
International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 09744304 Vol.9, No.1, pp 7989, 2016

Simultaneous Determination of Amoxicillin and Clavulanate Potassium in Dry Syrup by Derivative Spectrophotometry
Siti Morin Sinaga*, Fatimah Arinawati and Muchlisyam
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Sumatera Utara Jalan Tri Dharma No.5 Pintu 4, Kampus USU, Medan Indonesia, 20155
Abstract: The aim of this study was to test the validation of derivative spectrophotometric method in simultaneous determination the content amoxicillin and clavulanate potassium in dry syrup by derivative spectrophotometric method with zero crossing technique, in buffer phosphate pH 4,4methanol (91:9) mixture.
The research results were obtained the amoxicillin and clavulanate potassium content at the second derivative with ∆λ = 2 nm at the wavelength of 239.00 nm and 313.20 nm respectively. The samples Clavamox® dry syrup were (102.73 ± 8.95)% and Claneksi® (103.52 ± 8.88)% and clavulanate potassium content of the sample Clavamox® in dry syrup (97.64 ± 4.12)% and Claneksi® (95.75 ± 5.64)%. Based on the results of analysis determine the sample content of amoxicillin and clavulanate potassium compound in dry syrup supply amoxicillin fulfilled the requirements in United States Pharmacopoeia (USP) 30th edition (2007) and clavulanate potassium fulfilled the requirement in United States Pharmacopoeia (USP) 30th edition (2007). The results of validation test on the Clavamox® dry syrup, the percent recovery for the amoxicillin is 100.43%, relative standard deviation RSD = 0.98% and for clavulanate potassium, the percent recovery = 100.58%, RSD = 1.46%.
Keywords: Amoxicillin; Clavulanate Potassium; Derivative Spectrophotometry; Zero Crossing; Second Derivat; Dry syrup; Validation.
Introduction
Amoxicillin is a penicillin derivative antibiotics used to treat infections of the respiratory tract, gastrointestinal tract and urinary tract. Clavulanate potassium is a form of a salt of clavulanic acid. Clavulanic acid has antimicrobial working very weak, but can inhibit penicillinase of streptococci and βlactamase as gramnegative microbes to bind to the active center of the enzyme. Therefore, these compounds are used in combination along with βlactam antibiotics are not stable against βlactamase1,2. Structur formula of amoxicillin and clavulanate potassium can be seen in Figure 1 and Figure 2.
According to the United States Pharmacopoeia (USP) 30th3 for amoxicillin and clavulanate potassium suspension oral is not less than 90.0% and not more than 120.0% of the amount listed on the label.
Figure 1. Structural Formula of Amoxicillin
Figure 2. Structural Formula of Clavulanate Potassium
Determination the result of a mixture of amoxicillin and optimization clavulanate potassium by using high performance liquid chromatography and detected by ultraviolet spectrophotometer at a wavelength of 220 nm is obtained by comparison of the mobile phase pH 4.4 phosphate buffermethanol (91:9). Levels of amoxicillin and clavulanate potassium mixture has also been determined the result of simultaneous determination of amoxicillin and clavulanate potassium in combined tablets by nonderivative and derivative ultraviolet spectrophotometric techniques, using aqueous solvent obtained in first derivatives at each wavelength 244.9 nm and 272 nm. Level of amoxicillin has been determined the result comparative study of RPHPLC and UV spectrophotometric techniques for the simultaneous determination of amoxicillin and cloxacillin in capsules, using water as solvent obtained in first derivatives at each wavelength 258.0 nm4,5,6.
Previously, UVVIS Spectrophotometry was used preferably for quantitative estimations of concentrations of known substances at constant wavelength, because the fundamental spectra are mostly flat and are less characteristic than IR spectra, for example. However, higherorder derivatives now allow for an enhancement of the sensitiveness by a factor of 10100 or more as well as a characterization of the substances by providing fingerprints, even in complex mixtures. This is very important for ultramicroanalysis. Therefore, the bulk of papers concerning differentiation technique deals with UVVIS spectra. It is also the reason why in this book the field of UVVIS spectra is treated in detail7.
