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151 International Journal of Analytical and Bioanalytical Chemistry 2012; 2(2): 151-159
ISSN-2231-5012
Original Article
RP HPLC Method for the Simultaneous Quantification of Phenoxyethanol and
Potassium sorbate in Topical foam
Ajay Vairale *1, P Sivaswaroop
2, Prakash B Modi
1
1Dr. Reddy’s Laboratories Ltd., Dermatology, Innovation Plaza, Survey No. 42, 45, 46 & 54,Bachupalli, Qutubullapur, RR Dist 500 090, Andhra Pradesh, India.
Phone: +91 9959555701, Fax: +91 40 4434 6285
Email:[email protected] 2 IGNOU Regional Centre, Gyan Vatika Amravati Road Nagpur 440033, Maharastra India.
Received 24 April 2012; accepted 16 May 2012
Abstract
Preservative are substance that commonly added to various topical formulations in order to prolong their shelf life by inhibiting the microorganisms growth .The addition of preservative to topical formulations is to prevent them from alteration and
degradation by microorganisms during storage. Phenoxyethanol and potassium sorbate were used as antimicrobial preservative
in coal tar topical foam. Coal tars are complex and variable mixtures of phenols, polycyclic aromatic hydrocarbons (PAHs),
and heterocyclic compounds.
A unique stability- indicating HPLC method was developed for the simultaneous quantification of phenoxyethanol and potassium sorbate in topical foam containing coal tar in the presence of degradation products and excipients. Phenomenex
Luna, C18, 5 µm, 4.6 x 150mm, 100A column was used to achieve separation using gradient method. The mobile phase A
contains 0.1% phosphoric acid and the mobile phase B contains methanol. The flow rate was 0.7 mL min-1 and the detection
wavelength was 275 nm. The retention time of phenoxy ethanol and potassium sorbate is 6.3 and 7.5 minutes respectively. The
total run time was 42 minutes within which phenoxyethanol and potassium sorbate peaks were separated from degradation
products and excipients and coal tar peaks. Calibration showed that the response of potassium sorbate and phenoxyethanol, was
a linear function of concentration over the range 0.5-1.5 µg mL−1 (r ≥ 0.999) and 5.0-16.0 µg mL−1 (r ≥ 0.999) respectively.
The method was validated over this range for precision, intermediate precision, accuracy, linearity and specificity. The stability indicating method was developed and validated successfully and applied to the simultaneous quantitative determination of
phenoxyethanol and potassium sorbate in coal tar foam formulation.
© 2011 Universal Research Publications. All rights reserved
Keywords: Topical foam, Preservative, Phenoxyethanol, Potassium sorbate and Method
Validation, ICH guidelines.
1. Introduction:
Analysis of preservatives in pharmaceutical product is
most important as preservatives prevent the alteration and
degradation of the product by microorganisms during storage
[1-4].The minimum concentration of preservative required to inhibit the microorganisms growth through out the shelf life
of the product is called as minimum inhibitory concentration
Preservatives are not used indiscriminately, and preparations
that should not contain preservative include, injection into
cerebrospinal fluids, eye and hearts. Antimicrobial
preservatives are classified into two sub groups. Antifungal
preservatives include compounds such as benzoic and ascorbic acid and their salts, and phenolic compounds such as
methyl, ethyl, propyl and butyl p-hydroxybenzoate
(parabens). Antibacterial preservatives include compounds
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152 International Journal of Analytical and Bioanalytical Chemistry 2012; 2(2): 151-159
Fig. 1: Chemical structure of Potassium sorbate and Phenoxyethanol.
such as quaternary ammonium salts, alcohols, phenols,
mercurials and biguanidines.
Analytical methodologies developed for the quantification
of preservatives in the formulation matrices are usually
designed to overcome the problems associated with
interferences which are originated from other constituents
(active drug and its degradation impurities and individual
excipients). Because of the level they are used in
pharmaceutical products, preservative are usually present in
lower concentration in complex matrices. These preservatives
may be harmful to the patient due to their tendency to induce allergic contact. Stability indicating method must be used to
determine the preservative estimation during the stability
study.
