# An Isocratic Method for Quantification of Valproic Acid and Its Related Impurities Using Ion Pair Reagent by Ultraperformance Liquid Chromatography

Authors: Rakshit Thakkar; Hitesh Saravaia; Mrunal Ambasana; Madhavi Patel; Anamik Shah
Journal: ISRN Chromatography
 (2012)

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## Abstract

A selective ultraperformance liquid chromatographic (UPLC) method for the quantification of valproic acid and its known related impurities using ion pair reagent has been developed. The method includes reversed-phase Acquity HSS T3 column with 100 mm × 2.1 mm i.d. and 1.7μ particle size. The mobile phase consists of acetonitrile, 5 mM 1-hexanesulphonic acid sodium salt, flow rate is 0.6 mL/min, and UV detection is performed at 215 nm. A system suitability test (SST) was developed to govern the quality of the separation. The developed method has been validated further with respect to linearity, accuracy, precision, selectivity, LOD, LOQ, and Robustness.


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## Body

## 1. Introduction

Valproic acid is chemically known as 2-propylpentanoic acid. Chemical structures of valproic acid and its pharmacopeial-related impurities are given in Figure1. Valproic acid (VPA) is a chemical compound that has found clinical use as an anticonvulsant and mood-stabilizing drug, primarily in the treatment of epilepsy, bipolar disorder, and less commonly, it is also used to treat migraine headaches and schizophrenia [1–4]. To our knowledge, there is no paper describing an ultraperformance liquid chromatographic (UPLC) method that allows the separation of valproic acid and its related known impurities in bulk drugs. However, there are the UPLC-MS/MS method for the simultaneous determination of valproic acid and other 21 antiepileptic drugs for the routine therapeutic drug monitoring [5]. Furthermore we have used an ion pair reagent to resolute the valproic acid and its impurities more specifically, that is 2-isopropylpentanoic acid which is a structural isomer of valproic acid. Some articles are available on isocratic HPLC methods for the determination of valproic acid [6–10]. Gas chromatographic and colorimetric methods are also available in the literature for quantification of valproic acid [11–15]. Methods for determination of valproic acid by HPLC, LC-MS, or LC-MS/MS from biological matrix have also been published [16–26]. In addition, the capillary electrophoresis methods for the determination of valproic acid in different biological samples are also available [27–30].  Applying the above methods for quantification of impurities in a bulk valproic acid sample gives poor separation. Hence, the objective of this study is to develop a more selective LC method for the analysis of valproic acid and its related impurities. Besides this, it is accurate, precise, selective, robust, and linear with a reasonable analysis time. Furthermore, the developed method is applicable for the analysis of drug substances as well as commercially available drug products containing valproic acid. This work also deals with the validation of methods for the quantification of valproic acid and its impurities.Figure 1
Chemical structure of valproic acid and its related impurities.


## 2. Materials and Methods

### 2.1. Reagents and Chemicals

HPLC grade acetonitrile (ACN) was purchased from Fisher Scientific (Leicester, UK). 1-hexane sulphonic acid sodium salt anhydrous was procured from Spectrochem Pvt. Ltd. (India). Dematerialized water was further purified by filtering through Milli-Q Elix-3 purification system (Millipore, Milford, MA, USA). Valproic acid working standards and its related impurities were provided by Parth  laboratories Pvt. Ltd.(Rajkot, India).


### 2.2. Preparation of Standard Solutions

A mixture of acetonitrile and water (1 : 1) was used as sample diluent for all sample preparations. Stock solution of valproic acid and its related impurities were prepared by dissolving accurately 100 mg of each in 100 mL of sample diluent. Further pipette out 10 mL of this solution and dilute it up to 50 mL with sample diluent. This mixture contains 0.2 mg/mL valproic acid and its related impurities.


### 2.3. Instrumentation and Liquid Chromatographic Conditions

Waters Acquity UPLC (Switzerland) includes a binary solvent manager, a sample manager, PDA detector, and Empower 2.0 version software for data acquisition. A Waters Acqutiy HSS T3 column with 100 mm × 2.1 mm i.d. and 1.7μ particle size was used to achieve the best separation; flow rate was set at 0.6 mL/min while the injection volume was 10 μL. Based on the absorption maxima observed for all the four components, the detection wavelength was set at 215 nm. The Spinco. Ltd. ultrasonic bath was used for the mobile phase and sample degassing. The Sartorius microbalance was used for the weighing purpose.


