Analytical Method Development for Sodium Valproate through Chemical Derivatization

Background Sodium valproate has anticonvulsant activity and is structurally different to conventional antiepileptic drugs. The problem with valproic acid is the lack of a chromophore, which means that gas chromatography is the sole assay methodology. The introduction of benzoyl and phenyl groups to the molecule is a useful derivatisation, which enables the creation of detectable chromophores for HPLC analysis for pharmaceutical dosages as well as biological systems. Methodology. Sodium valproate was derivatised by the addition of a chromophore to its structure by introducing a methyl benzoyl or a phenyl group. Trichlorophenol and 2-hydroxyacetophenone were used to introduce phenyl and benzoyl groups to valproic acid, respectively. The reaction used was estrification reaction using coupling agents. An analytical method was then developed and validated using reverse-phase HPLC. The method was validated for parameters like linearity, range, accuracy precision, and robustness. Results The developed method was easy and feasible and can be applied to both routine analysis and bioanalysis. The method was very sensitive and could quantify valproic acid at a very low concentration of 0.75 × 10−5 mg/ml. The developed method was found to be linear (R2 = 0.997), accurate, precise, and robust. Conclusion The proposed chemical derivatisation and the developed analytical method are novel. The developed analytical procedure is the first of its kind; it is easy and feasible and can be used to quantify and detect sodium valproate at very low concentrations compared to other available methods in the literature.


Introduction
Valproic acid (VPA) has a broad spectrum of anticonvulsant activity but is structurally different to conventional antiepileptic drugs (Figure 1). Valproic acid is one of the antiepileptic drugs approved by the U.S. Food and Drug Administration (FDA) for migraine prevention [1][2][3][4].
Sodium valproate is the sodium salt of valproic acid (VPA). ere are several trade names for both materials, including Depalept (sodium valproate) in the form of an enteric coated tablet and Depalept Chrono (mix of both) in the form of a prolonged release tablet [15,16]. Sodium valproate is rapidly absorbed after oral administration, reaching peak blood levels within 1 to 4 hours [1]. e chemical structure of valproic acid lacks a chromophore, and hence, it has low absorption, which makes it more difficult to detect at low concentrations as it lacks a suitable chromophore. Valproic acid has only weak UV absorbance in the low wavelength range [17,18].
Chemical derivatisation techniques have been used for classical UV absorption or fluorescence analysis in solution.
ere are several reasons why chemical derivatisation is useful in liquid chromatography: derivatisation blocks hydrogen bonding sites and reduces the polarity of compounds. Converting alcohols or carboxylic acids to esters greatly improves chromatography. Derivatisation is also used in chromatography for the confirmation of sample identity [19].
During the past decades, multiple analytical methods have been developed to quantify valproic acid in dosage forms, plasma and animal tissues, including high-performance liquid chromatography. However, these methods lack sensitivity and are not capable of analysing the drug at very low concentrations [20][21][22]. e objective of this study is to derivatise sodium valproate to increase its detection in the UV detection range. An easy and feasible chemical derivatisation method will be capable of changing valproic acid or its sodium salt to be detected at very low concentrations. e developed method can be adapted in the routine analysis of valproic acid in pharmaceutical dosages as well as biological systems. e method will be validated for parameters like linearity, range, accuracy precision, and robustness. e proposed chemical derivatisation and the developed analytical method are novel. To the best of our knowledge, this procedure is the first of its kind. e developed method is intended to be used in the quantification and detection of sodium valproate at very low concentrations compared to other available methods in the literature.

Methodology
2.1. Chemical. Different reagents were used throughout the research project. All the reagents used were of analytical grade and were purchased from reliable resources. Sodium valproate was given as a gift from Birzeit Palestine pharmaceutical company (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP)) were purchased from Sigma, Germany. HPLC-grade acetonitrile and methanol were purchased from Sigma UK.
2,4,6-Trichlorophenol and 2-hydroxyacetophenone were purchased from Sigma USA. e Micropore Water System was used to generate HPLC-grade water.

Chemical Derivatisation.
Sodium valproate was derivatised by adding a chromophore to its structure by introducing a methyl benzoyl or phenyl group. e reaction followed the chemical reaction summarised in Schemes 1 and 2.

