A Rapid RP HPLC Method Development and Validation for the Analysis of Divalproex in Bulk and Pharmaceutical Dosage Forms

Dr. D. Samson Israel*, Shiny Ganji, B. Vinay Kumar

St Anns College of Pharmacy, Nayunipally (V),Vetapalem, Chirala, Prakasam (Dt),523187, Andhra Pradesh.

*Corresponding Author E-mail: samson.pharma@gmail.com

ABSTRACT:

The prime aim of the current work is to develop and validate a novel, specific, sensitive, precise, rapid and faster isocratic elution, RP HPLC method for estimation of Divalproex in bulk and pharmaceutical dosage forms. Chromatographic separation was achieved on Agilent SB C18, (50 x 4.6 mm, 5m) using an isocratic mode with mobile phase composed of Buffer and Acetonotrile are taken in 60:40%v/v(P^H 4) The flow rate was 1.0ml/min, temperature is maintained at30^OC and detection was made at 210 nm. The run time was 8 min. The developed method was validated according to the ICH guide lines and different analytical parameters such as linearity, precision, accuracy, specificity, limit of detection, limit of quantitation were determined. The linearity of calibration curve for each analyte is in concentration range of 25 – 150ppm. There exists good correlation between peak area and analyte concentration. Relative standard deviation values for Divaprolex is 1.24. LOD for drug is 2.3 and LOQ is 9.4. Hence the proposed method is highly sensitive, precise, accurate, robust and fast. The short retention time allows the analysis of large number of samples in short period of time and it is cost effective, so it can be successfully applied for routine analysis of active pharmaceutical ingredient and related impurities in bulk and pharmaceutical dosage forms.

KEYWORDS: Divalproex, method validation, RP-HPLC method, divalproex sodium

INTRODUCTION:

Divalproex sodium is an anticonvulsant drug. It is used to treat mania and help to prevent migraine headches¹. It is sold under brand names Depacon, Depakone, Depakote. It is effective in treatment of epilepsy. It is used to treat manic phase of bipolar disorder. It is chemically compounded from sodium valproate and valproic acid in 1:1 ratio¹. It works by increasing levels of brain neurotransmitter called GABA(gaba amino butyric acid).

It was believed that increasing GABA’s inhibitory action on brain neurons accounts for the ability of divalproex sodium to decrease seizures, curb manic behavior and decrease frequency of migraine headache¹. It was approved by USFDA in 1978. It was chemically called 2-propyl pentenoic acid². Molecular formula for the drug is C₁₆H₃₁NaO₄³.The chemical structure for the drug is as follows

Fig1. Chemical structure for Divalproex

Literature^4-7 reported few spectrophotometric, gas chromatographic and very few HPLC methods for analysis of Divalproex. The present research work is an attempt to develop a simple, economic, accurate, precise, and rapid method with short retention time for estimation of Divalproex in bulk and pharmaceutical dosage forms.

MATERIALS AND METHODS:

Chemicals and materials:

Divalproex was obtained as a gift sample from Hetero Drugs Ltd, Hyderabad. HPLC grade double distilled water, acetonitrile and all other chemicals were procured from Qualigens Fine chemicals Ltd, Mumbai.

Instrumentation:

The analytical separations were carried out on liquid chromatography equipped with UV detector and the output of signal was monitored and integrated using LC solution software. The analytical column used was Agilent SB C18, 50 x 4.6 mm, 5m. Mobile phase consisted of Phosphate buffer PH-4.0 and acetonitrile in ratio of 60:40.The flow rate was1.0ml/min and runtime was 8 min. The column was monitored at 30^oC. UV detection was measured at 210 nm and the volume of sample injected was 50µl.

Preparation of standard preparation:

Accurately Weighed and transferred 10mg of Divalproex Sodium working Standard into a 10 ml clean dry volumetric flask, and 7 ml of HPLC grade Water was added, sonicated for 5 minutes and made up to the final volume with diluent(standard stock).

Preparation of test solution:

5 tablets were weighed and crushed into powder, and transferred into a 250 mL volumetric flask, 130mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtrate, 2.0ml was pippeted out into a 100 ml volumetric flask and made up to 100ml with diluent.

Assay:

20µl of standard and sample solutions were injected into the chromatographic system and the areas of peaks were measured and the % assay was calculated using the formula

AT X WS X DT X P X Avg wt X 100 = % assay

AS X DS X WT X 100 X Label claim

Where:

AT = average area count of sample preparation

AS = average area count of standard preparation

WS = weight of working standard taken in mg

WT = weight of working sample taken in mg

DS = dilution of standard

DT = dilution of test sample

P = percentage purity of working standard Label claim in mg/ml. Label claim in mg/ml.

Method validation:

Validation parameters like system suitability, linearity, accuracy, precision, specificity, limit of detection, limit of quantitation and robustness were performed as per ICH guidelines⁸.

Linearity:

Linearity test solutions were prepared from the stock solution at different concentrations (25 -150 ppm). 50 µl of each solution was injected into HPLC system and the peak area of chromatogram was noted.

Accuracy:

To ensure accuracy of the method, recovery studies were performed by standard addition method at 50%, 100% and 150% level to pre analyzed samples and subsequent solutions were Pre analyzed. At each level, three determinations were performed.

System suitability parameters:

These tests are an integral part of method development and are used to ensure adequate performance of chromatographic system. Retention time (RT), number of theoretical plates (N), tailing factor (T) and resolution were evaluated.

Precision:

Precision of the method was determined in terms of repeatability as method and system precision. It was determined by analyzing six samples and assay was performed. % RSD values were also calculated.

Robustness:

The drug solution was subjected to small, deliberate changes like flow rate and temperature. The method was followed in accordance to ICH guide lines.

