International Journal of Scientific and Research Publications, Volume 2, Issue 12, December 2012 1

ISSN 2250-3153

A Validated, Specific Stability Indicating Reverse Phase

Liquid Chromatographic Method for the Simultaneous
Estimation of Phenylephrine HCL, Betamethasone
Valerate & Lignocaine HCL in Pharmaceutical Ointment
Safeena Sheikh, Suhail Asghar, Showkat Ahmad Patni

Unijules Life Sciences Ltd, Nagpur - 441501 (Maharashtra) India

Abstract- A validated, specific, stability indicating reverse phase

liquid chromatographic method has been developed for I. INTRODUCTION
simultaneous quantitative analysis of Phenylephrine HCl,
Lignocaine HCl and Betamethasone valerate in pharmaceutical
ointment base products. The method was optimized by analysis
T he product is an anti-hemorrhoid ointment. It is a topical
preparation that is applied directly to external hemorrhoids.
The ointment is helpful in soothing the inflamed veins and as
of the samples and sample solutions spiked with each analyte for well as relieving some of itchiness commonly associated with
recovery study. Good resolution between the analytes was hemorrhoids. It contains combinations of three main ingredients;
achieved in formulation and combined standards on Merck’ C18 Lignocaine hydrochloride, Phenylephrine Hydrochloride and
(250mm X 4.6mm, 5µ) column with mobile phase constituted of Betamethasone Valerate. An extensive literature survey revealed
phosphate buffer (0.01M) and acetonitrile (46: 54% v/v) further HPTLC, HPLC and colorimetric determination for Lidocaine and
the pH of the mobile phase was adjusted to pH=7.0(±0.05) with Phenylephrine hydrochloride in combination with other drugs; an
triethylamine. Detection was performed at 270nm.The method HPLC method utilized for the simultaneous determination of
was validated in accordance with ICH guidelines and validation Miconazole nitrate and Lidocaine and HPTLC method for
data showed that the assay is sensitive, specific and reproducible Lidocaine and Phenylephrine hydrochloride gel dosage form. But
for the simultaneous estimation of Phenylephrine HCl, there is no method which describes the simultaneous
Lignocaine HCl and Betamethasone Valerate in the presence of determination of Phenylephrine hydrochloride and Lignocaine
other pharmaceutical excipient. hydrochloride and Betamethasone Valerate from ointment
dosage form meant for external application. The objective of this
Index Terms- Estimation, Phenylephrine HCl, Lignocaine HCl, investigation was to develop simple, precise accurate and
Betamethasone Valerate, HPLC-UV, Assay, Method Validation. economical procedures for simultaneous estimation of
Phenylephrine HCl, Lignocaine HCl and Betamethasone
Valerate from an ointment dosage form.

Phenylephrine Hydrochloride is a vasoconstrictor that reduces swelling and relief itching and discomfort by tightening blood
vessels. Freely soluble in water and in ethanol (95%); practically insoluble in chloroform.

C9H13NO2, HCl Mol. Wt. 203.67

Figure 1: Chemical Structure of Phenylephrine HCl

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Betamethasone Valerate is an adrenocortical steroid that suppresses inflammation. It is freely soluble in chloroform; soluble in
ethanol (95%); practically insoluble in water and in light petroleum.

C27H37FO6 Mol. Wt. 476.58

Figure 2: Chemical Structure of Betamethasone Valerate

Lidocaine Hydrochloride is a local anesthetic to temporarily relieve pain. It is very soluble in water, freely soluble in ethanol (96
per cent). Melting point is 74 °C to 79 °C.