Preparation of Valproic Acid from Na Valproate.
Sodium valproate (Na-valproate) was converted to valproic acid by weighing Na valproate (0.5 g) and dissolving it in 25 ml water. To this solution, 1M HCl (4 ml) was added, and it was stirred using a magnetic bar for 30 min. e reaction was extracted with dichloromethane, and the organic layer was taken and dried under a vacuum using the Rota Evaporator.
e reaction was placed under vacuum argon. Dichloromethane (DCM) 8 ml was added to it, and the mixture was allowed to stir for 24 hr. e reaction was monitored using thin layer chromatography (TLC) with a mobile phase (7 : 1) of DCM : methanol. e product was purified by column chromatography using (7 : 3) hexane : ethylacetate. e same procedure was followed to derivatise valproic acid with 2-hydroxyacetophenone in which 27.58 mg (1 mmol) of it was added to the reaction.

Derivatisation of Sodium Valproate for HPLC Analysis
Using Trichlorophenol. Na valproate 33.5 mg (0.2 mmol) was dissolved in 1 ml acetonitrile. To this, 200 μl of HCl was added and allowed to stir for 30 minutes. DMAP solution (30 mg/0.5 ml acetonitrile) and EDC (40 mg/0.5 ml acetonitrile) were added to the Na valproate solution and were mixed together. Trichlorophenol (40 mg/0.5 ml acetonitrile) was then added to the mixture and stirred for 2 hours. e mixture was then dried under nitrogen. e dried powder was diluted to 10 ml with acetonitrile, which was then ready to be injected into the HPLC.

Derivatisation of Sodium Valproate for HPLC Analysis Using 2-Hydroxyacetophenone.
e same procedure followed for trichlorophenol was used for 2-hydroxyacetophenone. e procedure was performed using three levels of Na valproate. Na valproate (10 mg, 20 mg and 30 mg) was weighed, and each amount was dissolved in 1 ml acetonitrile in a separate tube. 2-Hydroxyacetophenone (9.45, 18.8 and 28.32 mg) was then added to it. To each tube, 30 mg DMAP and 40 mg EDC were added. Each mixture was allowed to stir for 1 hour and then dried under nitrogen. e dried powder was diluted to 10 ml with acetonitrile and was then ready to be injected into the HPLC. International Journal of Analytical Chemistry

HPLC Method Development.
e HPLC method development was performed using the 2-hydroxyacetophenol reaction.
is particular reaction was chosen to be the adapted analytical method due to its better separation. Moreover, this reagent introduces benzoyl to the valproic acid structure, which has an extended conjugation, so it will shift the absorbance of the parent drug to a more hyperchromic and bathochromic shift, while the reaction using trichlorophenol introduced a phenol group only. e reaction mixture was injected into the HPLC in different compositions of the mobile phase using methanol and acetonitril mixed with water in different percentage, and the percentage of organic solvent ranged from 50-90% (v/v). e detection wavelength was examined in the range of 230-254 nm. Moreover, the mobile phase was run at a flow rate in the range from 1-2 ml/min). e HPLC condition was set when the best peak shape and retention time were obtained.

HPLC Method Validation.
e method was validated for parameters like linearity, range, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), and robustness/ruggedness.
To evaluate the linearity and range of the method, 5 different test concentrations were prepared: 0.75 mg/1 ml, 1 mg/1 ml, 1.5 mg/1 ml, 2 mg/1 ml, and 3 mg/1 ml. ree separate injections were analysed under the same conditions, and the average reading was recorded. e obtained peak area was plotted against the concentration; the R 2 and regression line equation was recorded. e accuracy was assessed by adding 1 mg/1 ml valproic acid in addition to some widely used excipients that are usually added in oral dosage forms. e added excipients include starch, magnesium stearate, and carboxymethyl cellulose. e percentage recovery of the test was calculated.
Repeatability precision was established for 3 concentrations around the test concentration (0.7 mg/1 ml, 1.5 mg/ 1 ml, and 3 mg/1 ml). ree replicates of each concentration were prepared, and the relative standard deviation (RSD) of the result was calculated. e sensitivity of the method was established by measuring the LOD and LOQ. e LOD is expressed as a concentration that gives a signal-to-noise ratio of approximately 3 : 1, while the LOQ in a sample can be determined with acceptable precision and accuracy with a signal-tonoise ratio of approximately 10 : 1. e ruggedness/robustness of the method was determined by performing the same trial using small variations in different parameters, including: mobile phase pH, detection wavelength, and flow rate. e conditions of different parameters tested included the following: Mobile-phase composition, UV absorption (λ), flow rate, and measurement in different days [23].