A firstorder derivative is the rate of change of absorbance with respect to wavelength. A first order derivative starts and finishes at zero. It also passes through zero at the same wavelength as λmax of the absorbance band. Either side of this point are positive and negative bands with maximum and minimum at the same wavelengths as the inflection points in the absorbance band. This bipolar function is characteristic of all oddorder derivatives. The most characteristic feature of a secondorder derivative is a negative band with minimum at the same wavelength as the maximum on the zeroorder band. It also shows two additional positive satellite bands either side of the main band. A fourthorder derivative shows a positive band. The first and second derivatives may be generated using this technique. It is popular for dedicated spectrophotometer designs used in, for example, environmental monitoring. Firstderivative spectra may also be generated by a dual wavelength spectrophotometer. The derivative spectrum is generated by scanning with each monochromator separated by a small constant wavelength difference8.
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest concentration of analyte in a sample which can be quantitatively determined with suitable precision and accuracy9.
Accordingly, in this research will be conducted as the determination of amoxicillin and clavulanate potassium in dry syrup by derivative spectrofotometric method with zerocrossing method.
Instrumental
Apparatus
Tools used in this study is UVVisible spectrophotometer equipped with software Probe 2.42 UV (UV1800 Shimadzu), analytical balance (Boeco), cuvette, filter paper, rubber ball, spatula, toolsglassware and equipmentother tools required in sample preparation.
Reagent
Materials used were methanol, NaH2PO4, distilled water, amoxicillin trihydrate (BPFI) 98.64%, potassium clavulanate (Phiexia Company) stock 99.70%, Clavamox® and Claneksi® dry syrup.
Sampling
Sampling was done by purposive, which is determined on the basis of the consideration that the samples drawn have characteristics similar to those studied. The samples used are Clavamox® dry syrup (PT. Kalbe) and Claneksi® dry syrup (PT.Sanbe), each of which contains 125 mg of amoxicillin and clavulanate potassium 31.25 mg.
Preparation of the Stock Solution Amoxicillin
About 50 mg of amoxicillin were accurately weighed, then diluted with a solvent mixture of phosphate buffer pH 4.4methanol (91:9) in a 50 mL flask and paid back with the same solvent to obtain a solution with a concentration of 1000 µg/mL solution raw I (SS I). From this solution pipette 10 mL, was put into a 100 mL flask, diluted with a solvent mixture of phosphate buffer pH 4.4methanol (91:9) to mark the line, shaken until homogeneous in order to obtain a solution with a concentration of 100 µg/mL solution of the parent Raw II (SS II).
Preparation of the Stock Solution Clavulanate Potassium
About 50 mg of clavulanate potassium were accurately weighed, then diluted with a solvent mixture of phosphate buffer pH 4.4methanol (91:9) in a 50 mL flask and paid back with the same solvent to obtain a solution with a concentration of 1000 µg/mL (SS I). From this solution pipette 10 mL, was put into a 100 mL flask, diluted with a mixture of phosphate buffer pH 4.4methanol (91:9) to mark the line, shaken until homogeneous in order to obtain a solution with a concentration of 100 µg/mL (SS II).
Preparation Maximum Absorption Spectrum Amoxicillin
Taken as much as 2.0 mL of amoxicillin concentration 100 μg/mL was then inserted into a 10 mL flask and then diluted with buffer phosphate pH 4,4methanol (91:9) mixture solvent until the line mark, then shaken until to obtain a homogeneous amoxicillin solution with a concentration of 20 μg/mL. Absorbance was measured at a wavelength of 200400 nm.
Preparation Maximum Absorption Spectrum Clavulanate Potassium
Taken as much as 1.65 mL of clavulanate potassium concentration 100 μg/mL was then inserted into the 10 mL flask to be diluted with buffer phosphate pH 4,4methanol (91:9) mixture solvent until the line mark, then shaken until homogeneous to obtain a solution with a concentration of 16.5 μg/mL . Absorbance was measured at a wavelength of 200400 nm.