C14H22N2O, HCl, H2O Mol. Wt. 288.82

Figure 3: Chemical Structure of Lignocaine Hydrochloride

II. MATERIAL AND METHODS IV. MOBILE PHASE PREPARATION

All the reagents were of analytical grade or HPLC grade First the phosphate buffer (0.01M) was prepared by
unless stated otherwise. HPLC grade water was used throughout dissolving 1.36g of monobasic phosphate buffer in 1000ml of
the experiment. Potassium dihydrogen phosphate, acetonitrile distilled water. Then the solution was mixed with Acetonitrile in
and triethylamine were of Merck”. Betamethasone Valerate (B- the ratio 460:540 and then the pH of the mobile phase was
Val), Phenylephrine HCl (PH-HCl), Lignocaine HCl (L-HCl) adjusted to 7.0 (±0.05) with triethylamine.
and the formulation were of Unijules Life Sciences Ltd.

V. PREPARATION OF STANDARD SOLUTION

III. INSTRUMENTATION Standard Stock solution for Phenylephrine HCl
The HPLC system used was of Jasco LC-Net II/ADC Weigh accurately 10 mg Phenylephrine HCl and transfer it
(B211161095), pump, auto sampler, and an UV-2075 variable in to 100.0ml volumetric flask, add about 50.0ml of methanol
wavelength detector was controlled through Borwin software. and make up the volume to 100.0ml with methanol. (Solution A)
Injection volume was 20µl was used. Analytical column used for
this method is Merck” C18 (250mm X 4.6mm, 5µ).

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Standard Stock Solution for Betamethasone Valerate HCl, Lignocaine HCl and Betamethasone Valerate UV-detector
Weigh accurately 10.0 mg Betamethasone Valerate and were used. The chromatographic conditions used for the
transfer to 10.0 ml volumetric flask, add 5.0 ml purified estimation were given below.
methanol and allow it to dissolved with sonication and make up Column : Merck’ C18 (250mm
the volume with methanol to 10.0ml. Take 5ml stock solution in X 4.6mm, 5µ)
100.0ml volumetric flask and dilute with methanol up to the Wavelength : 270 nm
mark. (Solution B) Injection Volume : 20µl
Flow rate : 1.5 ml/min
Standard Stock solution for Lignocaine HCl Column Temperature : Ambient
Weigh accurately 250.0mg of Lignocaine HCl and transfer
it in to 100.0ml volumetric flask, add about 50.0ml of methanol
and make up the volume to 100.0ml with methanol. (Solution C).
VI. RESULT AND DISCUSSION
Combine Standard Method development
Take accurately 5.0ml of each of the above solutions (i.e. The primary target in developing this stability indicating LC
solution A, B and C) in 50.0ml volumetric flask and dilute up to method was to achieve the resolution between Phenylephrine
the mark with mobile phase. HCl, Betamethasone Valerate and Lignocaine HCl and the
formulation excipient. To achieve the separation of related
Sample Solution substances, stationary phase of C18 and a combination of mobile
Take an accurately 5.0g weighed amount of the sample in phase phosphate buffer with methanol and Acetonitrile were
100.0ml of beaker add 40.0ml of methanol and warm on water used. The separation of formulation excipient and all three active
bath at 60ºC for about 10-15minutes; cool with stirring. Transfer ingredients was achieved a Merck” C18 column and a mobile
the supernatant liquid to 100.0ml volumetric flask keeping the phase composed of [potassium dihydrogen phosphate buffer and
ointment in beaker. Repeat the procedure thrice with another Acetonitrile in the ratio 460:540 and Triethylamine was used to
fresh quantity of methanol. Allow to cool the volumetric in ice adjust the pH of the mobile phase to 7.0. Mobile phase flow rate
bath and make up the volume to 100.0ml with methanol. Dilute was maintained at 1.5ml/min and eluent were monitored at
5.0ml of the above solution to 50.0ml with mobile phase. 270nm. A 20µl of the sample was injected using a fixed loop.
The developed method/chromatograms showed that the method
Chromatographic Conditions was highly specific and all the actives were well resolved from
The mobile phase was filtered through 0.45µm, Nylon each other. The Chromatograms of all the actives and sample
membrane filter and degassed using vaccum before delivering it (ointment) are given below (Figure 4a & 4b).
in to the HPLC system. For detection of analyte Phenylephrine

Figure 4 (a): Typical chromatograms of combined standards and product.

