RP-HPLC stability-indicating method for

simultaneous determination of sodium

valproate, methylparaben and propylparaben in
oral solution
Acta Chromatographica
34 (2022) 2, 203–209 HISHAM ELREFAY1, OMNIA A. ISMAIEL2,
DOI: WAFAA S. HASSAN2, ABDALLA SHALABY2 and ALI FOUAD3p
10.1556/1326.2021.00907
© 2021 The Author(s)
1
Simco Pharmaceutical Industries, 6th of October, Egypt
2
Department of Analytical Chemistry, Faculty of Pharmacy, Zagazig University, Egypt
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, 71524,
Assiut, Egypt

ORIGINAL RESEARCH Received: March 20, 2021 • Accepted: May 18, 2021
PAPER Published online: June 15, 2021

ABSTRACT
A new, sensitive, stability-indicating reversed-phase HPLC method was validated and applied for the
simultaneous quantitation of sodium valproate and two paraben preservatives; methylparaben, and
propylparaben in the liquid dosage form. Stability tests were carried out through exposure of the
analyte’s solution to stress conditions. Separation of the analytes was achieved on (waters) C18 Column
(150 mm 3 3.9 mm, 5 mm). A mixture of 0.05 M monobasic potassium phosphate pH 3.5 and
acetonitrile (50:50; v/v) was applied at 1.5 ml/min flow rate and UV detection wavelength at 210 nm.
The degradation products and the analytes were completely separated. The linearity was performed in
the range of 50–150 % from a target concentration of 10 mg/ml propylparaben, 90 mg/ml methylpar-
aben, and 2.88 mg/ml sodium valproate with a coefficient correlation (R2) 1.0 for methylparaben,
propylparaben and sodium valproate. The validation results of the suggested method were in a good
agreement with ICH guidelines. Application of the proposed method for analysis of liquid dosage forms
was successfully carried out in the routine quality control process.

KEYWORDS
methylparaben, propylparaben, sodium valproate, stability-indicating RP-HPLC, stress degradatiocn

INTRODUCTION
Methylparaben [MP], methyl 4-hydroxybenzoate (Fig. 1a), has a molecular weight of 152.156
g/mol. propylparaben [PP], chemically is Propyl 4- hydroxybenzoate (Fig. 1b), with a mo-
lecular weight of 180.2 g/mol [1]. Both MP and PP are members of Parabens group which are
commonly used as preservatives in pharmaceutical formulation, as well as food and cosmetic
products due to their anti-fungal and antibacterial activity [2, 3]. The parabens proved ef-
ficiency over a wide pH range [4]. Addition of preservatives to liquid preparations; which are
susceptible to microbial growth; is needed to prevent the degradation and alteration of the
p
Corresponding author. Tel.: products [5]. MP and PP were assayed by titration methods in USP which require a long time
þ201004379068.
for sample preparation [6]. Parabens were assayed by RP-HPLC methods in pharmaceutical
E-mail: [email protected];
[email protected] products and cosmetics [3, 5, 7–9].
Sodium valproate [VLP] is chemically sodium-2-propyl pentanoate (Fig. 1c) [1]. It is the
first line drug used in the treatment of primary generalized, partial, and myoclonic seizures.
VLP mode of action is to increase the synthesis and decrease the metabolism of gamma-
aminobutyric acid, thus stabilizing the electrical activity in the brain [10, 11]. VLP is official

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204 Acta Chromatographica 34 (2022) 2, 203–209

Fig. 1. Chemical structure of (A): methylparaben, (B): propylparaben and (C): sodium valproate