### 2.4. Mobile Phase Preparation

Mobile phase A consisted of 5 mM 1-hexane sulphonic acid sodium salt anhydrous which was filtered through 0.22μ filter; mobile phase B consisted of acetonitrile (HPLC grade). The isocratic mode was set at 50 : 50 (V/V) ratio. The 5 mM 1-hexane sulphonic acid sodium salt solution was prepared by dissolving 0.94 gm of 1-hexane sulphonic acid sodium salt in 1000 mL of purified water.


## 2.1. Reagents and Chemicals

HPLC grade acetonitrile (ACN) was purchased from Fisher Scientific (Leicester, UK). 1-hexane sulphonic acid sodium salt anhydrous was procured from Spectrochem Pvt. Ltd. (India). Dematerialized water was further purified by filtering through Milli-Q Elix-3 purification system (Millipore, Milford, MA, USA). Valproic acid working standards and its related impurities were provided by Parth  laboratories Pvt. Ltd.(Rajkot, India).


## 2.2. Preparation of Standard Solutions

A mixture of acetonitrile and water (1 : 1) was used as sample diluent for all sample preparations. Stock solution of valproic acid and its related impurities were prepared by dissolving accurately 100 mg of each in 100 mL of sample diluent. Further pipette out 10 mL of this solution and dilute it up to 50 mL with sample diluent. This mixture contains 0.2 mg/mL valproic acid and its related impurities.


## 2.3. Instrumentation and Liquid Chromatographic Conditions

Waters Acquity UPLC (Switzerland) includes a binary solvent manager, a sample manager, PDA detector, and Empower 2.0 version software for data acquisition. A Waters Acqutiy HSS T3 column with 100 mm × 2.1 mm i.d. and 1.7μ particle size was used to achieve the best separation; flow rate was set at 0.6 mL/min while the injection volume was 10 μL. Based on the absorption maxima observed for all the four components, the detection wavelength was set at 215 nm. The Spinco. Ltd. ultrasonic bath was used for the mobile phase and sample degassing. The Sartorius microbalance was used for the weighing purpose.


## 2.4. Mobile Phase Preparation

Mobile phase A consisted of 5 mM 1-hexane sulphonic acid sodium salt anhydrous which was filtered through 0.22μ filter; mobile phase B consisted of acetonitrile (HPLC grade). The isocratic mode was set at 50 : 50 (V/V) ratio. The 5 mM 1-hexane sulphonic acid sodium salt solution was prepared by dissolving 0.94 gm of 1-hexane sulphonic acid sodium salt in 1000 mL of purified water.


## 3. Results and Discussion

### 3.1. Method Development

The column selection is the most important part in the method development to achieve maximum sensitivity, resolution, and speed. After performing several trials on different columns, the maximum separations  was achieved on Waters Acquity HSS T3 column with 100 mm × 2.1 mm i.d. and 1.7μ particle size column. The compatibility of the method to ICH guideline was  achieved by the isocratic elution at 50 : 50 (V/V) ratio of mobile phase A and B. The selectivity of the valproic acid and its related impurities can be altered by changing the pH of the buffer. The premethod validation experiments were performed to avoid uncertainty at the time of method validation. The chromatograms of the standard and sample are given in Figure 2.Chromatograms of working standards and sample of valproic acid with its related impurities, where (a) chromatograms of reference standard of valproic acid (0.2 mg/mL) and impurities (0.2 mg/mL). (b) Chromatograms of sample of valproic acid (0.2 mg/mL) and impurities (0.2 mg/mL).
(a)
(b)


### 3.2. Method Validation

#### 3.2.1. Accuracy

The accuracy experiment was performed by recovery study at three levels 150%, 100%, and 50% of concentration. The recovery was found between 98 to 102% which is suitable to the ICH guideline Q2 (A).


#### 3.2.2. Precision

The method precision was assessed using multiple preparations of a single sample. Six different preparations of valproic acid and its related impurities were analyzed in triplicate on the same day. The method precision data is shown in Table1. New solutions were prepared and analyzed on each of two successive days for intermediate precision study which is given in Table 2. The relative standard deviation values obtained for the peak areas of valproic acid, pentanoic acid, 2-ethylpentanoic acid and 2-isopropylpentanoic acid were not more than 2.0%.Table 1
Data for method precision.
Area
Injection
PA
2-EPA
2-IsoPPA
VPA
SET 01
224100
663931.5
672628.5
850128.5
SET 02
212925.5
631144
639404.5
821765
SET 03
213801
633675.5
641733.5
823815.5
SET 04
214931.5
635104
643164.5
824633
SET 05
218389.5
649573
654524
838073
SET 06
218432.5
642225.5
642627.5
830001
Mean
217096.7
642608.9
649013.8
831402.7
Std dev
4134.018
12416.7
12715.01
10893.97
% CV
1.904229
1.932233
1.959127
1.310312Table 2
Data for intermediate precision.
Area
Injection
PA
2-EPA
2-IsoPPA
VPA
SET 01
210954.0
623938.5
629944.0
813293.0
SET 02
208416.5
616521.5
623969.5
799387.0
SET 03
208213.0
617099.5
621781.0
800629.0
SET 04
208638.5
615928.5
622960.5
799801.5
SET 05
210549.5
626428.0
631619.5
813999.0
SET 06
211420.5
636014.5
636014.5
818790.0
Mean
209698.7
622655.1
627714.8
807649.9
Std dev
1431.1
7849.7
5673.5
8665.1
% CV
0.7
1.3
0.9
1.1