Preparation Derivative Absorption Spectrum Amoxicillin
Taken by 1.0 mL; 1.5 mL; 2.0 mL; 2.5 mL; 3.0 mL and 3.5 mL of stock solution amoxicillin concentration 100 μg/mL (SS II), then each put in a 10 mL flask to be diluted with the solvent buffer phosphate pH 4,4methanol (91:9) mixture. Then shaken until homogeneous to obtain a solution with a concentration of 10 μg/mL; 15 μg/mL; 20 μg/mL; 25 μg/mL; 30 μg/mL and 35 μg/mL Then made the absorption spectrum, then the absorption spectrum is transformed into a first derivative absorption spectrum and the second derivative at a wavelength of 200400 nm with Δλ=2nm.
Preparation Derivative Absorption Spectrum Clavulanate Potassium
Taken by 0.85 mL; 1.25 mL; 1.65 mL; 2.05 mL; 2.45 and 2.65 mL of stock solution clavulanate potassium concentration 100 μg/mL (SS II), then each put in a 10 mL flask to be diluted with the solvent buffer phosphate pH 4,4methanol (91:9) mixture. Then shaken until homogeneous to obtain a solution with a concentration of 0.85 μg/mL; 1.25 μg/mL; 1.65 μg/mL; 2.05 μg/mL; 2.45 μg/mL and 2.85 μg/mL. Then made the absorption spectrum, then the absorption spectrum is transformed into a first derivative absorption spectrum and the second derivative at a wavelength of 200400 nm with Δλ=2nm.
Determination of ZeroCrossing
Determination of the zerocrossing overlapping absorption spectrum obtained by each derived in different concentration of the solution. Zerocrossing each substance shown by the wavelength that has a zero uptake at various concentrations.
Determination of Wavelength Analysis
Created amoxicillin solution with a concentration of 35 μg/mL, clavulanate potassium solution with a concentration of 8.5 μg/mL, and a mixed solution of amoxicillin 35 μg/mL and clavulanate potassium 8.5 μg/mL. Each solution is then measured absorbance at a wavelength of 200400 nm. Then absorption spectrum is transformed into the first and second derivatives of each single substance from a mixture of amoxicillin and clavulanate potassium. The second derivative absorption spectrum from a single substance solution and a mixture of both overlay. Were chosen to be the wavelength analysis is that at a particular wavelength, the absorption single one of the compounds zero while single absorption partner compound and a mixture of both is almost the same or exactly the same. Because at these wavelength can selectively measure the uptake of one of the compounds without being bothered by the uptake of compounds partner.
Preparation and Determination Linearity Calibration Curves Amoxicillin and Clavulanate Potassium
Created amoxicillin stock solution with a concentration of 10 μg/mL; 15 μg/mL; 20 μg/mL; 25 μg/mL; 30 μg/mL and 35 μg/mL, then the second derivative absorption measured (Δλ = 2 nm) in wavelength analysis has been determined. Then do the analysis of the relationship between concentration and absorbance values thus obtained linear regression equation y = ax+b. And based on the absorption at a wavelength analysis, also conducted the calculation of the Limit of Detection (LOD) and the Limit of Quantitaion (LOQ). To determined the LOD and the LOQ can be used formula.
SD =
LOD =
LOQ =
Description:
SD = Standard Deviation
LOD = Limit of Detection
LOQ = Limit of Quantitation
Determination of Amoxicillin and Clavulanate Potassium levels in Dry Syrup
One bottle of dry syrup powder weighed. Then weighed carefully the amount of powder equivalent to 50 mg of amoxicillin and then the weight of which weighed analyte equivalent of 50 mg of amoxicillin clavulanate potassium is calculated equality contained therein (powder weighing as much as six times repetition), put in a 50 mL flask, added phosphate buffer pH 4.4methanol (91:9) to line sign while shaken. The solution is then homogenized with an ultrasonic stirrer for 15 minutes. The solution is then filtered,
approximately 10 mL of the first filtrate discarded. The filtrate subsequently accommodated. Then from this filtrate solution, 0.35 mL pipette and put into a flask and diluted with 10 mL of phosphate buffer pH 4.4methanol (91:9) to mark the line (concentration of 35 µg/mL for amoxycillin and concentrations of 8,5 µg/mL for clavulanate potassium). The solution is measured at the second derivative absorbance at a wavelength analysis has been determined to amoxicillin and clavulanate potassium. Furthermore, the absorbance was measured at a wavelength of 200400 nm, then the absorption spectrum is transformed into a second derivative absorption spectrum Δλ 2 nm in wavelength analysis of amoxicillin and clavulanate potassium respectively 239.00 nm and 313.20 nm.