in both USP and BP which have adopted GC and HPLC (Millipore Corporation, Billerica, MA, USA) ultra-pure
methods for quantitative analysis of this drug in formula- water was used.
tions, respectively [1, 6]. The literature reported different
analytical methods for the quantification of VLP in biolog- Instrumentation and chromatographic conditions
ical matrices either alone or in combination with other
The HPLC system (Shimadzu LC-10AD vp, Japan) was
drugs. These methods include HPLC [12–14], UV-spectro-
equipped with an auto-sampler, quaternary HPLC Pumps,
photometry [15] and fluorometry [16], and gas chroma-
Dual lamp Absorbance Detector and In-Line Degasser ISA
tography [17, 18].
Card. Data acquisition was performed on LC solution soft-
European Medicine Agency (EMA) mentioned that the
ware. The HPLC separation and quantitation were achieved
addition of antimicrobial preservatives in a medical
on Waters C18 Column (150 mm 3 3.9 mm, 5 mm particle
formulation requires special justification, so the release
size) was used for analytical separation. The mobile phase
specifications for a finished product should include both
consisted of 0.05M monobasic potassium phosphate pH 3.5,
identification and content determination tests for each
and acetonitrile (50:50; v/v). The flow was adjusted to 1.5
antimicrobial preservative included in the formulation
ml/min and the detector was set at 210 nm.
[19]. Parabens preservatives are considered as an integral
0.05M Monobasic potassium phosphate was prepared by
part of VLP oral solution. There is no reported HPLC
dissolving 6.8 g of potassium dihydrogen phosphate per liter
method for the simultaneous determination of VLP in its
of distilled water, the pH was adjusted into 3.5 using 0.1 M
oral syrup with parabens, so it was important to develop a
orthophosphoric acid and filtered through 0.45 mm filter
HPLC method capable of estimating both the analyte and
before use. Metrohm 827 pH meter (Switzerland) is used for
the preservatives.
adjusting the mobile phase pH.
Because of its unique features, as it is economically and
environmentally benign than other methods, RP-HPLC
method was used here [21–26]. The major advantage of the
Preparation of standard solutions
proposed method is that the assay of sodium valproate and The standard stock solutions of MP and PP were prepared
preservatives can be achieved in presence of their stress by transferring 90.0 mg MP and 50.0 mg PP into 100 ml
degradation products on a single chromatographic system volumetric flasks. 90 ml of water were added to each flask,
with UV detection without the need for prior derivatization. mixed and the volume was completed with water and
According to the obtained results, we suggest using the filtered using. 0.45 m filter to obtain a solution having a
proposed stability indicating method for quality control concentration of 0.90 and 0.50 mg/ml of MP and PP,
purposes. To assess the reproducibility and applicability of respectively.
the suggested method, validation was carried out as per ICH A working standard solution (containing 0.09, 0.01 and
guidelines [20]. 2.5 mg/ml of MP, PP, and VLP, respectively) was prepared by
transferring 250.0 mg of VLP (sodium valproate 288 mg
equivalent to 250 mg valproic acid) standard into 100 ml
EXPERIMENTAL volumetric flask. 30 ml of mobile phase were added, mixed, 10
ml of MP standard stock solution and 2 ml from PP standard
Reagents and chemicals stock solution were transferred into the volumetric flask
mixed and the volume was completed using mobile phase and
MP and PP were purchased from Merck (Germany) and
filter through a 0.45 m filter. Different concentrations ranging
VLP was purchased from (SCI) pharmatech, Inc Taiwan.
from 50 to 150% of the above solution were prepared.
Placebo was prepared in the lab without addition of active
ingredients and preservatives. 2-isopropyl pentanoic acid
Preparation of sample solutions
was purchased from El-Gomhouria Company for Trading
chemicals and medical supplies, Egypt. All chemicals used The sample solution was prepared by transferring 5 ml of
were of HPLC grade: Acetonitrile was purchased from Daviken oral syrup to 100 ml volumetric flask. 50 ml of
Scharlu (Barcelona, Spain), and orthophosphoric acid was mobile phase were added and mixed, the volume was
purchased from Merck. Daviken® (Amoun Pharma, Egypt) completed using mobile phase and the solution filtered
oral solution was purchased from the local market. Milli-Q through 0.45 m filter.

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Acta Chromatographica 34 (2022) 2, 203–209 205