#### 3.2.3. Linearity

The linearity of the method was evaluated by analyzing eight solutions in the concentration range between 50 to 400μg/mL for each solution of valproic acid and its related impurities. The peak areas obtained from different concentrations of the drugs were used to calculate linear regression equations. These equations are y=211.6x-1217,  y=238.6x+12028,  y=250.4x-8400, and y=321.8x-7153 with correlation coefficients of R²=0.998, R²=0.997, R²=0.999 and R²=0.99 for pentanoic acid, 2-ethylpentanoic acid, 2-isopropylpentanoic acid and valproic acid, respectively. The high values of the correlation coefficients are indicative of linear relationships between the analyte concentration and peak area.


#### 3.2.4. Limit of Detection and Limit of Quantification

The limit of detection (LOD) and limit of quantification (LOQ) were established by evaluating the minimum level at which the analyte could be readily detected and quantified accurately. The LOD and LOQ for each component are 0.5μg/mL and 2.0 μg/mL, respectively. Signal-to-noise ratio is more than 3.3 for LOD and more than 10 for LOQ.


#### 3.2.5. Selectivity

The selectivity of the method was evaluated by injecting blank matrix, each individual impurity, the valproic acid standard solution, and the spiked solution to check the interference of the diluent as well as the standard solution on each other. The method is proved to be highly selective that there is no interference on any component.


#### 3.2.6. Robustness

The influence of five (k) chromatographic parameters on the separation was investigated. The parameters examined were the amount of ACN in the mobile phases, the amount of buffer solution in the mobile phases, different column lots, different flow rates, and difference in pH of buffer. There is no such impact of the small changes on the above parameters observed which suggests that the method is highly robust. The results obtained are shown in Table 3.Table 3
Data for robustness.
% CV
Condition
PA
2-EPA
2-IsoPPA
VPA
Column change
0.1
0.1
0.2
0.1
MP ratio change (56 : 44)
0.9
0.05
0.1
0.4
MP ratio change (54 : 66)
0.5
0.8
0.3
0.1
Flow rate change (1.10 mL/min)
1.6
1.4
1.1
0.6
Flow rate change (1.3 mL/min)
1.4
0.3
0.2
0.1


## 3.1. Method Development

The column selection is the most important part in the method development to achieve maximum sensitivity, resolution, and speed. After performing several trials on different columns, the maximum separations  was achieved on Waters Acquity HSS T3 column with 100 mm × 2.1 mm i.d. and 1.7μ particle size column. The compatibility of the method to ICH guideline was  achieved by the isocratic elution at 50 : 50 (V/V) ratio of mobile phase A and B. The selectivity of the valproic acid and its related impurities can be altered by changing the pH of the buffer. The premethod validation experiments were performed to avoid uncertainty at the time of method validation. The chromatograms of the standard and sample are given in Figure 2.Chromatograms of working standards and sample of valproic acid with its related impurities, where (a) chromatograms of reference standard of valproic acid (0.2 mg/mL) and impurities (0.2 mg/mL). (b) Chromatograms of sample of valproic acid (0.2 mg/mL) and impurities (0.2 mg/mL).
(a)
(b)


## 3.2. Method Validation

### 3.2.1. Accuracy

The accuracy experiment was performed by recovery study at three levels 150%, 100%, and 50% of concentration. The recovery was found between 98 to 102% which is suitable to the ICH guideline Q2 (A).


