Micellar Determination of Amoxicillin in the Pharmaceutical Compounds by using Cloud Point Extraction

Batool Nassir Hamran, Ahmed Fadhil Khudhair*

Department of Chemistry, College of Science, Kerbala University, Karbala, Iraq

ABSTRACT:

A preconcentration and estimation method was developed for drug evaluation, with Amoxicillin being evaluated in this manner characterizing easy, fast, and high sensitivity. The method for extracting and estimating Amoxicillin tri hydrate. It is based on the interaction between the drug and the analytical reagent in the acid medium at pH equal 4.06 to form an intense yellowish brown colour, an ethanol-soluble consequence that Can also be easily extracted from micelles of Triton X-114 non-ionic surfactant and measured at a highest possible absorption of 415 nm.  The calibration curve specifies the concentration range [(0.059-1.07)*10^-3 mmol.L^-1] with r=0.9980 and a molar absorptivity 1.1204 L.mol^-1.cm^-1 .Limit of detection (LOD) and limit of quantification (LOQ) are  0.045 and 0.151 μg. mL^-1 respectively , The mean recovery percentage of 99.79%. The method was successfully applied in its pharmaceutical preparations to analyze the (Amoxicillin).

INTRODUCTION:

Amoxicillin chemically referred to as 6- (p-hydroxy-alpha-aminophenyl acetamido) penicillanic acid as shown in Figure(1). Amoxicillin is widely used as an antibacterial drug in the treat of infections generated by  grampositive and gramnegative bacteria [1]. it  used for  an animal and human treatment and has been considered one of the most commonly used β-lactam antibiotics in the globe [2]. There are many ways in which to work out Amoxicillin in bulk and quantity forms. Various ways in which to determine Amoxicillin such as: Titrimetric ways Simultaneous spectrophotometric and volumetric determinations of AMX depended on the mechanism of reaction in the catalyzed base hydrolysis followed by iodine oxidation in dilute acid solution [3],  Amoxicillin was determined  spectrophotometrically in pharmaceutical preparations using potassium iodate in acid medium [4], fluorescence [5].

Liquid phase micro-extraction [6], LC-MS [7], kinetic spectrophotometry [8], TLC-Densitometry method [2], HPLC [9,10], electrochemical determination [11], mass spectrometry [12], also determined by atomic absorption [13], Amoxicillin was also spectrophotometrically analyzed Without previous separation using UV derivative methods in conjunction with Clavulanic acid or antibiotic pharmaceutical mixtures [14], spectrophotometrically used a lot due to simplicity and cost effectiveness, sensitivity and selectivity, reasonable accuracy, precision and accessible in most quality control laboratories, visible spectrophotometry had already stayed sustainable in the field of pharmacological evaluation chromatography techniques. Molecular links between electron donors and acceptors were usually connected with the production of highly colored charge transfer complexes that absorb radiation in the visible region  [15,16]. A multitude of electron donating compounds have also been recorded with multiple acceptors to produce charge-transfer complexes. The fast formation of these complexes contributes to their usefulness in developing easy and convenient spectrophotometric techniques for these compounds [17].

Figure 1.structure of Amoxicillin tri hydrate

Cloud point extraction is one of the processes that had to extract metal chelates [18]. CPE (green method) has become the best extraction method compared to other extraction process due to its benefits such as simplicity, good recovery, high enrichment factor, low organic solvent production of organic solvents. And in addition, a wide range of compounds and metals can be extracted. In analytical techniques, surfactants and micelles are extremely used mainly in extraction and pre-concentration procedures [19,20]. A preconcentration process with the exception of the method of cloud point extraction depends on the use of non-ionic surfactant varieties in aqueous solutions and thus the creation of micelles and the heating of these solutions to a certain temperature will become cloudy or turbid this temperature is regarded as cloud point temperature [21, 22]. Cloud point extraction can also be combined with a turbidity method [23]. Cloud point extraction technique coupled with multiple methods such as: inductively coupled plasma mass spectrometry atomic absorption spectrophotometry, capillary electrophoresis of graphite furnace nuclear absorption spectrometry [21,24], flame atomic absorption [21,25], UV-visible spectrophotometer [21,26], fluorescence [22], high performance liquid chromatography with fluorescence detection [27] and flow injection [28]. there are deferent methods used to determine the trace element [29-40]. while there are  many pharmaceutical compounds by spectrophotometric methods [41-50].

In this study, Amoxicillin was extracted by reacting analytical reagents such as (NaIO₄) in a simple CPE process. After adding the appropriate surfactant compound that was extracted, the rich phase form determined the pharmaceutical compounds by coupling the CPE with other instrumental methods.