Forced degradation conditions accuracy and precision. The requirements for system suit-
ability are usually developed after method development and
Acid degradation: the mixture of MP, PP, and VLP was validation has been completed. The USP (2000) defines
prepared as previously described in the preparation of parameters that can be used to determine system suitability
working standard solution but 5 ml of 5N HCl were added prior to analysis. The system suitability parameters like
to the analytes. The mixture was shaken for 5 minutes and Theoretical plates (N), Resolution (R), Tailing factor (T)
left in the dark at room temperature for 1 hour, then were calculated and were compared with the standard values
neutralized by 5 ml of 5N NaOH before completing volume to confirm the suitability of the developed method. Theo-
into 100 ml with the mobile phase. retical plates, resolution and tailing factor values were rep-
Alkaline degradation: prepared by adding 5 ml of 5N resented for determination of MP, PP, and VLP are reported
NaOH to the mixture of MP, PP, and VLP standard solution, in Table 1.
containing the same amounts of working standard solution,
and shaken for 5 minutes and left in the dark at room tem-
perature for 1 hour, then neutralized with 5 ml of 5N HCl.
System repeatability
The volume was completed to 100 ml with the mobile phase. System repeatability results showed that RSD % was 0.11,
Oxidative degradation: prepared by adding 5 ml of 30% 0.14 and 0.05 for MP, PP, and VLP, respectively.
H2O2 to the same concentration of a mixture of MP, PP, and
VLP standard solution used in acid and alkaline degrada- Selectivity
tion. The mixture was shaken for 5 minutes and left in the
dark at room temperature for 1 hour, then the volume was Analysis of the oral solution confirmed selectivity of the
completed to 100 ml with the mobile phase. suggested method. No interference with the target analytes
was observed in presence of the excipients. The represen-
Validation tative chromatogram of MP, PP, and VLP in Oral solution
showed no interfering peaks from excipient components
The proposed analytical method was validated according to (Fig. 2).
ICH guidelines with respect to certain parameters such as
specificity, linearity, precision, accuracy, and system suit- Linearity, LOD and LOQ
ability[20].
Linearity of the method (within a given range) expresses its
Stability of standard solution ability to obtain a directly proportional relationship between
test results and the corresponding concentration of an an-
The Stability of standard solution was checked using stan- alyte. Under the optimum chromatographic conditions,
dard solutions of MP, PP, and VLP over a period of 22 h. linearity was established by injection of a series of sample
mixtures of MP, PP, and VLP at five different concentration
levels; 50, 75, 100, 125 and 150% of the target concentration
RESULTS range for MP, PP, and VLP (90, 10 and 2,500 mg/ml); into
the chromatographic system. Plotting the peak area against
Method development the corresponding concentration was used in construction of
The present investigation reported is a new RP-HPLC the calibration curves of each analyte. The Slope (b), inter-
method development and validation of simultaneous esti- cept (a) and correlation R2 were determined. LOD is the
mation of MP, PP, and VLP. The method developed was minimum concentration that can be detected by the pro-
proceeding with wavelength selection. The optimized wave- posed method. It was determined from the formula; LOD 5
length was 210 nm. In order to get the optimized RP-HPLC 3.3 σ/S, where (σ) is SD of the intercept and (S) is the slope
method, various mobile percent’s of monobasic potassium of regression equation. LOQ is the lowest concentration
phosphate and acetonitrile ranged from (90:10; v/v) to which can be measured with acceptable precision and ac-
(10:90; v/v) were tried. 0.05M monobasic potassium phos- curacy. It was calculated similarly from the equation LOQ 5
phate pH 3.5 and acetonitrile (50:50; v/v) as mobile phase 10 σ/S.
gave the best results. The column used was (waters) C18
Column (150 mm 3 3.9 mm, 5 mm particle size). The flow Table 1. System suitability parameters for MP, PP and VLP
rate was adjusted to 1.5 ml/min. The analysis was carried out
Parameters/Rt MP PP VLP Acceptance
at room temperature using 20 mL as an injection volume. 1.99 3.91 4.86 criteria
(min.)

Asymmetry 1.243 1.108 1.063 ≤2

METHOD VALIDATION Resolution (Rs) – 9.917 3.606 >2
Theoretical 18,023 29,765 29,171 >2000
plates (N)
System suitability HETP
p
0.0008 0.0005 0.0005 >0.012
p
System suitability parameters were defined as tests to Height equivalent of a theoretical plate (HETP) 5 L/N where – L is
confirm that the method can generate results of acceptable Length of the column.

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206 Acta Chromatographica 34 (2022) 2, 203–209

Fig. 2. Chromatograms of (A): Placebo and (B): a mixture of methylparaben, propylparaben and sodium valproate in pharmaceutical
formulation

Results of the regression statistics obtained for MP, PP, Table 3. The inter and intra-day precision (%RSD) data for MP, PP
and VLP are presented in Table 2. A significant correlation and VLP
between the analytes concentration and peak area was Intraday Inter-day Analyst to analyst
observed, R2 5 1.0 for MP, PP, and VLP. LOD and LOQ are Analyte RSD %
p p
RSD % RSD %
p

represented in Table 2.
MP 0.13 0.37 0.19
PP 0.14 0.14 0.26
Precision VLP 0.20 0.07 0.19
Method precision was determined both in terms of repeat- p
Average of three experiments for each concentration
ability; precision under the same operating conditions over a
short interval of time; and intermediate precision (Rugged-
ness) which shows the degree of reproducibility of test re- Accuracy
sults obtained by analyzing the sample under a variety of The accuracy of the methods was determined by injecting
normal test conditions such as different days, analyst or three concentrations of the sample solution into HPLC
instruments. In order to determine precision, six indepen- system in triplicate. 50, 100 and 150% of the target con-
dent sample solution preparations from a single lot of centration were used after spiking with standard concen-
formulation of MP, PP, and VLP solution (0.09, 0.01 and 2.5 tration of the analytes. The percentage recoveries for MP,
mg/ml, respectively) were injected into HPLC system in PP, and VLP were calculated.
triplicates on the same day (intraday precision), on three The results of accuracy studies were shown in Table 4;
different days (inter-day precision) by the same operator recovery values were within the accepted range.
and by another one (analyst to analyst precision). The
retention time and peak area were determined and expressed Robustness
as mean and %RSD calculated from the data obtained.
Intraday, inter-day and analyst to analyst precision re- The robustness of the proposed methods was indicated by
sults are shown in Table 3. %RSD values were less than 0.5% the constancy of the peak areas with minor changes in the
for the three analytes. experimental parameters. These parameters included: Flow
rate varied at three levels (± 6.6%), change in composition of

