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Radi & al. / Mor. J. Chem.

3 N1 (2015) 58-64

Optimization and Validation of a Method for Simultaneous Determination


of Amoxicillin and Clavulanic acid by HPLC in Different Pharmaceutical
Forms

Mohammed Radi*1, Youssef Ramli*2, Miloud El Karbane1, Aziz ElAlami1, Kalid


Karrouchi1, Saadia Issmaili1and Karima Bakhous1

1
National Medicines Control Laboratory, Rue LamfadalCharkaouiMadinat Al Irfane BP 6206/Rabat-
Morocco.
2
Medicinal ChemistryLaboratory, Faculty of Medicine and Pharmacy, Mohammed V University,Rabat-
Morocco

*Corresponding author. E-mail : mohammedradi63@yahoo.fr and yramli76@yahoo.fr


Received 05 Oct 2014, Revised 27 Oct 2014, Accepted 12 Dec 2014

Abstract: A simple, fast, economical, accurate, precise and reproducible RP HPLC method with
ultraviolet detection at 220 nm has been validated for the simultaneous determination of Amoxicilline and
Clavulanique acid. A good chromatographic separation between both compounds was achieved using a
reversed phase C18 column and a mobile phase, consisting of Acetonitril / NaH2PO4.buffer pH = 4.4). The
method was validated in terms of specificity, linearity, precision, accuracy, an drobustness.This fully
validated method, which allows the simultaneous measurement of amoxicillin and clavulanic acid in
different formulations, is rapid (total run time <7 min).This assay is suitable for routine quality control of
drugs.

Keywords: Amoxicilline, clavulanique acid, HPLC, method validation.

1. Introduction
Clavulanic acid or (2R, 3Z, 5R) -3-(2-hydroxyethylidene) -7-oxo-4-oxa-1azabicyclo [3.2.0] heptane-2-
carboxylic acid is abeta-lactamase inhibitor, administered on junction with some penicillins (suchas
amoxicillin and ticarcillin) to broaden the spectrum. It comes to a beta lactam antibiotic which is very low
antibiotic activity, but his liaison withbeta-lactamases is irreversible. It is a natural substance produced by
Streptomyces clavuligerus (Fig 1). Amoxicillin or [2-amino-2-(4-hydroxyphenyl) acetyl] amino-3,3-
dimthyl-6-oxo-2-thia-5-azabicyclo [3.2.0] heptane-4-carboxylic acid-7antibiotique-is a family-lactamine
bactericide amino penicillinin dicated for the treatment of bacterial sensitive infections bacteria. Amoxicillin
is the antibiotic most commonly used, especially in children because it has good oral absorption, abroad
antimicrobials spectrum and a cost (Fig 2).

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Radi & al. / Mor. J. Chem. 3 N1 (2015) 58-64

Fig. 1: Structure of clavulanic acid Fig. 2: Structure of amoxicillin

Based medicines amoxicillin and clavulanic acid are available in different pharmaceutics forms. Currently,
measurement of these molecules in the finished products is done through various methods [1-17]. In this
study, we propose a simple and rapid analytical method for the determination of these active ingredient sin
these pharmaceutics forms. The aim of such a move is to help laboratories and Drug specialists
pharmaceutical industry, to reduce the time and cost of analysis and subsequently minimize chemical
releases. In this work, we developed and validated according to ICH Q2B guidelines strategy[18]
(International Conference on Harmonization), a simple and rapid RP-HPLC method. It is specific, linear,
accurate, precise and robust. It is capable of simultaneously dosing amoxicillin and clavulanic acid with a
good resolution (> 3.5) in the different pharmaceutical forms: tablet, syrup, capsule and injectable solution.
This method also has the advantage of being used for the dosage of these pharmaceutical forms having only
amoxicillin as active ingredient.

2. Experimental section
2.1. Apparatus:
The chromatographic systems used is constituted by a Waters 2695 pump, auto sampler and Waters 2998
photodiode array detector (PDA) with Spectra Manager software and Empower Software data registration
were used for all absorbance measurements. Data acquisition was performed by the Totalchrom Software
data registration (USA), the Mettler Toledo scale made in Switzerland.

