World Journal of Pharmaceutical Sciences

ISSN (Print): 2321-3310; ISSN (Online): 2321-3086

Published by Atom and Cell Publishers © All Rights Reserved
Available online at: http://www.wjpsonline.org/
Original Article

A validated stability-indicating HPLC related substances method for salicylic acid in

bulk drug and dosage form
Sanjay A. Patila, Bhikan J. Khairnara, Dhananjay V. Maneb, Bhata R. Chaudharia*
a
Organic Research Laboratory, Dept. of Chemistry, JET’s Z.B. Patil College, Dhule (MS), India
b
Dept. of Chemistry, Shri Chhatrapati Shivaji College, Omerga Dist. Osmanabad (MS), India

Received: 17-04-2015 / Revised: 23-05-2015 / Accepted: 25-05-2015

ABSTRACT

A Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was developed for quantitative
determination of salicylic acid in bulk drugs and in pharmaceutical dosage form. The developed method is
stability indicating which separates the drugs from its degradation products and is also applicable for related
substances determination of salicylic acid in bulk drugs. The chromatographic method separation was achieved
on a Kromasil 60-5CN (250 X 4.6) mm, 5 µ column and the mobile phase containing the mixture of mobile
phase-A is the mixture of 1.0 mL of trifluoro acetic acid in 1000 mL purified water and mobile phase-B is
acetonitrile (70:30, v/v). The detection was carried out at wave length 230 nm and column oven temperature at
30°C. Forced degradation studies were performed for Salicylic acid using acid (0.1 N HCl), base (0.1 N NaOH),
Oxidation (2% H2O2) and thermal (80 oC) degradation. The degradation was observed for Salicylic acid in
Acidic, Basic and Oxidative degraded products were well resolved from main peak. The developed method
was validated with respective linearity, accuracy, precision, robustness, and forced degradation studies prove
stability indicating power of the method.

Key Words: Salicylic acid, Forced degradation, RP- HPLC, Stability indicating, Validation.

INTRODUCTION preventing pores from clogging up again by

constricting pore diameter, and allowing room for
Salicylic acid is known for its ability to ease aches, new cell growth. Because of its effect on skin cells,
and pains and reduce fevers. These medicinal salicylic acid is used in several shampoos to treat
properties, particularly fever relief, have been dandruff. Use of concentrated solutions of salicylic
known since ancient times, and it is used as an anti- acid may cause hyperpigmentation on unpretreated
inflammatory drug. In modern medicine1, salicylic skin for those with darker skin types 4,5
acid and its derivatives are used as constituents of (Fitzpatrick photo types IV, V, VI), as well as with
some rubefacient products. For example, methyl the lack of use of a broad spectrum sunblock.
salicylate is used as a liniment to soothe joint and Bismuth subsalicylate, a salt of bismuth and
muscle pain, and choline salicylate is used topically salicylic acid, is the active ingredient in stomach
to relieve the pain of mouth ulcers. Cotton pads relief aids such as Pepto-Bismol, is the main
soaked in salicylic acid can be used to chemically ingredient of Kaopectate, and “displays anti-
exfoliate skin. As with other hydroxy acids, inflammatory action (due to salicylic acid) and also
salicylic acid is a key ingredient in many skin-care acts as an antacid and mild antibiotic 6.
products for the treatment of seborrhoeic
dermatitis, acne, psoriasis, calluses, corns, keratosis EXPERIMENTAL SECTION
pilaris, acanthosis nigricans, ichthyosis, and
warts2,3 The standard treatment for calluses is a 6% Material and reagents: Salicylic acid was made
aspirin suspension in petroleum jelly, applied on available from Sigma Aldrich Ltd India (purity
the callus for one hour and then removed with ≥ 99.0 %), trifluoro acetic acid from Merck and
washing. Salicylic acid works as a keratolytic, Acetonitrile (HPLC grade) were obtained from
comedolytic, and bacteriostatic agent, causing the Qualigens fine chemicals, India Ltd. Hydrochloric
cells of the epidermis to shed more readily, opening acid, hydrogen peroxide and sodium hydroxide
clogged pores and neutralizing bacteria within, were obtained from rankem laboratories, India. All

