Analytical Letters

ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: http://www.tandfonline.com/loi/lanl20

Determination of Naphazoline Nitrate and

Antazoline Sulphate in Pharmaceutical
Combinations by Reversed-Phase HPLC

S. I. Sa‘sa’ , I. F. Al-momani & I. M. Jalal

To cite this article: S. I. Sa‘sa’ , I. F. Al-momani & I. M. Jalal (1990) Determination of Naphazoline
Nitrate and Antazoline Sulphate in Pharmaceutical Combinations by Reversed-Phase HPLC,
Analytical Letters, 23:6, 953-971, DOI: 10.1080/00032719008053438

To link to this article: http://dx.doi.org/10.1080/00032719008053438

Published online: 23 Oct 2006.

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 ANALYTICAL LETTERS, 2 3 ( 6 ) , 953-971 (1990)

DETERMINATION OF NAPHAZOLINE NITRATE AND

ANTAZOLINE SULPHATE IN PHARMACEUTICAL COMBINATIONS
BY REVERSED-PHASE nPLc
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KEY WORDS: Reverse Phase, Naphazoline Nitrate, HPLC,

Antazoline Sulphate, Stability, Analysis.

S.I. Salsa' and I.F. Al-Momani

Chemistry Department, Yarmouk University, Irbid, Jordan
I.M. Jalal
Al-Hikma Pharmaceuticals, P.O.Box 182400, Amman, Jordan

ABSTRACT
A high performance liquid chromatographic procedure
is presented for the simultaneous determination of
naphazoline nitrate and antazoline sulphate in pharma-
ceutical combinations. An aliquot of the sample is
dissolved in water containing N-butyryl-4-aminophenol
as an internal standard and is chromatographed on a
supelcosil LC-8 ( 5 w) (150 mm x 4.6 mm i.d.) column
using a mobile phase of acetonitrile/water/triethyl-
amine (40:59.75:0.25) adjusted to pH 4.5 by glacial
acetic acid. This proposed HPLC methodology has the
advantages of being fast and stability-indicating.

953

Copyright 0 1990 by Marcel Dekker, Inc.

 9 54 SA'SA' , AL-MOMANI. AND JALAL

It was verified for linearity, accuracy, precision and

was applied sucessfully to commercial products.

INTRODUCTION
Naphthazoline nitrate (I) has a potent vasocon-
strictor action. It has both rapid and prolonged
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action in suppressing conjunctival hyperaemia and is

used locally with antazoline sulphate (11) in nasal and
eye drops for nasal and occular allergies. 1 , 2

The development of efficient methods for the sim-

ultaneous analysis of naphazoline nitrate and anta-
zoline sulphate in pharmaceutical preparations has
received little attention in recent years. The analyt-
ical methodologies used are either colorimetric3 - 6 , by
forming colored compounds with the active ingredients,
or ultraviolet7 . These methods are of low sensitivity,
time-consuming and lack specificity.

Much work has been concerned with the use of

reverse-phase ion-pair HPLC technique to study the
retention behaviour of basic drugs and some of them
to determine naphazoline nitrate or antazoline sulphate
with other compound^^-'^. An excellent separation for
naphazoline with other compounds, such as tetrahydro-
zoline and chloropheniramin, was achieved on reverse
 NAPHAZOLINE NITRATE AND ANTAZOLINE SULPHATE 955

phase packing11r12. In these methods I and I1 were

separated from other active ingredients.

This paper describes a reversed-phase HPLC method

for the determination of I and I1 in commercial products.
The assay was applied successfully to two commercial
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products and proved to be accurate, pecise and free

of interferences from excipients.

I I1

EXPERIMENTAL

Materials
Generally, all chemicals used were the purest
available and used as received without further purifi-
cation. Water used was always distilled and deionized.
Acetontrile was HPLC grade from (May and Baker,
Dagenham, England). Glacial acetic acid was (99%) from
(Kock-Light, Haverhill, England). Ammonium acetate
(BDH, England) and triethylamine ( Chambrian Chemicals ,
England) were 99%. The active ingredients naphazoline
 956 SA'SA' , AL-MOMANI, A N D JALAL

nitrate and antazoline sulphate were obtained from

(Medilide, Italy). Both are USP reference standards.
The internal standard, N-butyryl-4-aminophenol was also
a USP reference standard.

Pharmaceutical Combinations (ophtazoline) and

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(antistin-privin)eye drops were purchased locally.