Phenoxyethanol (PE) and potassium sorbate (PS) were
used as preservative in the topical coal tar foam. PE is 2-
Phenoxyethyl alcohol used in dermatological products .The
molecular formula is C8H10O2 and molecular weight is
138.17.It is a colorless oily liquid. It is a bactericide and
reduce the use of other preservative use by 10-20 folds and is
non allergenic. US pharmacopeia [5] describes the GC
method for assay with external standard method. British
pharmacopeia [6] and European pharmacopeia [7] describes titration method for its estimation. Chromatographic purity
determination is done on GC with internal standard method.
The chemical structure of PE and PS shown in (Fig: 1).
PS is the potassium salt of sorbic acid and is white or
yellowish crystalline powder or granule. It is 2, 4-
Hexadienoic acid, potassium salt, C6H7KO2 and molecular
weight is 150.22. USP, BP and EP Pharmacopeias describes
the titration method for its qauntificaitons.It is effective in a
variety of applications including food, wine, and personal
care products and exhibits low toxicity .
Based on literature survey it was revealed that few methods are available for the estimation of PE and PS on HPLC but in
combination with other preservatives .Till date there is no
official or published method for the simultaneous
determination of both the preservatives by HPLC [8-12].
A unique stability- indicating RP-HPLC method was
developed for the simultaneous quantification of PE and PS
in Topical coal tar foam in the presence of active drug,
degradation products and excipients. The method was a
gradient elution method using Phenomenex Luna, C18, 5 µm,
4.6 x 150mm, 100A column with UV detection at 275 nm.
Samples are quantified using an external standard technique.
2. Materials and Methods
2.1 Instruments and apparatus:
HPLC analysis was performed with a Waters (Milford,
MA, USA) HPLC system equipped with a quaternary solvent
manager, sample manager, column-heating compartment,
Photodiode Array and UV detector. This system was
controlled by Waters Empower software.
Phenomenex Luna, C18, 5 µm, 4.6 x 150mm, 100A
(Phenomenex, USA) was employed for chromatographic
separation. Class A volumetric glassware, 10-mL Syringes, Transfer tube Harvester, Disposable tubes, Whatman Nylon
0,45 µm Syringe Needle, water bath, sonicator,
photostability chamber were used during the experimental
work.
2.2 Chemicals and reagents:
Deionized water, HPLC grade Acetonitrile (ACN), Methanol,
phosphoric acid, suitable reference standards of PE and PS.
PVDF membrane filters were used.
2.3. Method:
2.3.1 Chromatographic parameters
The analytes were separated on HPLC using
Phenomenex Luna, C18, 5 µm, 4.6 x 150mm, 100A column at oven temperature of 25oC with a gradient run program at a
flow-rate of 0.7 mL min−1. 0.1% phosphoric acid and
methanol were used as mobile phase A and B respectively
which was filtered through a 0.45 µm nylon filter, before use.
The separation was achieved by gradient elution starting with
a 15 minute isocratic run with the mobile phase ratio of A:B
as 50:50 (v/v). Then the ratio was changed linearly for A: B
as 15:85 (v/v) for next one minute, the system was run in the
isocratic run for 15 minutes. The initial ratio of 50:50 was
attained in one minutes and continued isocratically for 10
minutes. UV detection was performed at 275 nm. The sample injection volume was 20 µL.A mixture of Acetonitrile: Water
(50:50) was used as diluent for sample and standard
preparations.
2.3.2 Standard solution preparation:
Standard Stock solution
PE standard Stock solution (1,000 µg/mL): Accurately weigh
0.1 g of PE references standard/working standard into a 100
mL volumetric flask. Add 50 mL of diluent and vortex to
dissolve. Dilute to volume with diluent and mix well by
inversion.
153 International Journal of Analytical and Bioanalytical Chemistry 2012; 2(2): 151-159
Table I: Force degradation study of Phenoxyethanol in Topical foam.
Table II: Force degradation study of Potassium sorbate in Topical foam
PS standard stock solution (500 µg/mL): Accurately weigh
0.05 g of PS references standard into a 100 mL volumetric
flask. Add 50 mL of diluent and vortex to dissolve. Dilute to
volume with diluent and mix well by inversion.