### 3.2.2. Precision

The method precision was assessed using multiple preparations of a single sample. Six different preparations of valproic acid and its related impurities were analyzed in triplicate on the same day. The method precision data is shown in Table1. New solutions were prepared and analyzed on each of two successive days for intermediate precision study which is given in Table 2. The relative standard deviation values obtained for the peak areas of valproic acid, pentanoic acid, 2-ethylpentanoic acid and 2-isopropylpentanoic acid were not more than 2.0%.Table 1
Data for method precision.
Area
Injection
PA
2-EPA
2-IsoPPA
VPA
SET 01
224100
663931.5
672628.5
850128.5
SET 02
212925.5
631144
639404.5
821765
SET 03
213801
633675.5
641733.5
823815.5
SET 04
214931.5
635104
643164.5
824633
SET 05
218389.5
649573
654524
838073
SET 06
218432.5
642225.5
642627.5
830001
Mean
217096.7
642608.9
649013.8
831402.7
Std dev
4134.018
12416.7
12715.01
10893.97
% CV
1.904229
1.932233
1.959127
1.310312Table 2
Data for intermediate precision.
Area
Injection
PA
2-EPA
2-IsoPPA
VPA
SET 01
210954.0
623938.5
629944.0
813293.0
SET 02
208416.5
616521.5
623969.5
799387.0
SET 03
208213.0
617099.5
621781.0
800629.0
SET 04
208638.5
615928.5
622960.5
799801.5
SET 05
210549.5
626428.0
631619.5
813999.0
SET 06
211420.5
636014.5
636014.5
818790.0
Mean
209698.7
622655.1
627714.8
807649.9
Std dev
1431.1
7849.7
5673.5
8665.1
% CV
0.7
1.3
0.9
1.1


### 3.2.3. Linearity

The linearity of the method was evaluated by analyzing eight solutions in the concentration range between 50 to 400μg/mL for each solution of valproic acid and its related impurities. The peak areas obtained from different concentrations of the drugs were used to calculate linear regression equations. These equations are y=211.6x-1217,  y=238.6x+12028,  y=250.4x-8400, and y=321.8x-7153 with correlation coefficients of R²=0.998, R²=0.997, R²=0.999 and R²=0.99 for pentanoic acid, 2-ethylpentanoic acid, 2-isopropylpentanoic acid and valproic acid, respectively. The high values of the correlation coefficients are indicative of linear relationships between the analyte concentration and peak area.


### 3.2.4. Limit of Detection and Limit of Quantification

The limit of detection (LOD) and limit of quantification (LOQ) were established by evaluating the minimum level at which the analyte could be readily detected and quantified accurately. The LOD and LOQ for each component are 0.5μg/mL and 2.0 μg/mL, respectively. Signal-to-noise ratio is more than 3.3 for LOD and more than 10 for LOQ.


### 3.2.5. Selectivity

The selectivity of the method was evaluated by injecting blank matrix, each individual impurity, the valproic acid standard solution, and the spiked solution to check the interference of the diluent as well as the standard solution on each other. The method is proved to be highly selective that there is no interference on any component.


### 3.2.6. Robustness

The influence of five (k) chromatographic parameters on the separation was investigated. The parameters examined were the amount of ACN in the mobile phases, the amount of buffer solution in the mobile phases, different column lots, different flow rates, and difference in pH of buffer. There is no such impact of the small changes on the above parameters observed which suggests that the method is highly robust. The results obtained are shown in Table 3.Table 3
Data for robustness.
% CV
Condition
PA
2-EPA
2-IsoPPA
VPA
Column change
0.1
0.1
0.2
0.1
MP ratio change (56 : 44)
0.9
0.05
0.1
0.4
MP ratio change (54 : 66)
0.5
0.8
0.3
0.1
Flow rate change (1.10 mL/min)
1.6
1.4
1.1
0.6
Flow rate change (1.3 mL/min)
1.4
0.3
0.2
0.1


## 3.2.1. Accuracy

The accuracy experiment was performed by recovery study at three levels 150%, 100%, and 50% of concentration. The recovery was found between 98 to 102% which is suitable to the ICH guideline Q2 (A).