Experimental part:

Chemical materials:

All reagents and materials used are high purity materials and use distilled water to dissolve and prepare solutions and dilute them. 0.0024 M Amoxicillin (AMX) SDI stock solution has been prepared by dissolving 0.1g in 100ml  of distilled water and diluting to mark. 0.01 M stock solution of the reagent sodium periodate was prepared by dissolving 0.2 g in 50ml D.W then diluting to mark. 0.1 M of 36% hydrochloric acid solution was prepared 0.9ml  diluted in 100ml distilled water. 0.1M NaOH. (20% V/V) different of surfactants [TritonX-100, TritonX-114, SDS] prepared by take 5ml from each and diluting up to the mark by distilled water in 25ml volumetric flask. 0.1M stock solution of interferences was prepared by dissolving the 0.440g,.0.225g, 0.480g and 0.420g from the following [ascorbic acid, oxalic acid, citric acid and uric acid respectively] in distilled water and diluting to the mark in 25ml volumetric flask. The following salts out were also prepared by dissolving 0.1M of [ NaCl, NaNO₃, NH₄SO₄, NaF and pb(NO₃)₂ respectively] (0.146g, 0.212g, 0.330g, 0.104g and 0.828g) in 25ml distilled water.

Apparatus:

The primary tool used in this job is a Shimadzu double-beam UV-Vis spectrophotometer model UV-1800 (Kyoto, Japan) fitted with 1ml optical path cell to scan the absorption spectrum. Thermostatic water bath model. BS-11 NO.K109.125 (Korea) is used during all CPE studies. Used pH meter 4X Alkaline, AA size, 1,5 V (Germany) for solution pH readings. Hettich Centrifuge D-78532 (Germany) to separate the equase phase from the rich phase. vortex mixer 945307 (THE.U.S.A). FT-IR-8400S, Japan, HPLC Shimadzu LC-20AB, HA/SIL 100A C18, 5µm, 150×4.6mm, P/N:HS-1546-M185. 

CPE Procedure for Amoxicillin:

Placed 10mL of the drug in a centrifuge tube and added 1mL of Sodium period. Added 0.5mL of hydrochloric acid. Following the formation of a yellowish brown color added 0.5ml of 20 percent V/V Triton X-114 blended the solution very well in a vortex mixer. Transfer the tube to the water bath for 30 minutes at 85 Co for half an hour. The phases were separated by centrifugation at 4000rpm for 5 min. The aqueous phase was easily removed by Pour. The surfactant-rich phase containing the colored product was dissolved in 0.5mL ethanol and the product absorbance measured at 415nm against a blank reagent prepared under similar conditions. This method is demonstrated in equation 1 and Figure 3.

Preparing pharmaceutical samples:

One form of amoxicillin pharmaceutical, namely capsules. The powder of thirteen capsules was mixed, homogenized, and the content of one capsule (0.6708g) equivalent to 500mg of active drug was dissolved with continuous shaking in sufficient quantity of water. The solution was transmitted to a 100mL volumetric flask and diluted to label with water.

HPLC Procedure for Amoxicillin:

Prior the measure the series of standards Amoxicillin solutions in the High Performance Liquid Chromatography instrument (HPLC). The series of the standard solution were varied from (25to700)μg.mL-1 for Amoxicillin solutions and to determine in HPLC technique. As for the applications, the measurements were made on five samples of local pharmacies and were prepared at a concentration of 300 ppm.The chromatographic analysis was performed on Shimadzu UFLC equipped with a SPD-20A prominence diode arrayand a single wavelength UV–Vis detector (190–1100 nm) and Phenomenex C18 column (4.6 × 150 mm, particle size 5μm). The mixture of mobile phase consists of (65:35 % v/v) acetonitrile : methanol. 2μL of sample was injected with a flow rate of 1 mL/min and retention time was 1.75 min. AMX was detected at 226 nm using UV–Vis wavelength detector [51, 52]. 

Micelles [Amoxicillin-NaIO4-HCl-Triton X-114]

Figure 3. Proposed reaction mechanism for complex formation of Amoxicillin.

RESULTS AND DISCUSSION:

Absorption spectra:

Absorption spectrometry of Amoxicillin and sodium period and complex were measured. It was observed that there was a significant shift towards the visible region when the Amoxicillin 226nm wavelength complex was created. The wavelength of the reagent was 235nm, while the reagent was added to the drug with a shift of 380nm, the complex was at the greatest wavelength of 415nm when acid was added. This are shown in Figure 4.

Figure 4  UV Vis spectrophotometry A: spectrum of Amoxicillin ,B: spectrum of Amoxicillin – NaIO4, C: spectrum of complex of Amoxicillin – NaIO4 – HCl

Optimisation of the experimental conditions:

To obtain the best performance for the cloud point extraction (CPE) procedure, the effects of the experimental conditions studied include: order of addition, pH effect, reagent concentration, surfactant types, surfactant effect concentration, heat time, temperature effect, salt out and interference effect had been evaluated and optimized.

Order of addition:

The most significant variables investigated were the order of components where the order of AMX solution (0.1w/v percent,10mL) and NaIO₄ reagent (0.2 w/v %,1mL) (0.1M HCl 0.5mL) was then added to the complex formation by adding 0.5ml from 20 percent V/V Triton X-114, then heated for half an hour at 85C^o, then separate and use the centrifuge, then add 0.5ml of ethanol to the rich solubility stage after measuring the UV. Vis spectra. The highest absorbance outcome was selected in the third test.