*Corresponding Author Address: Bhata R. Chaudhari, Organic research laboratory, Dept. of Chemistry, JET’s Z.B. Patil College,
Dhule (MS), India
 Chaudhari et al., World J Pharm Sci 2015; 3(6): 1184-1190
chemicals and reagent were used as HPLC ), oxidation ( 2 % H2O2) and to evaluate the
grades, and Milli-Q- water was used throughout ability of the proposed method to separate Salicylic
the experiment. acid from its degradation products .For acid 4.0
hour, for base 4.0 minutes and for oxidative study
Chromatography Conditions: A chromatographic period was 3.0 hours . The peak purity test was
system (Water, Japan) consisting of quaternary carried out for Salicylic acid peak by using
solvent delivery pump, a degasser an auto injector, photo diode array detector (PDA) detector in
column oven and UV detector, 10A-VP series with stress samples. Related substances studies were
Chromeleon software. The chromatographic carried out for stress samples against Salicylic
column of 250 mm length and internal acid standard and the mass balance ( % Related
diameter of 4.6 mm filled with Kromacil-CN C- substances = % Related substances + %
18 stationary phase with particle size 5µm and degradation ) was calculated10,11 .
pore size 100 A° was used. The instrumental
settings were a flow of 1.0 mL/min; the RESULT AND DISCUSSION
injection volume was 10 µl. column oven
temperature 30 °C. Optimization of chromatographic condition:
The primary objective in developing this
Mobile phase: stability-indicating HPLC method was to
Mobile phase preparation: Buffer preparation: 1.0 achieve the resolution between Salicylic acid
mL of trifluoro acetic acid in 1000 mL water. and its impurity along with degradation
Mobile phase: mixture of Buffer and Acetonitrile products. In C18 and C8 stationary phase using
(70: 30) ammonium acetate, phosphate buffer and
1.0 mL trifluoro acetic acid in 1000 mL of purified different ion pairing reagent ,sodium
water and acetonitrile (70: 30 v/v). The mobile dodecasulphate (SDS), Octane sulfonic acid ) at
phase was premixed and filtered through a 0.45 µm different pH. The resolution between the
nylon filter and degassed. Typical chromatograph Salicylic acid and degradation products could
is shown in Fig.2. not achieve and the retention time of Salicylic
acid and degradation product about 10 min by
Preparation of Standard stock solution: Standard using C18 and C8 stationary phase. It was
stock solution 500 ppm of Salicylic acid in mobile observed that the analyte has strong interaction
phase solution was prepared in volumetric flask. with non-polar stationary phases such as
octadecyl silane co-valently bound on silica in
Sample solution: Ten tablets of Salicylic acid particular with high carbon loading which
(1300.0 mg) were finely ground using agate results in peak tailing. Mobile phase was
mortar and pestle. The ground material, which selected in terms of its components and
was equivalent to 50 mg of the active proportion. The separation of degradation
pharmaceutical ingredients, was transferred product and Salicylic acid was achieved on
accurately in to a 100.0 ml calibrated volumetric observed at the retention time of main peak and its
flask containing mobile phase the drug was impurities shown Kromacil CN ODS ( 250 X 4.6 )
extracted from tablet by vortex mixing mm 5 µ column and the mobile phase
followed by ultra-sonication for 10 min. The containing the mixture of mobile phase 1.0 mL
resultant mixture was filtered through 0.45 µ trifluoro acetic acid in 1000 mL of purified water
membrane filter, The desired concentration for and acetonitrile (70 : 30 v/v). Forced degradation
drug was obtained with diluents 7,8 . study showed the method is highly specific and
the entire degradation products were separated
Selectivity: Selectivity is the ability of the proposed from the main peak . The developed method
method to access unequivocally the analyte in the was found to be specific and method was
presence of components which may be validated in accordance with International
expected to be present typically impurities, Conference on Harmonization (ICH) guidelines.12
related substances, these might be includes After that method development need to validate as
degradants matrix etc9 .The specificity of the per ICH guidelines, as specificity is passes, no
developed liquid chromatography(LC) method for interference was observed at the main peak and
Salicylic acid was carried out in the presence of impurities shown in table no. I , method is linear
its related impurities. Forced degradation studies correlation coefficient (r) is greater than 0.990
were also carried out for Salicylic acid to shown in table-II, Method is accurate and accuracy
provide an indication of the stability indicating between 98 to 102 % shown in table no.III and IV
property and specificity of proposed method. and method is robust is shown in table No.V.
Intentional degradation was attempted to stress
condition. Acid ( 1.0 N HCl ), base ( 1.0 N NaOH

