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:vairale_ajay@rediffmail.com 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

Available online at http://www.urpjournals.com

International Journal of Analytical and Bioanalytical Chemistry

Universal Research Publications. All rights reserved

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