Intermediate Standard solution (100 µg/mL PE, 10 µg/mL PS): Pipette 10.0 mL of PE standard stock solution and 2.0
mL of PS standard stock solution into a 100 mL volumetric
flask. Add 50 mL of diluent and vortex to disperse. Dilute to
volume with diluent and mix well by inversion.
Working Standard solution (10 µg/mL PE, 1 µg/mL PS):
Pipette 10.0 mL of the intermediate standard solution into a
100 mL volumetric flask. Dilute to volume with diluent and
mix well be inversion.
2.3.3 Sample preparation:
Remove and discard the plunger from the Transfer tube
Harvester. Attach the clear plastic tube from the transfer tube Harvester to the tip of the foam can. Shake foam can
vigorously for at least 15 seconds. Remove the plunger from
a 10 mL plastic syringe and fill it with foam by dispensing it
through the clear tube of the Harvester. Attach the syringe
plunger and the needle onto the 10 mL syringe filled with
the product. Dispense the product through the syringe and
Degradation
pathways Area response Wt.(g) %Recovery % control Peak angle
Peak
threshold
1N HCL, 24 hrs 140814 0.5155 0.5012 98.6 0.368 0.416
1N NaOH, 24 hrs 128849 0.5173 0.4570 89.9 0.508 0.549
3%H2O2, 6 hrs 103216 0.5300 0.3573 70.3 0.517 0.520
Heat 80°C, 24 hrs 77311 0.5086 0.2789 54.9 0.702 0.622
Control 142529 0.5145 0.5083 100 0.500 0.511
UV 140365 0.5088 0.5072 99.5 0.361 0.402
UV Control 139542 0.5032 0.5098 100.3 0.325 0.413
Visible 144498 0.5110 0.5199 101.7 0.373 0.414
Visible control 138244 0.4974 0.5110 100.7 0.410 0.470
Degradation pathways
Area response Wt.(g) %Recovery
% control
Peak angle
Peak threshold
1N HCL, 24 hrs
191203 0.5155 0.0474 97.5 0.364 0.467
1N NaOH, 24 hrs 194852 0.5173 0.0482 99.0 0.405 0.482
3%H2O2,6 hrs 132902 0.5300 0.0321 65.9 0.497 0.582
Heat 80°C,24 hrs 103906 0.5086 0.0261 53.7 0.601 0.695
Control 195667 0.5145 0.0486 100 0.327 0.471
UV 164155 0.5088 0.0413 99.5 2.817 0.568
UV Control 191175 0.5032 0.0486 99.8 0.337 0.462
Visible
176348 0.5110 0.0442 101.7 0.984 0.490
Visible control
190179 0.4974 0.0489 99.3 0.302 0.418
154 International Journal of Analytical and Bioanalytical Chemistry 2012; 2(2): 151-159
Table III: System Precision
S.No Phenoxyethanol Potassium sorbate
1 149376 194947
2 150143 196091
3 150158 195950
4 149736 195837
5 150053 195964
6 149881 195960
Mean 149891 195792
%RSD 0.2 0.2
Table IV: Method Precision
Table V: Accuracy of Phenoxyethanol in Topical foam.
S.No Phenoxyethanol Potassium sorbate
1 0.500 0.0494
2 0.508 0.0501
3 0.506 0.0520
4 0.501 0.0496
5 0.504 0.0497
6 0.502 0.0496
Mean 0.50 0.050
% RSD 1% 1%
Nominal
% Target
Theoretical
Cone.(µg/ml)
R/C Ratio
Recovered
Cone. (µg/ml)
% Recovery
Mean
(n=3)
%RSD
(n=3)
80-1 8.318 14385 8.437 101
102 1 80-2 8.318 14406 8.449 102
80-3 8.318 14455 8.478 102
100-1 10.397 14380 10.543 101
101 1 100-2 10.397 14385 10.520 101
100-3 10.397 14400 10.558 102
120-1 12.477 14385 12.655 101 101 0
120-2 12.477 14357 12.631 101
120-3 12.477 14380
12.652 101
Mean: 14388 101
% CV: 0
R: 1.0
155 International Journal of Analytical and Bioanalytical Chemistry 2012; 2(2): 151-159
Table VI: Accuracy of Potassium sorbate in Topical foam.
