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Validated Simple, Rapid and Accurate HPLC Methods for
determination of Metamizole Sodium in Solid and Liquid Dosage
Forms
Hamide Z. SENYUVA*, Sureyya OZCAN, Burak Veli KABASAKAL
TUBITAK-Ankara Test and Analysis Laboratory
Abstract:
The aim of the study was to develop and validate high performance liquid
chromatography (HPLC) assay for the rapid determination of Metamizole Sodium in
solid and liquid dosage forms. The experimental procedure involved reversed-phaseHPLC with a Zorbax SB C18 column (5 µm particle size, 4.6 ID x 250 mm), metanolwater volumetric solution (80 : 20, v/v) mobile phase, UV detection at 254 nm for
Metamizole Sodium. The flow rate of the, mobile phase was 1 mL/min. The retention
time of Metamizole Sodium is ca. 3.1-3.3 min. Formulations components did not give
rise to any interfering peaks. Calibration curve was linear over the range 0.5-100 µg/mL
for analyte.The metamizole recovery range is at three level (10µg/mL, 15µg/mL and
25µg/mL) 93-100 %. The method was validated with respect to linearity, precision,
accuracy, specificity and robustness. Due to its simplicity and accuracy, the assay
method is suitable for routine analysis of both solid and liquid formulations.
Key words: metamizole, dipyrone, hplc, validation
1-Introduction:
Metamizole (synonym: dipyrone) is sodium sulfonate derived from aminopyrine, a
pyrazolone non-steroidal anti-inflammatory drug (NSAID). Pharmacologic actions are
similar to asprin which is able to inhibit the cyclooxygenase pathaway. this drug
produces a relatively high incidence of allergy and intolerance[1].
Metamizole has been determined by titrimetric[2], uv-spectrophotometric [3-4-5],
colorometric [6], polarographic [7], TLC, HPTLC[5], gas liquid chromatography (GLC)
[8] and HPLC[9] method.Reversed phase HPLC is the seperation method of choice for
most pharmaceutial compounds both hydrophilic and hydrophobic, due to the stable,
reproducible nature of the HPLC columns, the largely aqueous composition of mobile
phase , and relative ease of reproducing the methods in variety of laboratories.
A simple, quick, specific and accurate reversed phase HPLC method to assay
metamizole in solid and liquid dosage forms.Methot was validated with the parameters
of linearity range, precision, recovery and accuracy.
2-Experimental:
2.1- Materials
Methanol (HPLC grade J.T. Baker), Ultra pure water obtained from Millipore water
purification unit. Certificated Metamizole Sodium obtained from Fine Chemical.
2.2- Instrumentation
An Agilent 1100 series High-Performance Liquid Chromatography equipped with
DAD, ALS auto sampler and thermostated column module. The HPLC column was a
Zorbax SB C18 column (5 µm particle size, 4.6 ID x 250 mm)
2.3- Analytical Method
HPLC system was performed by using Zorbax SB C18 column (5 µm particle size, 4.6
ID x 250 mm) column and a mobile phase composed of Methanol:Water (80:20). The
injection volume was 100 µL. 25 oC was column temperature. DAD wavelength is 254
nm.All samples were prepared in water.
* Corresponding Author: Hamide Z. SENYUVA
Fax: +90 312 212 37 49
e-mail: [email protected]
Calibration Solvents 100 µg/mL metamizole stock solutions were prepared and diluted
with water.Calibration Solvents were prepared freshly at the concentrations of 0.5, 1.0,
5.0, 10.0, 15.0, 25.0, 50.0, 100.0 µg/mL by diluting stock solution with water
(r2>0.999).
2.4 Validation of Developed Method
Linearity of the method was established by dublicate injections of solutions containing
metamizole in the range 0.5-100 µg /ml. Determination of intra-day precision, eight
samples of each three different concentrations (5,15,25 µg/ml) were given in one day.
For determination of inter-day precision same series of samples were injected during
three days.The relative standart deviations and correlation coefficients were calculated
for these analyses.Table 1 shows inter-day and intra-day precision.The accuracy of the
method was provided by spiking three different concentrations; 10µg/mL,15µg/mL and
25µg/mL of metamizole into placebo.
3- Result and Discussion
The simple, quick, specific and accurate method was developed to determine
metamizole in solid and liquid dosage forms.
3.1-Linearity Range:
Linearity of the method was established by injecting solutions containing 0.5-100 µg
ml-1 of metamizole (r2>0.999).
3.2 Detection and Quantification Limit:
The detection limit was determined by adding metamizole standart solutions to the
placebo which signal/noise ratio was 3. LOD was 0.4 ng/mL for metamizole. LOQ was
1.3 ng/mL at which signal-to-noise ratio was 10.
3.3 Precision:
Repeatibility studies were performed by spiking of metamizole at three different levels
(5,15,25 µg /ml) in same day dublicate injection (r2 >0.999) . Precision for replicate
analyses in placebo ranges from 93 to 100%.Table 1 shows recovery.
3.4- Accuracy:
The accuracy of the method was provided by spiking three different concentrations;
10µg/mL, 15µg/mL and 25µg/mL of metamizole into placebo. The placebo contained
sodiummetabisulphide, benzyl alcohol and deionized water. .The metamizole recovery
range is 93-100 % for each level.Good recoveries were obtained for each
concentrations, confirming that the method was accurate.Figure 2 shows the
chromatograms of blank and 25 µg/mL spiked sample.
Table1.Recovery of spiked Samples
Conc.(µg/mL)
10
15
25
Validation Recovery
Within-batch (n=8)
Mean %
s%
RSD %
98,6
5,1
5,2
97,4
2,8
2,8
96,4
6,9
7,2
Between Batch (n=3)
Mean %
s%
96,1
3,1
100,0
2,7
92,8
6,3
RSD %
3,2
2,7
6,8
DAD1 A, Sig=254,16 Ref=off (METVALID\MET00173.D)
mAU
400
A
300
blank
200
100
0
2
3
400
B
300
4
3.327 - Ametamizole
re
a:
20
81
.1
9
0
1
DAD1 A, Sig=254,16 Ref=off (METVALID\MET00176.D)
mAU
min
25 ug/mL spike
200
100
0
0
1
2
Figure 2. A- Blank Sample
3
4
min
B- 25µg/mL Spiked Sample
3.5- Applicability of the method to marketed formulation
Seven different samples which three of them are in solid dosage form and four of them
are in liquid dosage form, were analyzed by using proposed method for two
concentration and each was double injected.Table 2 shows the concentrations of
metamizole in marketing drugs.
Table2. Resuls of marketing drugs
Part No
1
2
3
4
5
6
7
Sample ID
Novalgin Ampul
Andolor Ampul
Adepiron Ampul
Novapyrine Ampul
Novalgin Tablet
Andolor Tablet
Devaljine Tablet
1 mg metamizole/2mL
0,99
1,08
1,06
0,99
500 mg metamizole/Tablet
501,17
545.02
460.00
4. Conclusion
The method was validated as linearty, precision, accuracy, specificity and was also
applied to real marketed samples. The assay can be used to asses stability of the liquid
and solid dosage forms. Data obtained for seven products represent two different sample
types liquid and solid .The recovery of the drug was essentially quantitative.
References
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