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Info:
STD – 3-0028E1201
Group: UNIJULES
Control Method:

# Name RT Area [µV.Sec] Factor Plates

1. Phenylephrine HCl 2.652 110362.695 1.000 4952.03

2 Lignocaine HCl 4.815 384922.210 1.000 5962.41
3 Betamethasone Valerate 5.421 89510.674 1.000 9263.25

Total Area of Peak = 584795.579 [µV.Sec]

Figure 4 (b): Typical Chromatogram of Sample

Info:
OINTMENT
Group: UNIJULES
Control Method:

# Name RT Area [µV.Sec] Factor Plates

1 Phenylephrine HCl 2.633 110212.253 1.000 4985.36

2 Lignocaine HCl 4.862 385422.104 1.000 5962.14
3 Betamethasone Valerate 5.421 89523.514 1.000 9245.17

Total Area of Peak = 585157.871 [µV.Sec]

impurities, degradation-products, matrix, etc. The specificity of

VII. METHOD VALIDATION the developed method for Phenylephrine hydrochloride,
Specificity Betamethasone Valerate and Lignocaine hydrochloride. The
Specificity is the ability of the method to asses placebo was not interfering with the actives.
unequivocally the analyte in the presence of components, which
may be expected to be present. Typically these might include

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Linearity and range hydrochloride at different levels. The data were subjected to
The linearity of detector response to different concentrations statistical analysis using a linear model; the calibration curves of
of actives was studied in the range of 1.0 to 12.0mcg/ml for PH-HCl, L-HCl and B-Val are shown in (figure 5(a), 5(b), 5(c)).
Phenylephrine hydrochloride, 1.0 to 8.0mcg/ml for Regression characteristics of the proposed HPLC method are
Betamethasone Valerate and 40.0 to 320.0mcg/ml for Lignocaine given in (Table 1).

Linearity of Phenylephrine HCl y = 13490x

R2 = 0.9993
180000
160000
Area under curve

140000
120000 Series1
100000
Series2
80000
60000 Linear (Series2)
40000
20000
0
0 2 4 6 8 10 12 14
Concentration (mcg/ml)

Figure 5(a): Calibration Curves of Phenylephrine HCl.

Series1
Linearity of Betamethasone Valerate
Series2
Linear (Series2)
160000
140000
Area Under Curve

120000 y = 17457x
100000 R2 = 0.9995
80000
60000
40000
20000
0
0 1 2 3 4 5 6 7 8 9
Concentration (mcg/ml)

Figure 5(b): Calibration curves of Betamethasone Valerate

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Linearity of Lignocaine HCl

600000 y = 1525.5x
R2 = 0.9995
500000
Area under curve

400000 Series1
300000 Series2
200000 Linear (Series2)

100000
0
0 50 100 150 200 250 300 350
Concentration (mcg/ml)

Figure 5(c): Calibration curves of Lignocaine HCl

Table 1: Regression characteristics of the proposed HPLC method.

Linearity Experiment Phenylephrine Betamethasone Lignocaine

Hydrochloride Valerate Hydrochloride
Range (mcg/ml) 1 to 12 40 to 320 40 to 320
Regression coefficient (r2) 0.9993 0.9995 0.9995
Slope 13490 17457 1525.5

Precision to be 0.909, 0.804 and 1.093 for PH-HCl, L-HCl and B-Val
Precision was measured in terms of repeatability of respectively (Table.2). This shows that the precision of the
measurement, performed by injecting the standard solution six method is satisfactory as relative standard deviation is not more
times (n=6) and measuring the peak areas. The RSD was found than 2.0%.