Apparatus
A liquid chromatograph comprised of a Du pont 8800
pump, Rheodyne 7125 loop-injector, Du pont variable
W-absorbance detector, spectra-physics (SP-4100)
integrator and a reverse-phase column (150 mm long x
4.6 mm i.d., dp = 5 v). supelcosil LC-8 from (Supelco
Inc., Pennsylvania, U.S.A.) was used.

Chromatographic Conditions
The mobile phase used consists of acetonitrile/
water/triethylamine (40:59.75:0.25, v/v) adjusted to
pH 4 by glacial acetic, acid. The mobile phase was
filtered using 0.45 microporous filters and was
degassed by vacuum prior to use. Also the sample
solutions were always filtered using 0.45 membrane
filters to prolong pump life and to avoid colum
blockage by any particulate matter.
The chart speed was 0.5 cm/min. The flow rate was
2 mL/min. The wavelength 285 run. the detector sensit-
 NAPHAZOLINE NITRATE AND ANTAZOLINE SULPHATE 957

ivity was ( 0 . 1 6 ) AUFS. The injection volume was 10 ILL

and the internal standard was N-butyryl-4-aminophenol.

Preparation of the Standard Solutions

Internal standard solution - The stock internal
standard solution was prepared by dissolving 4 mg of
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N-butyryl-4-aminophenol in 100 mL of water.

Standard solutions for linearity - Six different

concentrations have been prepared for each compound
covering the range of 25-150% of the expected working
range. These solutions were prepared by dissolving
6.25 mg of I in 250 mL internal standard solution.
The following concentrations of I in the internal
standard solution were prepared by proper dilutions:
0.0025, 0.0050, 0.0075, 0.0100, 0.0125 and 0.0150 mg/mL.
A l s o 50 mg of I1 was dissolved in 1 00 mL internal
standard solution and the following concentrations of
I1 in internal standard solution were prepared by
proper dilutions: 0.050, 0.100, 0.150, 0.200, 0.250

and 0.300 mg/mL.

Preparation of the Sample Solutions

The sample stock solutions were prepared by direct
dilution of the drug with the internal standard solution
to the required concentration.

Percent recovery study - This study was performed

by standard addition method in which standard solutions
 958 SA'SA' , AL-MOMANI, A N D J A L A L

of I (0.0020, 0.0035 and 0.0050 mg/mL) and the following

amounts of I1 (0.040, 0.070 and 0.100 mg/mL) were added
to a known amount of the drug. The resulting mixtures
were assayed and the results obtained were compared
with the expected ones.
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Content uniformity - The uniformity tests for both

compounds were performed on a commercial product
(ophtazoline) by the developed method, one mL of the
drug was diluted to 25 mL with internal standard
solution. Ten bottles of the drug were treated by the
same manner.

Stability Study
The stability of both compounds was studied by
keeping each compound in a dark bottle in a thermostat
at 7 O o C for a period of 30 days. Then accurately
weighed samples were taken at different intervals of
time and dissolved in100 mL of the internal standard
solution. The solution was sonicated for 5.0 minutes,
filtered through 0.45 wn membrane filter, then 4.0 mL
was diluted to 10 mL with the internal standard
solution.

Assay Method
Equal volumes of (10 vL) and approximately equal

concentrations of freshly prepared standard and sample

 NAPHAZOLINE NITRATE AND ANTAZOLINE SULPHATE 959

solutions were injected into the HPLC and were chroma-

tographed under the same conditions described above.
The standard and sample solutions contained the same
concentrations of the internal standard. The quantity
of each component injected was always within the linear
range.
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Calculations
The results were calculated using response ratios
(RR) relative to internal standard based on peak areas
or peak heights.
Percent of the label claim found = -x
RRX
100
RRS

where RRx = sample response ratio, and RRs = standard

response ratio.

RESULTS AND DISCUSSION

In order to optimize the chromatographic para-
mete the effects of acetonitrile concentration,
pH, and the triethylamine concentration on the capa-
city factor (k') were studied (Figs. 1 - 3 ) .

The capacity factor (k') for I, I1 and the internal

standard were affected by the variation of acetonitrile
concentration in the mobile phase (Fig. 1). At high
acetonitrile concentration all compounds gave very
sharp peaks and were very close to each other with low
 960 SA'SA', AL-MOMANI, A N D JALAL

t
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k'

'

2 5% 3 0% 35% 4 0% 45%

ACETONITRILE CONC. (VIV%)

Figure 1. Plots of the capacity factor (k') versus

the acetonitrile percentage in the mobile
phase.

k' values. Decreasing the acetonitrile concentration

gave better resolution with suitable analysis time.
Lowering the acetonitrile concentration below 35% led
to broader peaks with longer retention time. Therefore
a mobile phase of 40% acetonitrile was selected because
it provided baseline separation and sharp peaks in a
reasonable time.
 NAPHAZOLINE NITRATE AND ANTAZOLINE SULPHATE 961

I1 L 1 A -

2
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k' l o - -
" " J

1.s- * *-

Figure 2 . Plots of the capacity factor versus the pH

of the mobile phase.