Chemical Derivatisation.
e chemical derivatisation was successful completed using the coupling regents DMAP and DCC. e TLC results showed a spot that was clearly visualised under UV light. e spot was the reactant used in the derivatisation reaction to introduce conjugation to valproic acid by either trichlorophenol or 2-hydroxyacetophenol. e spot was a dark black with an R f value of approximately 0.6. e spot was clearly seen under the UV light compared with valproic acid, which was not seen under International Journal of Analytical Chemistry 3 UV because it lacks conjugation. e R f of the synthesised compound was higher than all the reacting regents due to its increased lipophilicity. e IR data of the derivatised compound showed a successful esterification reaction of the valproic acid.

Method Development and Validation.
e developed methods for HPLC separation were run using reverse-phase chromatography. e HPLC conditions are illustrated in Table 1.
Linearity and range was performed on a sodium valproate in a concentration range of 0.75-0.30 mg/ml. e concentration was plotted against the peak area under the curve (AUC). e calibration curve was linear with R 2 � 0.997, and the regression line equation was y � 8E +06 x + 2E +06 (Figure 2 and Table 2). e accuracy of the method was performed by adding commonly used excipients, which include starch (10 mg), carboxymethyl cellulose (10 mg), and Mg stearate (1 mg) in the tested reaction. e resulted peak area of this mixture and the peak area of the standard were used to calculate the percentage assay. e result of the % assay was found to be 99.91 (Table 3). e generated peak of the derivatised valproic acid was highly separated with high resolution from other interfering peaks, which elute early due to their high polarity (Figure 3). e results of repeatability and precision showed that the developed method was repeatable (intermediate precision) over the tested range of valproic acid concentrations. e RSD was in the range from 0.3-2.1 (Table 4). e method was found to be robust under the tested variations mentioned in the methodology section; the injected concentration of sodium valproate was 1 mg/1 ml. e    International Journal of Analytical Chemistry results showed no variability among the generated peaks at the above-mentioned conditions. e RSD of the AUC was found to be 0.807 (Table 5). e LOD and LOQ of the method provide an indication of the method sensitivity. e minimum quantity of sodium valproate in which the method can detect is expressed as an LOD and was determined by injection-diluted samples of the derivatised valproic acid. e noise : peak ratio of 3 : 1, which represents the LOD, was found to be 0.75 × 10 − 6 , while the noise : peak ratio of 10 : 1 was determined as the LOQ and was found to be 0.75 × 10 − 5 .
Finally, the method was found to be selective for the generated peak of the derivatised valproic acid. e peak was well separated from the other eluting peaks, which have a shorter retention time due to its hydrophilicity compared to the more lipophilic derivatised valproic acid. Moreover, the derivatised peak was symmetrical with an acceptable theoretical plate ( Figure 3).
As expected, the developed method increased the absorption of the valproic acid when derivatised compared with the underivatised valproic acid. We can clearly notice the huge difference in absorbance between the derivatised and underivatised valproic acid. e absorbance of underivatised valproic acid at concentration of 1 mg/ml showed an absorbance less than 0.02 (Figure 4). e method is simple and feasible and can be applied to both routine analysis as well as bioanalysis, and it is also capable of measuring very small quantities of valproic acid in biological systems.

Conclusion
In this research project, we could successfully develop an analytical method for Na valproate through the introduction of conjugation. e conjugation was introduced by reacting valproic acid with 2-hydroxyacetophenone to form an ester with the drug. e introduced benzoyl ring made the drug more lipophilic. e eluted peak showed sufficient absorbance to be quantified at much lower concentrations compared to the parent drug. e developed method is easy and feasible and can be applied to both routine analyses as well as bioanalysis.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
ere are no conflicts of interest regarding the publication of this paper.  Figure 4: e HPLC chromatogram of underivatised Na valproate. 6 International Journal of Analytical Chemistry