Table VII: Detector Linearity
accurately weigh 0.5 g of sample into a 250 mL volumetric
flask. Add 100 mL of diluent and vortex to disperse. Sonicate
for 5 minutes and vortex to disperse. Dilute to volume with
diluent and mix well by inversion. Filter through a 0.45 µm
nylon filter into an HPLC Vial.
The typical representative chromatograms of blank, standard
and sample shown in Fig 2,3 and 4.
3. Results and Discussion
3.1 Optimization of chromatographic conditions: A greater challenge in developing method for nature
product iorigini is iemance. As idiscussed iearlier ithei liquid
carbonis detergent composed of coal tar. The greater
challenge is to separate other component with preservative
from the peaks of coal tar which is nature product in origin
[16]. Development of HPLC method was carried out with the
test for solubility of PE and PS in a mixture of organic
solvent and aqueous solvent in different ratio. The ratio of
water: acetonitrile at 50:50 was selected as diluent.
Chromatographic separations of individual peaks including
unknown peaks were established on reversed-phase at 275.0 nm based on the absorbance maximum of both the
components. The wavelength maxima of PE are 270 nm and
Nominal
% Target
Theoretical
Cone. (µg/ml)
R/C Ratio
Recovered
Cone. (µg/ml)
% Recovery
Mean
(n=3)
%RSD
(n=3)
80-1 0.829 193684 0.823 99
99 0 80-2 0.829 193799 0.823 99
80-3 0.829 194960 0.824 99
100-1 1.036 193353 1.027 99
99 0 100-2 1.036 193980 1.030 99
100-3 1.036 193707 1.029 99
120-1 1.243 193464 1.233 99
99 0 120-2 1.243 193896 1.236 99
120-3 1.243 193635 1.234 99
Mean: 193731 99
% CV: 0
R: 1.0
Phenoxyethanol.
Potassium sorbate.
Concentration (µg/mL) Response Concentration (µg/mL) Response
5.345 75271 0.505 98201
8.018 112528 0.758 147279
10.690 149220 1.010 195008
13.363 186550 1.263 244055
16.035 223029 1.515 291382
Slope (m) 13827 Slope (m) 191342
Y-intercept 1504 Y-intercept 1930
Co-rrelation coefficient R 1.00 Co-rrelation coefficient R 1.00
156 International Journal of Analytical and Bioanalytical Chemistry 2012; 2(2): 151-159
Table VIII: Robustness
Table XI: Ruggedness
Fig. 2: Typical chromatogram of blank
Phenoxyethanol Potassium sorbate
Parameters Precision
Tailing
%
w/w
Purity
angle
Purity
Threshold Precision
Tailin
g
%
w/w
Purity
angle
Purity
Threshold
Flow Rate
0.6 0.69 1.1 0.46 0.302 0.427 0.55 1.2 0.045 0.329 0.433
0.7 0.30 1.1 0.46 0.275 0.412 0.25 1.1 0.045 0.349 0.471
0.8 0.48 1.1 0.46 0.269 0.430 0.52 1.1 0.045 0.343 0.472
Column
Temperatur
e
22.5° 1.0 1.1 0.46 0.253 0.407 0.83 1.1 0.046 0.342 0.453
25.0° 0.30 1.1 0.46 0.275 0.412 0.25 1.1 0.045 0.349 0.471
27.5° 0.40 1.1 0.46 0.379 0.385 0.61 1.1 0.046 0.301 0.436
Buffer conc.