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Result of forced degradation experiments: Accuracy (Recovery test): Accuracy of the method
The Singh and Bakshi13 suggested that target was studies by recovery experiment. The recovery
degradation of 10-20 % when establishing experiment were performed by adding known
stability indicating properties of analytical amounts of the drugs in the placebo. The
methods, because even intermediate degradation recovery was performed at five levels, 50%,
products should not interfere with any stage of 100% and 150 % of the label claim of the
drug analysis. Although conditions used for tablet 300 mg . Three samples were prepared
forced degradation were adjusted to achieve for each recovery level, and the percentage
degradation in this range, this could not be recoveries were calculated from amount found
achieved for condition than expose to acid, base and amount added. The accuracy study of
and oxidizing agent even after long exposure. impurity was carried out in triplicate at three
Peak purity test result confirmed the Salicylic levels 50%, 100%, and 150% of 0.3% ( 500 µg
acid peak was homogeneous under all the % mL-1 ) p-hydroxy benzoic acid and phenol. Three
for drug substances and drug products at stress samples ere prepared for each recovery level,
condition tested. The mass balance of Salicylic and the percentage recoveries were calculated
acid in stress samples was close to 100% and from amount found and amount added. The
moreover, related substances of unaffected average recoveries of three levels nine
Salicylic acid in the tablets conformed the stability determination from Salicylic acid were 96.0-
- indicating nature of the method. The result
forced degradation studies are summarized in Calibration and linearity: Linearity test solution
Table-I for the method prepared from Salicylic acid
impurity stock solution at concentration levels
Chromatographic peak purity data were from Limit of detection (LOQ) to 150% of the
obtained from the spectral analysis report. Peak impurity. Standard solution containing Salicylic
purity greater than 990 is indicative of peak acid impurity in each linearity level were
homogeneity. The peak purity for Salicylic acid prepared . Linearity solution was injected in
from degradation studies was in the range 999- triplicate. The calibration graphs were obtained
1000, indicating homogeneous peaks and thus by plotting peak area verses the concentration
establishing the specificity of the method. data treated by least-suare linear regression
Chromatograms obtained from famciclovir analysis, The calibration graph were found to
tablets solutions obtained after stress testing be in the mentioned concentration the slopes
under acidic, basic and oxidative conditions are and correlation coeffients are shown in Table-II
shown in fig. (2b-j) respectively. No peaks co-
eluted with the Salicylic acid peaks, suggesting Robustness: To determine the robustness of the
the method which is enabled for specific developed method experimental condition were
analysis of famciclovir in the presence of its purposely altered and the resolution between
degraded products and impurity. Salicylic acid and its impurity were evaluated
the flow rate of the mobile phase was 1.0 mL
METHOD VALIDATION min -1. To study the effect of flow rate on the
resolution, it changed by 0.2 unit from 0.8 to
System suitability: For system suitability the 1.2 mL min -1 . While the other mobile phase
resolution impurity and Salicylic acid were components were held constant as stated in
evaluated if the resolution was found greater chromatography conditions. .While other mobile
than 1.5 hence studies six replicate injection of phase components were held constant as stated in
salicylic acid standard. solution were used and chromatography conditions. The effect of column
the RSD of peak area ratio , and number of oven temperature at 25°C and 35°C instead of
theoretical plates of the peak were calculated 30°C while the other mobile phase component
.The system suitability result are shown in were held constant stated in chromatography
Table II. condition . The result are shown in Table-V

Precision: The precision of the related Stability of analytical solution: The stability
substances method was checked by injecting of the standard solution and the sample solution
six individual preparation of Salicylic acid ( was tested at intervals of 0, 12,16 and 24 h
500 µg mL -1 ) spiked with 1.5 µg mL-1 of .The stability of the solution was determined
impurity. The intermediate precision ( inter-day by comparing results of the related substances
precision ) of the method was also evaluated of the freshly prepared standard solution . The
using two different analyst, different HPLC RSD for the related substances determined up
system and different days in the same laboratory. to 48 h for Salicylic acid was 0.40 % .The
related substances values were ± 2% after 24 h.

