromatographic separation is called the system suitability test (SST). The results obtained in these analyses performed according to the United States Pharmacopeia (USP) (McNally, 2000) are given in Table II.

TABLE II
System suitability results

Parameter	Phenytoin	Rivastigmine IS	Valproic acid	Oxacillin IS	Recommended values
Retention time (tR)	5.425	7.578	7.746	12.190	-
Tailing factor	1.113	1.622	1.138	1.153	≤ 2
Capacity factor (k)	1.277	2.181	1.249	2.541	≥ 1
Resolution (Rs)	-	6.664	-	8.708	≥ 2
Theoretical plates (N)	10320	8805	10483	9128	> 2000
Selectivity (α)	-	1.707	-	2.033	> 1
RSD% (tR)	0.147	0.088	0.091	0.123	≤ 1
RSD% (area)	0.267	0.505	0.129	0.511	≤ 1

Therefore, the results showed that the conditions are adequate and the method can be used in routine analysis for these antiepileptic drugs. The standard mixture chromatogram showing the separation of the compounds is given in Figure 3.

FIGURE 3
Standard mixture chromatograms showing the separation of compounds 1) A) Phenytoin and B) Rivastigmine (IS), 2) C) Valproic acid and D) Oxacillin(IS).

The purpose of chemical analysis is quantitative analysis in which the amount of a substance in the sample is determined. Careful sample preparation is critical to accurately calculate the concentration of an unknown substance from the sample. Some of the samples may be lost with each preparation. However, there are some strategies to minimize sample loss. The internal standard (IS) method is widely used to deal with sample loss and still make accurate concentration measurements. The concentrations of the internal standards rivastigmine tartrate and oxacillin sodium selected in this study were kept constant at 20 μg/mL and 1.0 μg/mL throughout the study. The working ranges of the developed method have been determined. Calibration graphs were drawn using the peak area (mAu) ratio values obtained as a result of the analysis of phenytoin and valproic acid at six concentration levels and solutions containing IS at a fixed concentration against varying concentrations. Correlation coefficient (r) values are greater than 0.999. The results show an excellent correlation between the peak area ratio and the concentration of the studied compounds (Table III).

TABLE III
The calibration data of analysed compounds

Calibration Data	Phenytoin	Valproic acid
Linear equation	y = 0.142x - 0.067	y = 0.0098x - 0.0272
Standard error of the slope	2.16.10-2	3.20.10-4
Standard error of the intercept	5.47.10-2	1.34.10-1
Correlation coefficient (r)	0.999	0.998
Linearity range (μg/mL)	10-40	200-600

To determine the precision of the developed RPLC method, intraday (repeatability) and interday (reproducibility) studies were carried out. For this, phenytoin and valproic acid solutions containing a constant concentration of IS were prepared at two different concentrations within the linear working range determined in the calibration. Independent solutions prepared were analyzed three times a day. These prepared solutions were kept in a refrigerator at +4 ^oC by cutting off contact with air. After the intraday analysis, the retention times and peak area values of the compounds were recorded in the analysis performed on the 3rd day. Measured concentration and RSD% values calculated using the calibration function according to these data are given in Table IV.

TABLE IV
Precision data of method

Compound	Theoretical concentration (μg/mL)	Intraday mean concentration (μg/mL)	RSD% (μg/mL)	Interday mean concentration (μg/mL)	RSD% (μg/mL)
Phenytoin	15.000	14.838	0.300	15.226	0.455
	30.000	31.154	0.474	30.726	0.734
Valproic acid	300.00	300.024	0.835	302.122	0.934
	500.00	488.510	0.747	504.486	0.897

According to ICH guidelines, the RSD% value should be 1% and below in intraday repeatability data, and the RSD% value should be 2% and below in interday repeatability data.

For quantitative analysis, tablets containing phenytoin active ingredient (Eptandoin^®, 100 mg) and valproic acid (Convulex CR^® 300 mg) were prepared as stated in the material and method section. As a result of the HPLC analysis, when the peak area ratio values of the compounds were substituted in the calibration function, the amounts in the tablet samples for studied compounds were calculated. The data are presented in Table V. According to the data obtained from the table, average values very close to the amount of studied compounds active ingredient in the tablet samples were obtained. The calculated RSD% values show that the precision of the results is good, and the accuracy is high since the bias percentage value is below 1%.

TABLE V
The amount of phenytoin and valproic acid in the tablet samples and recovery results

Parameters	Phenytoin	Valproic acid
Labeled claim (mg)	100	300
Amount found (mg)1	98.477	298.195
RSD %	0.603	0.517
Bias%	-1.523	-0.601
Recovery%	98.477	99.398
RSD %	0.115	0.427

• ¹
  Five experiments

A recovery study was carried out to express the accuracy of the developed method. For this purpose, the tablet samples prepared from the standard solution of phenytoin and valproic acid, not exceeding the calibration working range, were added. As a result of the HPLC analysis, when the peak area ratio values of the compounds in the additive sample were substituted in the calibration functions, their amounts in the samples were calculated. Using these results, the recovery values of the method could be calculated (Table V). According to the ICH guideline, the average % recovery should be between 95-105% (Singh, 2015). This result shows that the accuracy of the method is high.