Mobile
phase A
0.05% 0.45 1.1 0.46 0.348 0.421 0.32 1.1 0.046 0.471 0.480
0.1% 0.30 1.1 0.46 0.275 0.412 0.25 1.1 0.045 0.349 0.471
0.2% 0.75 1.1 0.46 0.276 0.404 0.53 1.1 0.046 0.338 0.464
Sample Day 1 /Analyst 1 % w/w
Day 2/ Analyst 2 % w/w
Day 3/ Analyst 3 % w/w
Potassium
sorbate
Phenoxy
ethanol
Potassium
sorbate
Phenoxy
ethanol
Potassium
sorbate
Phenoxy
ethanol
1 0.0494 0.500 0.0486 0.498 0.0481 0.490
2 0.0501 0.508 0.0490 0.506 0.0485 0.496
3 0.0500 0.506 0.0494 0.508 0.0470 0.485
4 0.0496 0.501 0.0489 0.505 0.501 0.513
5 0.0497 0.504 0.0490 0.505 0.0494 0.509
6 0.0496 0.502 0.0489 0.506 0.0493 0.502
Mean
0.050 0.50 0.049 0.50 0.049 0.50
% RSD 1.0% 1.0% 0.0% 1.0% 2.0% 2.0%
157 International Journal of Analytical and Bioanalytical Chemistry 2012; 2(2): 151-159
Fig. 3: Typical chromatogram of Standard Fig. 4: Typical chromatogram of Sample
that of PS is 261 nm. Buffer selection of phosphoric acid at
0.1% concentration was used to achieve the separation
between PE and PS. To achieve the chromatographic
separations various column make with different chemistry
were evaluated. Phenomenex Luna, C18, 5 µm, 4.6 x 150mm
was selected for the chromatography .The gradient method
was used to reduce the run time as coal tar components were
mostly soluble in organic solvent and late eluting.
3.2 Validation of analytical method
The method was validated for specificity, precision, accuracy, sensitivity and linear range as per the International
Conference on Harmonization (ICH) guidelines [13-15].
3.2.1 System suitability
System suitability parameters were measured so as to verify
the system performance. Injected five injections of working
standard solution into HPLC. The relative standard deviation
of each analyte (PE and PS) peak area in five injections
should not be more than 2.0%. The tailing factor for each
analyte peak should not be more than 2.0. The resolution
between the PE and PS peaks should be not be less than 2.0.
All these system suitability parameters covered the system, method and column performance. The system suitability
parameters were verified before each parameter of method
validation and achieved at each method validation
parameters.
3.2.2 Specificity
Samples of coal tar foam placebo PS, Foam placebo PE, PE
and PS and bulk product of foam were each subjected to
stress conditions. This was done by subjecting individual
reference materials and the coal tar products to acid and base
hydrolysis, heat, peroxide oxidation and photo degradation.
Foam solution and Placebo solution were used to eliminate
any background peaks. Acid-treated solutions: 0.5g each of placebo and bulk
product solution was treated with 1.0mL of 1N HCl. The
mixture was allowed to stand for 24 hours. It was then
neutralized with 1.0mL 1N NaOH.
Base-treated solutions: 0.5g each of placebo and bulk
product were treated with 1.0mL 1N NaOH. The mixture was
allowed to stand for 24 hours. It was then neutralized with
1.0mL of 1N HCl.
Heat-treated solutions: 0.5g each of placebo and bulk
product were weighed into a 250 mL volumetric flask and
placed in 80°C oven for 24 hours.
H202 treated sample solutions: 0.5g each of placebo and bulk
product was weighed into a 250 mL volumetric flask
followed by addition of 1mL of 3% H202 . The mixture was
allowed to stand for 6 hours.
UV-visible treated: Light degradation was performed by
exposing samples of the product analyte and placebo to UV
and visible light for 141.5 hours. The average visible light intensity was 869 lux and the average UV intensity was 336
µW/cm2 resulting in 1.23 million lux hours of visible
exposure and 475.44W-hrs /m2 UV exposure.
The results revealed that the PE was unstable in heat, base
and oxidation conditions, however the peak purity of PE was
achieved in all degradation conditions as purity angle was
always less than purity threshold. The results of force
degradation study were summarised in Table1 .The results
revealed that the PS was unstable in heat and oxidation
conditions, however the peak purity of PS was achieved in all
degradation conditions as purity angle was always less than purity threshold. The results of force degradation study were
summarised in Table2.
3.2.3 System Precision and Method Precision
A working standard was prepared according to the method
and injected six times in succession. The relative standard
deviation of the peak area responses for PS and PE was
calculated. Six assay specimens of product as foam were
prepared and analysed according to the method. The % RSD
for PE and PS peak was 0.2% for six replicate injections. The
% RSD for PS and PE for method precision was less than 2%
for both the components. The results of system precision and
method precision were summarized in Table 3 and Table 4.
3.2.4 Accuracy
To confirm the accuracy, two product placebo were
prepared by omitting PE and second omitting PS. All other
ingredients were added at the normal formulation ratios.