Validation Test
Accuracy Test
Accuracy test was conducted by the addition of raw materials is to make three samples with the analyte concentration of a specific range of 80%, 100%, 120%. Where in each specific range is used 70% and 30% of raw samples to be added and then mix the sample and standard absorbance was measured at a wavelength of 200400 nm, then the absorption spectrum is transformed into a second derivative absorption spectrum Δλ 2 nm in wavelength analysis of amoxicillin and clavulanate potassium respectively 239.00 nm and 313.20 nm. Percentage recovery can be calculated by the formula10.
% Recovery = 100 %
Description:
CF = concentration of the substance after the addition of raw materials
CA = concentration of the substance before adding the raw materials
C*A = number of raw added
Precision Test
Precision is measured as relative standard deviation or coefficient of variation. Precision measured indicates the degree of fit between the individual test results when a method is repeated for a homogeneous sample. Relative standard deviation value which meets the requirements showed a precision method performed.
Based on the results of recovery prescribed amoxicillin and clavulanate potassium standard deviation amoxicillin and clavulanate potassium of the formula:
SD =
Description:
X = The number of substances in the sample
= Number of substances sample average
n = Number of repetitions
Standart Deviation (SD) obtained based on the value, calculated relative standard deviation of amoxicillin and clavulanate potassium by the formula:
RSD = x 100%
Description:
X = Number of substances sample average
SD = Standard deviation
RSD = Relative Standard Deviation
Results and Discussion
Results Determination of the Maximum Absorption Curves
Determination of maximum absorption spectra performed at a wavelength of 200400 nm. Measurement of the concentration of amoxicillin in the 35 μg/mL, where as for clavulanate potassium at a concentration of 8.5 μg/mL and. Based on the research results, obtained the maximum wavelength amoxicillin and clavulanate potassium at 239.00 nm and 313.20 nm respectively.
Results Determination of ZeroCrossing at First Derivatives
Absorption spectrum of amoxicillin concentration of 35 µg/mL and clavulanate potassium concentration of 8.5 µg/mL was transformed into a first derivative absorption spectrum with Δλ = 2 nm. Results of the determination of the zero crossing in the first derivative of the absorption spectrum obtained by overlap first derivatives on each substance. Zero crossing in the first derivative spectrum of each wavelength is shown by agents who have zero absorption. Overlapping absorption spectrum amoxicillin and clavulanate potassium in the first derivatives can be seen in Figure 3.
Amoxicillin 35 µg/mL
Clavulanate Potassium 8.5 µg/mL
Figure 3. Overlapping absorption spectrum of amoxicillin and clavulanate potassium at first derivative at wavelength 239.00 nm and 313.20 nm respectively.
Determination of Absorption Zero crossing the Second Derivatives
Results of the second derivative absorption spectrum determination is made by first making the absorption spectrum of amoxicillin solution with a concentration of 35 µg/mL and clavulanate potassium with a concentration of 8.5 µg/mL at a wavelength of 200400 nm. Absorption spectra have been obtained is transformed into a second derivative absorption spectrum with Δλ = 2 nm.
The second derivative absorption spectrum of respectively of these substances overlay. The results indicate zero crossing at a wavelength of 239.00 nm, 246.80 nm, and 249.60 nm to amoxicillin, whereas for clavulanate potassium was obtained at a wavelength of 263.40 nm, 313.20 nm and 320.60 nm. Wavelength and absorbance analysis can be seen in Table 1 and overlapping absorption spectrum of amoxicillin and clavulanate potassium on the second derivative in Figure 4.
Table 1. Wavelength Analisys and Absorbance at Second Derivative
Wavelength (nm) 
Absorbance 