Variation of pH (Fig. 2 1 , has no effect on the k'

value for all compounds, except the internal standard
where the k' value slightly decreased as pH decreased.
Thus pH = 4.5 was selected as the working pH value.

The capacity factor values of I, I1 and the inter-

nal standard have been affected by the variation of
triethylamine percent in the mobile phase. At lower
concentrations of triethylamine k' for I and I1 in-
creased to large value (Fig. 3 1 , while k' values for
 962 SA'SA', AL-MOMANI, A N D J A L A L
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TRIETHYL AMINE (V/V%)

Figure 3 . Plots of the capacity factor versus the

triethylamine percentage in the mobile
phase.

the internal standard were slightly affected. At

higher concentrations of triethylamine ( 0 . 5 % ) , k' f o r

I and I1 also decreased with complete interference
between the peaks of internal standard and I. Therefore
the selected concentration of triethylamine was 0.25%,
since this concentration provides optimum separation
among the peaks of I, I1 and the internal standard
 NAPHAZOLINE NITRATE AND ANTAZOLINE SULPHATE 963

and because it provides better separation of the

internal standard from I.

To dqtermine the linearity of the detector response,

calibration standard solutions of I and I1 were prepared
as described in the text. A plot of the detector res-
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ponses as peak area,ratios versus amount injected was

linear in the range of 0.0025 - 0.0180 mg/mL for I,
with a correlation coefficient of 0.9999, and 0.0500 -
0.3000 mg/mL for 11, with a correlation coefficient of
0.9998

To determine the accuracy of the method, a recovery

study was conducted by the standard addition method
using HPLC analysis. In all cases, satisfactory
recoveries and reproducibility of peak heights and
areas were obtained. A linear regression of the data
shows excellent linearity over the analysis range
studied (Tables I and 11). No interferences due to
excipients were detected in the chromatograms produced.

The chromatograms shown in Figures ( 4 , s ) indicate

that it is possible to ac.%eve a baseline separation of
I, I1 and the internal standard from other pharmaceuti-
cal excipients usually used in formulation.

The analytical results of two commercial products

(Table 111) indicate that the proposed assay can be
 964 SA'SA', AL-MOMANI, A N D J A L A L

Table I: Recovery of Naphazoline Nitrate by the Standard

Addition Method.

Total mg Present mg Founda % Recoverya

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0.0100 0.0101f2.1 100.5f2.1

0.0120 0.012121.1 100.4fl. 1
0.0135 0.0134t1.0 99.621.0
0.0150 0.0151f1.6 100.721.6

Slope = 0.9872 Intercept = 0.0002 R = 0.9990

aMeanfRSD for 6 determinations.

Table 11: Recovery of Antazoline Sulphate by the

Standard Addition Method.

Total mg Present mg Founda % Recoverya

0.2000 0.2006f2.0 100.3f1.9

0.2400 0.243420.6 101.4f0.8
0.2700 0.2720f1.2 100.7f1.2
0.3000 0.3039f1.2 100.3fl. 1

Slope = 1.027 Intercept = -0.0043 R = 0.9998

aMeanfRSD for 6 determinations.
 NAPHAZOLINE NITRATE AND ANTAZOLINE SULPHATE 965

a-Std.
b-Oph'
- 01 ine
-
I

1.s
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Figure 4. a) A typical chromatogram of a standard 10 pL

injection containing 0.1 ug naphazoline
nitrate (I) (tR = 2.03 min) and 2.0 pg
antazoline sulphate (11) (tR = 3.42 min)
in the I . S . Column, 150 mm long x 4.6 mm
i.d. supelcosil LC-8, 5 w; Eluting
solvent, ACN/water/TEA (40:59.75:0.25)
adjusted to pH 4.5 by glacial acetic acid;
Temperature, ambient; Flow rate, 2 mL/min;
0.16 AUFS; h = 285 ~ n .
b) A chromatogram for 1 mL ophtazoline sample,
25 mL internal standard under the same
conditions.

used for the quantitation of both active compounds.

The results show that the proposed method is accurate,
precise and fast.