In this quantitative analysis study, excipients commonly used in tablets did not hurt the analysis results. Chromatograms showing the tablet samples are given in Figure 4. No interfering peaks were found in the chromatograms.

FIGURE 4
Chromatogram of drug dosage forms A) 1) Phenytoin and 2) Rivastigmine (IS) (Epdantoin 100 mg) B) 1) Valproic acid and 2) Oxacillin(IS) drug dosage form (Convulex CR 300 mg).

There are many RPLC studies on the separation of phenytoin and valproic acid alone or simultaneously with different compounds in different samples. In these studies, qualitative and quantitative analyses of the compounds were carried out using classical trial and error methods. In phenytoin analysis, the stationary phases used in the studies are generally classical C8, C18 and the organic solvents used are common solvents in RPLC (Rahman et al., 2020; Kole, Parameswaran, 2023; Sakaguchi et al., 2023; Sungthong et al., 2019; Nair, Vinayan, Mangalathillam, 2021). In the study conducted by Kole and Paremeswaran (2016), the analysis of the phenytoin compound was performed using a C18 column in a mixture of acetonitrile-5 mM potassium phosphate buffer (50:50%, v/v). It is 10% (v/v) more than the amount of acetonitrile used in our study. The compound was eluted from the HPLC column at approximately the same time in our study. Sakaguchi and his colleagues selected a C18 column, which is used especially for the analysis of polar compounds, in their study. They performed the analysis in a binary mixture containing 0.1% formic acid in water and 0.1% formic acid in methanol (50:50, v/v). In this study, the mobile phase pH value for the ionized compound phenytoin was not measured. In the study conducted by Rahman et al. (2020), the physical and chemical stability of phenytoin and phenytoin sodium was determined. In this study, the determination was made in the binary mixture of acetonitrile-20 mM phosphate buffer (40:60, %v/v) on the classical C18 column without any optimization study when used in different techniques as well as the RPLC technique. There is no chromatographic data for the compounds in the study. Sungthong et al. (2019) determined the effect of methanol and acetonitrile on phenytoin analysis by trial-and-error method. Acetonitrile with high elution power was selected for this study and used in the mobile phase at 60% (v/v). Again, the optimum mobile phase pH value was not given in this study. Nair et al. (2021) performed the analysis of phenytoin in a binary mixture of methanolphosphate buffer (pH 7.3) (70:30, %v/v) on a C18 column. In the literature research, a high concentration of organic solvent in the mobile phase was generally used to determine valproic acid by the RPLC method (Soukhon et al., 2023; Kishore et al., 2003). Soukhon et al. (2023) performed valproic acid analysis in a binary mixture of water-acetonitrile (55:45, %v/v) on a C18 column. Kishore et al. (2003) analyzed valproic acid in a 45:55 v/v mobile phase consisting of acetonitrile and 0.05 M phosphate buffer (pH 3.0) on a C18 column at 50 ^oC. The analyses performed for these two compounds are higher than the amount of solvent used in our study. In addition, the retention times of the compounds differed according to the chromatographic conditions. When evaluated in terms of cost and environmental impact, our study has made an important contribution to the literature for RPLC analyses of compounds.

CONCLUSION

In this study, the RPLC method for the qualitative and quantitative analysis of the antiepileptic drugs phenytoin and valproic acid, which are widely usedin the treatment of epilepsy, one of the neurological disorders, was developed. This study, in which the optimum condition is determined depending on the pK_a value of phenytoin and valproic acid, is far from trial and error. First of all, the pH value measured in the binary mixtures prepared to determine the retention time of studied compounds was measured by pH standardization. According to the LSS method, the logk_w value, known as the hydrophobicity indexof the compound, was calculated using the logk-φ linear relationship. Chromatographic conditions were determined for the analysis of phenytoin and valproic acid according to the suitability of the chromatographic parameters. This developed method is the first study in the literature with this optimization. In this study,an analytical procedure suitable for routine use was developed, and the method was validated for the determination of the amount of studied compounds in single-active ingredient pharmaceutical dosage forms. Method validation showed excellent results for linearity, precision, accuracy, limit of quantitation, and limit of detection parameters. These analyses are a pioneering study for method optimization and validation without any trial and error.

DATA AVAILABILITY STATEMENT

Not Informed.

ACKNOWLEDGMENTS

This work was supported by Süleyman Demirel University Scientific Research Projects Coordination (Project 4934-YL2-17).

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Edited by

• Associated Editor:
  Carlota Rangel Yagui

Publication Dates

• Publication in this collection
  12 Jan 2026
• Date of issue
  2025

History

• Received
  12 Apr 2024
• Accepted
  04 July 2024