Amoxicillin 35.0 μg/mL 
Clavulanate Potassium 8,5 μg/mL 
Amoxicillin and Clavulanate Potassium 

239,00 
0,0004 
0,0000 
0,0003 
246,80 
0,0005 
0,0000 
0,0004 
249,60 
0,0003 
0,0000 
0,0005 
263,40 
0,0000 
0,0004 
0,0004 
313,20 
0,0000 
0,0005 
0,0005 
320,60 
0,0000 
0,0001 
0,0001 
Figure 4. Overlapping absorption spectrum of amoxicillin and clavulanate potassium on the second derivative at wavelength 239.00 nm and 313.20 nm respectively.
Determination of Wavelength Analysis
Figure 5. Wavelength analysis of amoxicillin λ = 239.00 nm
Figure 6. Wavelength analysis of clavulanate potassium λ = 313.20 nm
Determination of the wavelength of the analysis done by making a solution of amoxicillin 35 µg/mL, a solution of clavulanate potassium 8.5 µg/mL and mixed solution of amoxicillin 35 µg/mL and clavulanate potassium 8.5 µg/mL. Then made the absorption spectrum of the first and second derivatives, then overlaid. To determine the wavelengths of the absorption spectrum analysis on each derivative is done by observing the absorption wavelength shows zero partner compounds and other compounds uptake and absorption thereof has a value equal or nearly equal. Amoxicillin wavelength spectrum analysis, and the wavelength spectrum analysis each clavulanate potassium can be seen in Figures 5 and 6.
Based on the above image, obtained by the wavelength used for the determination of the mixture of amoxicillin and clavulanate potassium uptake is on the second derivative, is 239.00 nm for amoxicillin, and 313.20 nm for clavulanate potassium. It is known based wavelength selection for each derivative analysis. The wavelength of the analysis is obtained by determining the zero crossing for amoxicillin and clavulanate potassium. At first derivative absorption, wavelength analysis for amoxicillin can be found. However, the wavelength analysis for clavulanate potassium was not found, so the assay mixture of amoxicillin and clavulanate potassium performed on the second derivative.
Determination Results Linearity Calibration Curves
The linearity of the calibration curve showed a linear relationship between the absorbance with concentration. Amoxicillin regression equation, Y = (7X + 0.25).106 with a correlation coefficient, r = 0.9995 and clavulanate potassium, Y = (21X + 1.35).106 with a correlation coefficient, r = 0.9997. R values > 0.995 showed a linear correlation relationship between X and Y11. The calibration curve amoxicillin and clavulanate potassium for each wavelength of 239.00 nm and 313.20 nm can be seen in Figures 5 and 6.
Figure 5. Calibration curve amoxicillin at a wavelength of 239.00 nm
Figure 6. The calibration curve of clavulanate potassium at a wavelength of 313.20 nm
Determination Results of Amoxicillin and Clavulanate Potassium Levels in Dry Syrup
Determination is done by using Clavamox® and Claneksi® in dry syrup containing amoxicillin 125 mg and 31.25 mg potassium clavulanate. Measurement of amoxicillin and clavulanate potassium raw on both substances each amoxicillin and clavulanate potassium 35µg/mL and 8.5 µg/mL, which is adapted to the content ratio of the two substances in the preparation, namely 125:31.25 or 4:1.
The prepared sample is then measured at a wavelength of 200400 nm. Furthermore, the results of the absorption spectrum is transformed into a second derivative absorption spectrum with Δλ = 2 nm. Can be determined based on the absorbance spectrum of amoxicillin and clavulanate potassium at a wavelength analysis has been obtained previously, is wavelengths 239.00 nm and 313.20 nm. Levels of amoxicillin and clavulanate potassium in Clavamox® and Claneksi® can be seen in Table 2.
Table 2. Levels of amoxicillin and clavulanate potassium in Clavamox® and Claneksi
No 
Drug 
Clavamox® 
Claneksi® 
Label claim (mg) 
Requirements (%) 
1. 
Amoxicillin 
(102.73 ± 8.95)% 
(97.64 ± 4.12)% 
125 
90120 
(121.27 – 135.55) mg 
(118.76 – 125.34) mg 