2.2.Reagents and standards:


The standard used for the determination of amoxicillin is a working standard having a purity of 100.3% and
the water content is 13.0% and that of clavulanic acid was 99.2% and water content is 0.0%. All reagents
used for the preparation of the mobile phase are reagent grade except the Acetonitril is HPLC
grade.Potassium phosphate monobasicdihydrate(NaH2PO4, 2H2O)and Acetonutril weresuppliedfromSigma -
Aldrich (Germany). The placebo used in the validation process consists of the usual excipients present in the
commercial formulation (Silica gel "amorphoussilica" Collodal anhydroussilica, magnesiumstearate,
hydroxypropyl cellulose, macrogol 4000, macrogol 6000, dioxidetitanium, crospovidone, hydroxypropyl
LS, XanthanGum, sodium Benzoate, Aspartame, aroma of red fruits, propylparaben, methylparaben.

2.3. Chromatographic conditions:


The chromatographic conditions are gathered in the table 1. The mobile phase was filtered through a 0.45-
m Millipore filter and degassed by vacuum prior to use.
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Radi & al. / Mor. J. Chem. 3 N1 (2015) 58-64

Tab. 1 :Chromatographic conditions of the method


Colomn Column symmetric C18, 250 mm 4.6 mm, 5um.
Flow 1 ml/min
Temperature Ambient Temperature.
Wavelength 220nm
Injection volume 20 l
Mobile phase (Acetonitril / buffer pH = 4.4): (10% / 90%). pH = 4.4 (5.15g
NaH2PO4, 2H2O in 1000 ml of distilled water).
Retention time 8.0 min
dilution medium Distilled water

3. Results and discussion


3.1 The specificity of the chromatographic method:
The selectivity of the method was confirmed by the absence of potential interference caused by the
excipients with one hand amoxicillin and clavulanic acid of the other part, by comparing the chromatograms
of the blank, placebo, active ingredient alone (AIA), and that of the reconstituted pharmaceutical form (RPF)
(Fig 2a). Comparison of chromatograms of peak purity of amoxicillin and clavulanic acid with that of the
baseline was considered better (stackable) indicating that no additional peaks were eluted with the active
ingredients (Fig2b).
This shows that the method is capable of easily assayed amoxicillin and clavulanic acid in the presence of
these excipients.

Fig.2a: Chromatograms of Blank, Placebo, AIA and RPF.

Fig.2b: Chromatographic purity of amoxicillin and clavulanic acid


3.2 Linearity
The linearity of the method was determined by preparing 3 series of five minimum concentrations (70% -
85% -100% -115% -130%) of the target concentration (500g / ml of amoxicillin and 62,5g/ml clavulanic
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Radi & al. / Mor. J. Chem. 3 N1 (2015) 58-64

acid) of the active ingredients alone (AIA) and reconstituted pharmaceutical form (RPF). Take the average
of each injection zone and graphing the average peak relative to the actual concentration of each solution
(active ingredients alone: Fig 4 and reconstituted pharmaceutical form: Fig5).

Fig. 3 : Chromatograms of the linearity of the method

20000000 20000000
y = 22916x + 47825 y = 23060x + 35351
15000000 R = 0,992 15000000 R = 0,995

10000000 10000000

5000000 5000000

0 0
0,0000000
20,0000000
40,0000000
60,0000000
80,0000000 0,0000000
20,0000000
40,0000000
60,0000000
80,0000000

Fig4: Linearity of active ingredient alone Fig 5: Linearity of Reconstituted form

Table 2:Study of the linearity of the method.


Amoxicillin Clavulanic Acid
AIA RPF AIA RFP
Regression equations y=229167x+478258 y=230602x+353513 y=27894x+30854 y=27881x+26483
Slope :a 229167 230602 27894 27881
Intercept: b 478258 353513 30854 26483
2
Correlation coefficient: r 0,993 0,995 0,992 0,998

Confidence interval of a Max=238706,89 Max=238339,22 Max=29143,01 Max=28469,02


Min= 219627,14 Min=222865,29 Min=26645,43 Min=27293,90
Confidence interval of b Max=968911,25 Max=750261,19 Max=110963,95 Max=64074,76
Min= -12394,66 Min= -43235,90 Min= -49255,98 Min= -11109,40

The regression equations of active ingredient alone and reconstituted pharmaceutical form are linear for
amoxicillin and clavulanic acid (Table 2).