## 3.2.2. Precision

The method precision was assessed using multiple preparations of a single sample. Six different preparations of valproic acid and its related impurities were analyzed in triplicate on the same day. The method precision data is shown in Table1. New solutions were prepared and analyzed on each of two successive days for intermediate precision study which is given in Table 2. The relative standard deviation values obtained for the peak areas of valproic acid, pentanoic acid, 2-ethylpentanoic acid and 2-isopropylpentanoic acid were not more than 2.0%.Table 1
Data for method precision.
Area
Injection
PA
2-EPA
2-IsoPPA
VPA
SET 01
224100
663931.5
672628.5
850128.5
SET 02
212925.5
631144
639404.5
821765
SET 03
213801
633675.5
641733.5
823815.5
SET 04
214931.5
635104
643164.5
824633
SET 05
218389.5
649573
654524
838073
SET 06
218432.5
642225.5
642627.5
830001
Mean
217096.7
642608.9
649013.8
831402.7
Std dev
4134.018
12416.7
12715.01
10893.97
% CV
1.904229
1.932233
1.959127
1.310312Table 2
Data for intermediate precision.
Area
Injection
PA
2-EPA
2-IsoPPA
VPA
SET 01
210954.0
623938.5
629944.0
813293.0
SET 02
208416.5
616521.5
623969.5
799387.0
SET 03
208213.0
617099.5
621781.0
800629.0
SET 04
208638.5
615928.5
622960.5
799801.5
SET 05
210549.5
626428.0
631619.5
813999.0
SET 06
211420.5
636014.5
636014.5
818790.0
Mean
209698.7
622655.1
627714.8
807649.9
Std dev
1431.1
7849.7
5673.5
8665.1
% CV
0.7
1.3
0.9
1.1


## 3.2.3. Linearity

The linearity of the method was evaluated by analyzing eight solutions in the concentration range between 50 to 400μg/mL for each solution of valproic acid and its related impurities. The peak areas obtained from different concentrations of the drugs were used to calculate linear regression equations. These equations are y=211.6x-1217,  y=238.6x+12028,  y=250.4x-8400, and y=321.8x-7153 with correlation coefficients of R²=0.998, R²=0.997, R²=0.999 and R²=0.99 for pentanoic acid, 2-ethylpentanoic acid, 2-isopropylpentanoic acid and valproic acid, respectively. The high values of the correlation coefficients are indicative of linear relationships between the analyte concentration and peak area.


## 3.2.4. Limit of Detection and Limit of Quantification

The limit of detection (LOD) and limit of quantification (LOQ) were established by evaluating the minimum level at which the analyte could be readily detected and quantified accurately. The LOD and LOQ for each component are 0.5μg/mL and 2.0 μg/mL, respectively. Signal-to-noise ratio is more than 3.3 for LOD and more than 10 for LOQ.


## 3.2.5. Selectivity

The selectivity of the method was evaluated by injecting blank matrix, each individual impurity, the valproic acid standard solution, and the spiked solution to check the interference of the diluent as well as the standard solution on each other. The method is proved to be highly selective that there is no interference on any component.


## 3.2.6. Robustness

The influence of five (k) chromatographic parameters on the separation was investigated. The parameters examined were the amount of ACN in the mobile phases, the amount of buffer solution in the mobile phases, different column lots, different flow rates, and difference in pH of buffer. There is no such impact of the small changes on the above parameters observed which suggests that the method is highly robust. The results obtained are shown in Table 3.Table 3
Data for robustness.
% CV
Condition
PA
2-EPA
2-IsoPPA
VPA
Column change
0.1
0.1
0.2
0.1
MP ratio change (56 : 44)
0.9
0.05
0.1
0.4
MP ratio change (54 : 66)
0.5
0.8
0.3
0.1
Flow rate change (1.10 mL/min)
1.6
1.4
1.1
0.6
Flow rate change (1.3 mL/min)
1.4
0.3
0.2
0.1


## 4. Conclusions

The proposed isocratic ion-pair UPLC method shows a good separation of valproic acid from its impurities present in bulk drug. The robustness study indicates that mainly the amount of ion pair reagent in the mobile phase solution should be monitored carefully to ensure the best chromatography since this has a significant effect on the separation. The method was shown to be selective, precise, sensitive, and linear which is proven by the result of the validation summary shown in Table4. The method can be used for the quantification of valproic acid and their pharmacopeial-related impurities. It can be applicable for the analysis of drug substances and drug products in pharmaceutical dosage form also.Table 4
Summary of validation results observed by the proposed method.
Parameter
PA
2-EPA
2-IsoPPA
VPA
LOD (μg/mL)
0.5
0.5
0.5
0.5
LOQ (μg/mL)
2.0
2.0
2.0
2.0
Accuracy (%)
99.12–99.80
98.27–101.1
100.01–101.9
98.56–101.35
Precision (RSD, %)
Method precision
0.61
1.21
0.90
1.07
Intermediate precision
0.91
0.33
0.56
1.34
Linearity
Range (μg/mL)
50–400
50–400
50–400
50–400
Correlation coefficient
0.998
0.997
0.999
0.995
Slope
211.6
238.6
250.4
321.8
Intercept
−1217
12028
−8400
−7153
Theoretical plate
35426
21329
17200
31128
Asymmetry
1.06
1.23
1.02
1.31



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*Source: 836132-2012-12-17.xml*