Limit of quantification(LOQ) and limit of detection(LOD):

It was calculated based on signal to noise ratio as described in ICH guide lines Q2(R1)

RESULTS AND DISCUSSION:

Method development and optimization:

To optimize the chromatographic conditions, the effect of mobile phase is studied with various solvent system combinations for the determination of Divalproex in bulk and pharmaceutical dosage forms. A mixture of phosphate Buffer and Acetonotrile (60:40%v/v) was selected as it gave best resolution. The effect of flow rate was studied in the range of 0.9 to 1.2 ml/min and 1.0ml/min was preferred to be effective. Under these conditions, the analyte peak obtained was well defined and free from tailing. The retention time (RT) was found to be 3.582min. The optimized parameters were listed in Table 1. Chromatograms for blank, placebo and standard solution were depicted in Fig 2, 3 and 4.

Table 1 Optimized chromatographic parameters

Flow rate	:	1.0 ml/min
Column	:	Agilent SB C18, 50 x 4.6 mm, 5m.
Detector wave length	:	210 nm
Column temperature	:	30°C
Injection volume	:	50mL
Run time	:	8min
Standard Concentration	:	100ppm
Diluent	:	Water: ACN(1:1)

Fig2 Chromatogram for blank solution

Fig 3 Chromatogram for placebo

Fig 4 Chromatogram for divalproex standard

Table 2 System suitability parameters

S.No	Retention time	Area	plate count	Asymmetric factor
1	3.582	1170889	3260	1.04
2	3.529	1168745	3187	1.03
3	3.482	1166845	3167	1.04
4	3.441	1163697	3162	1.04
5	3.395	1159830	3265	1.03
6	3.41	1156249	3264	1.01
Avg	3.473	1164376
%RSD	1.24

Fig 5 Chromatogram for system suitability studies

Method precision:

The results were shown in Table 3. The results obtained were within acceptable criteria.

Table 3 Method precision results

S.No	% Assay
1	101.91
2	98.98
3	98.64
4	98.12
5	100.14
6	99.27
Avg	99.51
SD	1.23
%RSD	1.24

Linearity:

A calibration graph was obtained by plotting graph between peak area versus concentration. Excellent correlation was obtained between peak area and concentration with R² = 0.999 for active ingredient Results are shown in Table 4 and the calibration curves for linearity are shown in Fig 6

Table 4 Linearity results for Divalproex

Concentration (ppm)	Peak area
25	253652
50	5812157
75	852432
100	1138749
125	1418765
150	1744940

Fig 6 Calibration curve for Divalproex

Accuracy:

The closeness of obtained value to true value indicates that the proposed method is accurate. The recovery data for accuracy studies was shown in Table 5. The accuracy chromatograms for the respective concentrations were shown in Fig 7,8 and 9

Table 5 Accuracy studies of Divalproex

S.No	%spiked level	% recovery	SD	%RSD
1	50%	100.28
2	50%	100.9	0.332	0.33
3	50%	100.8
1	100%	98.84
2	100%	101.51	1.437	1.43
3	100%	101.1
1	150%	100.57
2	150%	100.01	0.605	0.601
3	150%	100.6

Fig 7

Fig 8

Fig 9

Table 7 Robustness study for Divalproex

Parameters studied	Retention time	Peak area	Aymmetric factor	theoretical plates
Flow rate	0.9ml/min	4.219	1444021	1.03	3054
(1.0ml/min)	1.1ml/min	2.704	938358	1.01	3002
Column temp.	25^OC	3.195	1152248	1.02	3010
(30^oC)	35^OC	3.14	1158748	1.06	3049

Fig 12 Chromatogram showing flow decrease

Fig 13 Chromatogram showing flow increase

Fig 14 Chromatogram showing temperature decrease

Fig 15 Chromatogram representing temperature increase

CONCLUSION:

The method proposed for the analysis of Divalproex in bulk and pharmaceutical dosage forms was found to be specific, precise, accurate, rapid and economical. The developed method was validated in terms of accuracy, linearity, robustness and precision in accordance with ICH guidelines. The method is cost effective due to short retention time which enabled analysis of Divalproex with small amount of mobile phase. The method was found to be precise and accurate from the recovery studies. The method is sensitive due to low detection and quantitation limits. Robustness data indicate that the method is not susceptible to small changes in chromatographic conditions. This method was successfully applied for estimation of drug as well as impurity in bulk and dosage forms. Hence, this method can be used for routine analysis and quality control of Divalproex in pharmaceutical industries.

ACKNOWLEDGEMENTS:

The authors are grateful to Management, Principal of St. Anns College of Pharmacy for providing necessary research facilities to carry out research work and to Hetero Drugs Ltd, Hyderabad for providing the gift sample of drug.

REFERENCES:

1. www.minddisorders.com/del-fi/divalproex

2. www.wikipedia.org

3. http://pubchem.ncbi.nih/gov/compound/divalproex

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5. Rakshit Takkar et.al, “An isocratic method for quantification of valproic acid and its related impurity using ion pair reagent by ultra performance liquid chromatography”, ISRN Chromatography, Vol 2012(2012)

6. S. Ramanjaneyulu, “Development and validation of RP HPLC method for estimation of Divalproex sodium in tablets”, Int. J. of Innovations in Pharmaceutical Research”, 3(1), 2015, 927 – 934

7. Hu M, Huang S, Xiao B. Determination of magnesium valproate in serum by gas chromatography. CJC.1987; 5(6):362-364.

8. ICH Harmonized Tripartite guideline, validation of analytical procedures: Text and Methodology Q2 (R1) Current Step 4 version, November (2005).

Received on 30.12.2015 Accepted on 28.01.2016

Asian J. Pharm. Ana. 6(1): January- March, 2016; Page 15-22

DOI: 10.5958/2231-5675.2016.00003.X