The specificity of the method is confirmed by the

content uniformity study of both compounds which show
 966 SA'SA', AL-MOMANI, A N D J A L A L

a-Std.
I b-Antistin-Privin

1.s
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i h.
Figure 5 . a) A typical chromatogram of a standard 10 uL
injection containing 0.10 ug naphazoline
nitrate ( I ) and 2.0 ug antazoline sulphate
( 1 1 ) in the internal standard (I.S.).
Column, 1 5 0 mm long x 4.6 m i.d. Supelco-
sil LC-8, 5 pm; Eluting solvent, ACN/water/
TEA (40:59.75:0.25) adjusted to pH 4 . 5 by
glacial acetic acid; Temperature, ambient;
Flow rate, 2 mL/min; 0.16 AUFS; A = 285 nm.
b) A chromatogram for 1 mL antistin-Privin
sample.25 mL internal standard under the
same conditions.

compliance to specifications of all bottle forms and

support the specificity of the HPLC results (Table IV)
and (Fig. 6 1 .

The results obtained for the stability study of

both compounds shown in (Table V ) reveal no signifi-
 NAPHAZOLINE NITRATE AND ANTAZOLINE SULPHATE 967

Table 111: Results for the Analysis of Naphazoline

Nitrate and Antazoline Sulphate.

Product Label Claim Percent Label Claim

(mg/mL1 Founda

Naphazoline Antazoline Naphazoline Antazoline

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Nitrate Sulphate Nitrate Sulphate

Ophtazoline 0.25 5.00 100.3t1.0 98.1fl.l

(Lot 72004)
103.0t1.3 101.3f1.8
101.5t2.3 101.4k2.0

Ant istin- 0.25 5.00 99.9k1.4 98.421.8

privin
(Lot 6234) 99.8+0.8 99.4f0. 7
100.0k0.5 99.321.2
~~

aMeanfSRD for 5 determinations.

cant loss in activity of I1 throughout the study,

but a considerable degradation was noticed through the
period of study of I.l3 However, no new peaks were
noticed.

The assay presented here has been shown to be

applicable to commercially available products. The
method is quite simple, accurate, precise, rapid and
easy to perform.
 968 SA'SA', AL-MOMANI, A N D J A L A L

Table I V : Content U n i f o r m i t y of I and I1 i n

O p h t a z o l i n e Drug.
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B o t t l e No. P e r c e n t Label Claim Found

I I1

1 97.7 96.3

2 99.6 100.1
3 99.6 101.4

4 100.7 100.9

5 102.1 101.4

6 100.6 99.0

7 102.7 101.4

8 98.1 99.0

9 97.1 97.0

10 102.1 99.8

Average 100.0 99.6

RDS 2.0 1.8

High 102.7 101.4

LOW 97.1 96.3

 NAPHAZOLINE NITRATE AND ANTAZOLINE SULPHATE 969
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Figure 6. Content uniformity chromatograms for 10

bottles of ophtazoline for I and 11.

Table V: Stability of I and 11.

~ ~ ~~

Period Elapsed, Days mg ra mg I I ~

0 0.75fl. 3 3 2.72k1.47
5 0.73f1.36 2.71f0.74
11 0.73k1.37 2.71f0.37
16 0.6721.43 2.71f0.74
20 0.66k3.03 2.71k1.48
28 0.64f1.56 2.7020.74

a
MeanfRSD for 5 determinations.
 970 SA'SA' , AL-MOMANI, AND JALAL

ACKNOWLEDGEMENTS
The financial support provided by Yarmouk
University is gratefully acknowledged. The authors
would also like to thank Al-Hikma Pharmaceuticals,
Amman, Jordan, for providing the raw materials and
standards. The technical help by Mr. Hamed Khalil
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is highly appreciated. Also, the authors would like

to thank Dr. Khamis Abbas for his assistance.

REFERENCES

1. The Pharmaceutical Codex, 11th ed., The Pharmaceu-

tical Press, London, pp, 50, 585 (1979).
2. W. Martindale, The Extra Pharmacopoeia, 27th ed.,
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3. L. Szabolcs, Acta Pharm. Hung., 50(3), 130 (1980).
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30(6), 18 (1980).
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@(9), 1135 (1979).
 NAPHAZOLINE NITRATE AND ANTAZOLINE SULPHATE 971

11. J . A . De Schutter, W.B. Van Den and P. De Moerloose,

J . Chromatogr., =(1), 303 (1987).
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Received November 19, 1989

Accepted !larch 29, 1990