Table 2. Five level calibration graphs for MP, PP and VLP

Table 4. Accuracy (% recovery) data for sodium valproate,
Analyte methylparaben and propylparaben in pharmaceutical formulation
sodium MP PP VLP
Parameter methylparaben propylparaben valproate Nominal concentration % % %
p p p
90, 10 and 2,880 mg/ml recovery recovery recovery
Linearity (mg/ 45–135 5–15 1,440–4,320
MP, PP and VLP. ± SD ± SD ± SD
ml)
LOD (mg/ml) 0.34 0.01 30.38 50% 100.60 ± 100.23 ± 100.15 ±
LOQ (mg/ml) 1.03 0.03 92.04 0.14 0.15 0.07
Slope 51,362 45,867 405.6 100% 100.04 ± 100.53 ± 100.30 ±
Intercept 49,620 920.4 5,814 0.13 0.19 0.09
r2 1.0 1.0 1.0 150% 99.47 ± 100.43 ± 99.48 ±
tR± SD (min.) 2.004 ± 0.013 3.983 ± 0.020 4.8904 ± 0.02 0.04 0.02
0.011 p
Average of three experiments for each concentration.

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Acta Chromatographica 34 (2022) 2, 203–209 207

mobile phase water ± 5%, change in column oven temper- under oxidative conditions at room temperature. All
ature (± 58C), and change in different brand of filter paper. degradation products were chromatographically resolved
These minor changes did not affect the peak areas of both from target analytes the acidic degradation products were
drugs. chromatographically resolved from target analytes; resolu-
tion between every two successive peaks was greater than 2.
Stability of standard solution
The Stability of standard solution indicates the possibility of DISCUSSION
using standard solutions of MP, PP, and VLP over a period
of 22 h without degradation as the RSD % of the standards
was less than 1.0 for MP, PP, and VLP. The present investigation reported is a novel RP-HPLC
method development and validation of simultaneous esti-
mation of MP, PP, and VLP. Mixture of MP, PP, and VLP
Forced degradation studies
did not estimate simultaneously before. The method devel-
Forced degradation studies were established by subjecting oped was proceeding with optimized wavelength 210 nm
samples of MP, PP, and VLP standard solutions to degra- and mobile phase consisting of 0.05M monobasic potassium
dation in NaOH, HCl and H2O2. The degradation samples phosphate pH 3.5 and acetonitrile (50:50; v/v) In order to get
were analyzed using the proposed method. Minor degrada- the optimized RP-HPLC method. The column used was
tions of MP, PP, and VLP (∼3.5%) were observed under (waters) C18 Column (150 mm 3 3.9 mm, 5 mm particle
acidic conditions at room temperature. Several small size). Analysis of the oral solution confirmed selectivity of
degradation product peaks were observed as shown in Fig. 3. the suggested method.
At room temperature, MP, PP, and VLP peaks showed A significant correlation between the analytes concen-
approximately 13%, 11% and 11% degradation under the tration and peak area was observed, R2 5 1.0 for MP, PP,
alkaline condition, respectively. The elution profiles of and VLP. The Stability of standard solution indicates the
degradation products are shown in Fig. 3. Degradations of possibility of using standard solutions of MP, PP, and VLP
(MP ∼ 12%), (PP ∼ 8%) and (VLP ∼7%) were also observed over a period of 22 h without degradation.

Fig. 3. Degradation chromatograms of methylparaben, propylparaben and sodium valproate using (a): 5N NaOH, (b): 5N HCl and (c): 30%
H2O2

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208 Acta Chromatographica 34 (2022) 2, 203–209

Fig. 4. Degradation chromatogram of 2-isopropylpentanoic acid, the reported degradation product of VLP

Application of the proposed method for analysis of liquid ACKNOWLEDGMENTS

dosage forms was successfully carried out in the routine
quality control process. Separation of the analytes from their
The authors are grateful to Simco Pharmaceutical Industries,
degradation products was successfully achieved with high
6 of October City, Egypt, for providing laboratory facilities
selectivity, accuracy, precision and robustness of the devel-
to carry out this work.
oped method.
Minor degradations of MP, PP, and VLP (∼3.5%) were
observed under acidic conditions at room temperature. At
room temperature, MP, PP, and VLP peaks showed SUPPLEMENTARY MATERIAL
approximately 13%, 11% and 11% degradation under the
alkaline condition, respectively The raw chromatograms of Supplementary material to this article can be found online at
MP, PP, and VLP were added as additional data (supple- https://doi.org/10.1556/1326.2021.00907
mentary material).
Standard solution of 2-isopropylpentanoic acid, the
reported degradation product of VLP under the alkaline
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