3.3 Precision (repeatability and intermediate precision).


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Radi & al. / Mor. J. Chem. 3 N1 (2015) 58-64

The repeatability and intermediate precision were validated as described in the ICH guidelines Q2B [10] by
performing 3 series (3 different operators) six samples each containing 100% of active ingredients in a
reconstituted pharmaceutical form (Fig 6).

Fig. 6: Chromatogram of the repeatability of the method

The relative standard of deviation of repeatability and intermediate precision of the two active ingredients are less than
2% (Table 3), we can conclude that the analytical method chosen is faithful.

Tab. 3: Study of the precision of the method.


Amoxicillin clavulanic Acid
Repeatability RSDr=1,14% RSDr=1,12%
Inter mediate precision RSDIP=0,96% RSDIP=0,62%

3.4 Accuracy
The accuracy of the method was determined on 3 series (3 different operators) of the reconstituted
pharmaceutical form having five concentration levels (70% -85% -100% -115% and 130%) of the target
concentration (500g/ml of amoxicillin and 62,5g/ ml of clavulanic acid). The average recovery and the
confidence interval of the two active ingredients are reported in Table 4.
They are all included in the standards fixed by the European Pharmacopoeia (95% and 105%), therefore the
method of assay is accurate.

3.5 Robustness
The robustness of the method has been studied by the changes in the method such as changing the rate of 0.1
ml / min that is to say a value of 0.9 ml / min and 1.1 ml / min), the temperature of the column (5 C of the
set point namely : 20 C and 30 C), the composition of the mobile phase (Acetonitrile / pH 4.4) (5% / 95%
and 15/85%), detection wavelength (210 and 230 nm) and column (old column and new one ). (Table 4)
From the results of the number of theoretical plates, asymmetry factor, the resolution, the content and the
RSD, due to the variation of these parameters, we can conclude that the method is robust.

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Radi & al. / Mor. J. Chem. 3 N1 (2015) 58-64

Tab.4:Robustness of the method.


Number of Asymmetry Rsolution Content RSD
Theoretical Factor
Plates
N > 3000 1.1%< F< 1.4% > 3,5 (95% 105%) < 2.5%
Ac.
Amox Ac.Clav Amox Ac.Clav Amox Ac.Clav Amox
Clav
Temperature 20C 4964 5955 1,5 1,5 5,7 101,86 104,86
Temperature 25C 4037 4878 1,26 1,26 4,1 99,91 99,9 1 2,4
Temperature 30C 5628 6978 1,36 1,36 5,1 100,65 102,68
Mobile
5340 5410 1,5 1,5 3,9 99,22 102,65
phase95/05%
Mobile 1,1
4037 4878 1,26 1,26 4,1 99,91 99,9 1,3
phase90/10%
Mobile
5988 8120 1,42 1,42 3,7 101,44 101,1
phase85/15%
Flow = 0,9 ml/min 3578 3344 1,46 1,46 3,9 100,15 98,97
Flow = 1,0 ml/min 4037 4878 1,26 1,26 4,1 99,91 99,9 0,5
0,7
Flow = 1,1ml/min 3649 3849 1,47 1,47 4,0 100,79 100,31
= 210nm 3977 4876 1,25 1,25 4,2 99,12 100,44
0,6
= 220nm 4037 4878 1,26 1,26 4,1 99,91 99,9
0,3
= 230nm 4084 4871 1,44 1,44 4,1 100,34 99,93
New column 7276 8984 1,43 1,43 5,9 101,98 100,94
Old column 4037 4878 1,26 1,26 4,1 99,91 99,9 1,5 0,7

Conclusion
The assay method proposed RP-HPLC was demonstrated as a simple, rapid and economical method for the
simultaneous determination of amoxicillin and clavulanic acid in pharmaceutical forms: tablets, syrup,
capsule and injectable solution.
The mobile phase consisted of 90% of pH4.4 buffer, simple to prepare and the analysis time is less than
8min consumes less than 8ml of mobile phase, the flow rate is 1 ml/min, the temperature is ambient, the
preparation of the samples is carried out in water only.
The validation of the method is based on a statistical study (Cochran's test, Student's test, Fisher test, Dixon's
test...). The method is specific, linear, accurate, faithful and robust. So this method can be easily adopted for
routine analysis of these active ingredient sin pharmaceutical forms studied.

References
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Radi & al. / Mor. J. Chem. 3 N1 (2015) 58-64

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