Triplicate placebo were spiked at 80%, 100% and 120% of
respective PE and PS. The method concentration level (10
µg/ml PE and 1.0 µg/ml for PS). About 0.5 gram of the
placebo was weighed out into ten 250-mL volumetric flasks.
158 International Journal of Analytical and Bioanalytical Chemistry 2012; 2(2): 151-159
Known concentrations of PE and PS were spiked into each of
the 250-mL volumetric flasks. Each volumetric flask was
diluted to volume with diluents and prepared as per method.
The results of accuracy for PS and PE at all the levels were
within the range of 98%-102%. Table 5 and Table 6 illustrate
the recovery of the method for PE and PS.
3.2.5 Linearity of Detector Response
Linearity studies were performed using PE and PS
reference standard at concentrations corresponding to 50%,
75%, 100%, 125% and 150% of the method target levels (1.0
µg mL-1 of PS and 10.0 µg mL-1 of PE). The results of
linearity experiment revealed that the method was linear in
the range of 50-150% of test concentration of both the
actives. The Co-relation coefficient for both the active was
found to be 1.0. The actual concentrations of the analyte were
presented with the data and linear regression parameters in
Tables 7.
3.2.6 Standard and sample solution stability A working standard was prepared according to the method
and stored at ambient conditions for 24 hours and 6 days. The
stored working standard was assayed against a freshly
prepared working standard (control) at both test points. The
area response for PS and PE from the stored standard was
compared to the response from a freshly prepared working
standard. The response of the both PS and PE was compared
up to 6 days.
One set of six replicate samples were prepared and analysed
as per the method. The same samples were re-analysed after
storage at room temperature at 24 and 48 hours. This was done to simulate unexpected instrument delays. The % w/w
of PE and PS were calculated at each test point and was
compared to the initial results. The result of solution stability
revealed that the standard and sample solution was stable up
to 48 hrs.
3.2.7 Filter interference
A filter study was conducted on a sample solution of the
foam. The sample solution was passed through a Whatman
0.45 µm nylon filter, before dispensing the filtrate into an
HPLC vial. An unfiltered bulk product sample solution was
also used as control. The filtered and unfiltered solutions were assayed as per the method. The results revealed that
there was no interference from nylon filter in quantitation of
PS and PE.
3.2.8 Robustness
To demonstrate method robustness, working standards and
foam samples were analysed by deliberate variation of the
following parameters. Robustness evaluations were
conducted for PE
and PS in foam by varying the following method conditions:
Flow Rate- 0.7 mL/minute ± 10%. Column Temperature-
25°C ± 10%.Change in concentration of phosphoric acid in
mobile phase A. The result of robustness revealed that the change in flow, temperature and % of phosphoric acid in
mobile phase A was not impacting the quantitation of PS and
PE. The results of robustness are summarized in Table 8.0.
3.2.9 Ruggedness Intermediate precision was also studied using different
column and performing analysis on different day with
different analysts. The result of ruggedness are summarised
in Table 9.0. The results of ruggedness study indicated that
the developed method was rugged.
3.2.10 Application of Developed Method
Developed method is stability indicating and can be used
for the simultaneous quantification of PE and PS in coal tar
topical foam in presence of degradation products in stability
by the industry. The quantification of PE and PS help in
evaluation the actual concentration of these in evaluation the
preservative efficacy though out the shelf life of the product.
The hplc method can also be used in other pharmaceutical
formulations containing PE and PS separately or in
combination.
4. Conclusion
The RP- HPLC method for simultaneous determination of PE and PS proves to be simple, linear, precise, accurate and
specific. The total runtime is 42 min within which the PE and
PS peaks and degradation products were separated. The
method was validated showing satisfactory data for all the
method validation parameters tested. The developed method
is stability indicating and can be used for the simultaneous
determination of the PE and PS in formulated products in
quality control and stability studies by the industry. The
method can also be used in evaluating the preservative
efficacy level throughout the shelf life of the formulation.
5.0 Acknowledgement The authors wish to thank the management of Dr.Reddy’s
Laboratories Ltd. for supporting this work. Co-operation
from Formulation and Analytical Development colleagues of
Dermatology Division is highly appreciated for supporting
this work.
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Source of support: Nil; Conflict of interest: None declared