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The result indicate that the solution were stable dosage form and used for dissolution of tablets
for 24 h at ambient temperature. containing Salicylic acid in quality control
laboratory.
CONCLUSION
ACKNOWLEDGEMENT
The developed method was completely validated
showing satisfactory data for all method We thank to Head, Department of Chemistry
validated parameters tested . The developed and Principal, JET’s Z.B Patil College, Dhule,
method can be conveniently used for the India for providing necessary facilities for this
related substances in bulk drugs, pharmaceutical research work.

Table I: Results of analysis of forced degradation study samples using proposed method,
indicating percentage degradation and peak purity of Salicylic acid
_________________________________________________________________________
Stress conditions/duration (%) Degradation Peak purity data*
_________________________________________________________________________
Acidic/1.0 N HCl/ 4h 0.0006 1.000
Basic/1.0 N HCl/ 4h 0.0010 1.000
Oxidative/ 2% H2O2 /4 h 0.0006 0.999
Thermal/80 oC/24h 0.0001 0.999
_________________________________________________________________________
*Peak purity values in the range of 0.990-1.000 indicate a homogeneous peak

Table II: Regression Characteristics and System Suitability Parameters of Proposed RP-HPLC
Method
______________________________________________________________
Parameters Results
______________________________________________________________
Retention time (min) salicylic acid 8.5
Tailing factor- Salicylic acid 1.2
Theoretical plates- Salicylic acid 12256
Linearity range-Impurity (ugmL-1) LOQ-150
Limit of detection-Salicylic acid (LOD) (ugmL-1) 0.0007
Limit of Quantification- Salicylic acid (LOQ) (ugmL-1) 0.002
Slope (m) 13425
Intercept (c) 31
Correlation coefficient (r) 0.999
Method precision (RSD %) (n=6) 0.30
_________________________________________________________________________

Table III: Recovery result of Salicylic acid

____________________________________________________________________
Level of addition Amount added (mg) Amount found (mg) % Recovery)
______________________________________________________________________
LOQ 2.97 3.09 104.0
50 5.01 5.01 99.8
100 9.97 9.98 100.2
150 14.69 14.52 98.8
_______________________________________________________________________

Table IV: Recovery result of Impurity

_________________________________________________________________________
Level of addition Amount added (ug) Amount found (ug) % Recovery
_________________________________________________________________________
80 2.0 1.92 96.00
90 2.25 2.21 98.22
100 2.5 2.48 99.20
110 2.75 2.71 98.54
120 3.0 2.92 97.33

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Table V: Results of robustness study

_________________________________________________________________________
Parameter Variations Resolutions*
________________________________________________________________________
As such sample - 3.4
Tempreture 28oC Not applicable
32oC Not applicable
Flow Rate 0.8 mL/min Not applicable
1.2 mL/min Not applicable
_________________________________________________________________________
*Resolution between Salicylic acid and Impurity (Phenol)

Fig.2. (a). Chemical structure of Salicylic acid

Fig.2. (b). A typical blank chromatogram of the Salicylic acid

Fig.2. (c). A typical chromatogram of the Salicylic acid standard sample

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fig.2. (d). A typical Chromatogram of Salicylic acid as such sample

Fig.2. (e). A typical Chromatogram of Salicylic acid in acid degradation (0.1 N HCL)

Fig.2. (f). A typical Chromatogram of Salicylic acid in base degradation (0.1 N NaOH)

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fig.2. (g). A typical Chromatogram of Salicylic acid in oxidative degradation (2.0 % H2O2)

Fig.2. (h). Chromatogram of Salicylic acid in Thermal degradation at 80 °C

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