2. 
Clavulanate Potassium 
(103.52 ± 8.88)% 
(95.75 ± 5.65)% 
31.25 
90120 
(30.57 – 34.12) mg 
(28.80 – 31.04) mg 


Amoxicillin and clavulanate potassium levels obtained in the above table shows that the dry syrup preparation Clavamox® and Claneksi® on the market meet the requirement in the Farmakope Indonesia Edisi V12, which is not less than 90% and not more than 120% of the amount listed on the label.
Test Results Validation
Validation parameters tested were accuracy, precision, limits of detection and quantitation limits. Accuracy is expressed in percent recovery were determined using standard addition method. Precision test done using parameters Relative Standard Deviation (RSD)10.
Accuracy Test
Accuracy test with parameter percent recovery is done by using Clavamox® in dry syrup with standard addition method, which is made by adding a certain amount of standard solution. Then the solution is measured in accordance absorbance wavelength analysis, which is 239.00 nm and 313.20 nm. Results recovery of amoxicillin and clavulanate potassium by standard addition method standard Clavamox® in dry syrup can be seen in Table 3.
Table 3. Test Results Recovery Amoxicillin and Clavulanate Potassium
Specific ranges (%) 
Amoxicillin recovery (%) 
Clavulanate Potassium recovery (%) 
80 
102.28 
99.65 
101.43 
98.96 

100.59 
98.00 

100 
101.07 
101.98 
100.40 
101.35 

99.66 
100.50 

120 
100.05 
101.98 
99.49 
101.35 

98.93 
100.50 

Ratarata % recovery 
100.43 
100.58 
Standard Deviation (SD) 
0.98 
1.46 
Relative Standard Deviation (RSD) (%) 
0.98 
1.46 
Based on the results obtained in Table 4 shows that the average percent recovery obtained for amoxicillin is 100.43% and 100.58% for clavulanate potassium. The results obtained are eligible for the accuracy of the validation of analytical procedures because the average is between the range of 98102%9.
Precision Test
Precision test is done by calculating the relative standard deviation. Based on the calculation of data on levels of amoxicillin and clavulanate potassium, obtained relative standard deviation is 0.98% for amoxicillin and clavulanate potassium 1.46%. The relative standard deviation of the results of the two substances which meet the requirements of ≤2%9.
Table 5. Validation parameters for derivative spectrophotometric
Parameters 
Amoxicillin 
Clavulanate Potassium 
Corr.Coef (r) 
0.9995 
0.9997 
Slope 
0.000007 
0.000021 
Intercept 
0.00000025 
0.00000135 
Accuration (%) 
100.43 % 
100.58 % 
LOD (µg/mL) 
1.57 µg/mL 
5.26 µg/mL 
LOQ (µg/mL) 
0.70 µg/mL 
2.30 µg/mL 
Conclusion
Based on the research conducted, it can be concluded spectrophotometric method with zero crossing derivatives can be used to set the levels of amoxicillin and clavulanate potassium. Levels of amoxicillin and clavulanate potassium in dry syrup preparation Clavamox® and Claneksi® meet the requirements of an oral suspension levels according to the Farmakope Indonesia edisi V11. Validation test conducted on dry syrups Clavamox® showed that the spectrophotometric method of derivatives meet the requirements validation, which includes parameters of accuracy and precision.
References
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