Table 2: Results of Precision

Number of Area’s of Active ingredients % Assay

Injections Phenylephrine Lignocaine HCl Betamethasone
HCl (PH-HCl) (L-HCl) Valerate (B.Val)
(PH-HCl) (L-HCl) (B-Val)
Injection 1 110010.253 385422.104 89823.514 99.91 99.63 101.36
Injection 2 110209.223 385099.809 90890.098 100.09 99.54 102.57
Injection 3 109989.833 391999.981 87987.500 99.89 101.32 99.28
Injection 4 110201.199 384997.986 89535.098 100.08 99.51 101.04
Injection 5 112521.562 390899.900 89529.498 102.19 101.05 101.03
Injection 6 111218.259 386886.910 88890.395 101.01 100.01 100.24
Mean 100. 52 100.17 100. 92
LIMIT
NMT 2. 0% SD 0. 913 0. 805 1. 104
%RSD 0. 909 0. 804 1. 093

Intermediate Precision: Accuracy

Intermediate precision also called as ruggedness of the The accuracy of the method was determined by recovery
method it was determined by analyzing standard solutions by two study carried out using standard addition method at three
different analysts, using different instruments, in two different different concentration levels. The resulting spiked sample
labs and on different days. The values of RSD obtained under solutions were assayed in triplicate and the result obtained were
set-I conditions were 0.747, 0.959 and 0.594 for PH-HCl, L-HCl compared with the expected results and expressed as percentage
and B-Val respectively. As the values of RSD for the two sets of recovery. The mean percentage recoveries of PH-HCl, L-HCl
conditions are below 2.0% for all the drugs, intermediate and B-Val were found to be 100.94, 100.01 and 100.98
precision of the method is established. respectively which are within the acceptance limit.Table.3

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Table 3: Results of Accuracy of Experiment

Amount of Sample Amount of drug added Amount recovered % Recovery

PH- L-HCl B-Val PH- L-HCl B-Val PH- L-HCl B-Val PH-HCl L-HCl B-Val
HCl µg/ml µg/ml HCl µg/ml µg/ml HCl µg/ml µg/ml µg/ml µg/ml µg/ml
µg/ml µg/ml µg/ml
10 250 0.5 8 200 0.4 18.19 449.75 0.908 101.055 99.94 100.88
10 250 0.5 8 200 0.4 18.20 449.55 0.915 101.11 99.90 101.66
10 250 0.5 8 200 0.4 18.10 450.55 0.910 100.55 100.04 101.11
10 250 0.5 10 250 0.5 20.33 500.38 10.08 101.65 100.10 100.80
10 250 0.5 10 250 0.5 20.25 500.29 10.20 101.25 100.10 102.00
10 250 0.5 10 250 0.5 20.29 500.45 10.11 101.45 100.09 101.10
10 250 0.5 12 300 0.6 22.10 549.98 11.01 100.45 99.99 100.09
10 250 0.5 12 300 0.6 22.10 549.97 11.08 100.45 99.99 100.72
10 250 0.5 12 300 0.6 22.11 550.00 11.05 100.50 100.0 100.45
Mean % Recovery 100.94 100.01 100.98

Robustness phase composition and pH change were studied and retention

It is a measure of its capacity to remain unaffected by small time of PH-HCl, B-Val and L-HCl was noted. The factor selected
but deliberate variations in the method parameters and provides were flow rate, pH and % Acetonitrile in the mobile phase. It was
an indication of its reliability during normal usage. To determine observed that there were no deliberate changes in the
the robustness of the developed method, typical variations in chromatogram, which demonstrated that the RP-HPLC method
analytical conditions were tested. Influence of flow rate, mobile developed, are robust .Results describe in Table V.

Table 4: Robustness data

Factor Level Retention time

PH-HCl L-HCl B-Val

Flow rate ml/min

1.4 -0.1 2.821 4.955 5.766
1.5 0 2.675 4.852 5.425
1.6 +0.1 2.524 4.732 5.310
pH of the mobile phase
6.9 -0.1 2.670 4.845 5.560
7.0 0 2.675 4.852 5.425
7.1 +0.1 2.723 4.861 5.586
% Acetonitrile in the mobile phase
53 -1 2.635 4.822 5.400
54 0 2.675 4.852 5.425
55 +1 2.680 4.873 5.455

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