Beruflich Dokumente
Kultur Dokumente
3 N1 (2015) 58-64
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
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.
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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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.
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).
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
The regression equations of active ingredient alone and reconstituted pharmaceutical form are linear for
amoxicillin and clavulanic acid (Table 2).
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).
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.
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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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
[1] USP34-NF29, (2011), 1882-1889.
[2] USP34-NF29, (2011) 2366-2369.
[3] Pharmacope europenne 7.0 (2011) 5231-5233
[4] Pharmacope europenne 7.0 (2011) 2971-2973.
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