Effect of pH

Measuring acidity is one of the most significant variables influencing the effectiveness of processing that, based on the CPE technique because of the pH, plays a crucial part in the creation of complexes and the chemical stability of alternatives. The pH of the solution was adapted from 1 to 12 using a solution of 0.1M HCl and 0.1M NaOH and measuring using a pH meter. Results in Figure 5. show increased absorbance when the pH value was equivalent to 4.06.

Figure 5.effect of the pH on the absorbance of AMX-NaIO₄ complex

Effect of concentration of reagents:

The reagent concentration effect (NaIO₄) on absorbance value was studied using different NaIO₄ concentration and held the other chemical and physical parameter constant. Figure 6. discribed that the absorbance value was enhanced with the addition of the concentration reagent. The best outcome was relying on when the reagent concentration was 0.9 mmol.L^-1.

Figure 6. Effect of Conc. of NaIO₄ on AMX-NaIO₄ – HCl-Triton X-114 complex

Type of surfactants:

Different forms of surfactant were studied and it absolutely was found that Triton X-114 is that the best one utilized in CPE for the present method. Concentration of the Triton X-114 has a very important influence on the extraction efficiency under the cloud point extraction technique, because of its necessary effect to extend extraction efficiency through diminish the phase volume quantitative relation. The impact of the Triton X-114 concentration on the absorbance of the extracted complex was studied within the surfactant concentration of (20%v/v) Triton X-114, the concentration and volume used for Triton X-114 very high, so that they must be used under heating at a temperature regarding fifty C^o because of a high viscosity. The results are given in Figure 7. Can be indicated that a volume of one ml (0.035M )of (20%V/V) Triton X-114 gave the best absorbance and was utilized in all the subsequent experiments.

Figure 7.  Impact of concentration of Triton X-114

When using separate surfactant kinds, it was discovered that the highest outcome was when using Triton X-114, as shown in Figure 8.

Figure 8. Impact of surfactant form on absorption

Effect of temperature equilibrium and time of incubation:

To achieve an associated degree of effective phase separation and simple preconcentration, it is vital to optimize the impact temperature and moment of incubation.

Table4. Temperature impact on the absorption of AMX-NaIO₄-HCl –TX-114 complex

Figure9. Over lapping between Abs, log D, log K _ex against temperature

Figure10. Overlapping between Abs, E%,K_ex, D against Temperature

A variety of incubation temperature ranging from 50 to 85C^o at incubation time from 5 to 30 min was implemented to study the impact of temperature on the absorbance product processing. It was absolutely demonstrated that the maximum complicated absorbance was achieved at 85C^o (Figure9, Figure10 and Table4) and the best time at 15 min Table 5 and Figure 11, due to the increased rate of molecular movements, the phase separation becomes difficult at higher temperatures. Therefore, the stabilization temperature of 85C^o was chosen for additional experiments.

Table5. Effect of time on the absorbance of AMX-NaIO₄ complex

Figure11. Time impact on the absorption of AMX-NaIO₄ complex

Effect of Salt out   

Study the effects of salts on CPE by adding 0.5ml of 0.1 M of  NaF, NaCl, NH₄SO₄ and NaNO₃ to the solution after completion of the procedure the product acquired observe. It was found that some salts such as NaCl and NaF, were added. Absorption values reduced while sodium nitrate was added to raise slightly. However, when adding ammonium sulphate, the reduction was tiny relative to the value with the addition of salt. This is due to the reality that some salts cause an increase in bonding, which improves absorption.

Table 6. Impact of Salt out on the absorbance of AMX-NaIO₄-HCl-TritonX-114 complex

Effect of interference:

The effect of interference expected to be present in the medication (AMX) was studied to know the method selectivity under study by adding 0.5ml (0.1M) from each interference [ascorbic acid, oxalic acid, citric acid and uric acid] with 10ml (0.001M) from the drug and the remainder of the sample were as ideal circumstances. Note that some interference often interfere with drug and cause a decrease in absorption such as oxalic acid and ascorbic acid and uric acid and either citric acid did not affect absorption as in Table 7. 

Table 7. Impact  of interferent on absorption of AMX-NaIO₄-HCl –TX-114 complex

Calibration Curve of  Amoxicillin:

When the optimum conditions for all the above factors were developed, the calibration curve of the drug was evaluated by altering the concentration of the drug, with distinct levels varying from 0.059*10^-3 to 1.07*10^-3mmol.L^-1. It was noted that absorption improves with growing concentration as it is a linear line The calibration curve was plotted between absorbance and concentration of Amoxicillin as shown in Figure 12

Figure12.Calibration curve of Amoxicillin

Table 8. Statistical data analysis of Amoxicillin's spectrophotometric determination by cloud point extraction.

parameter

Amoxicillin

Color of product

Yellowish brown

λmax(nm)

415

Reagent

Sodium periodate

Medium

Acidic

Surfactant

Triton X-114

Regression equation

y=1.1204x+0.7174

Correlation coefficient(r)

0.9980

Coefficient of determination (R2)

0.9962

Molar absorptivity (L.mol-1.cm-1)

1.1204

Intercept

0.7174

Concentration rang (μg.ml-1)

25-450

Composition of product

1:1

Recovery%

99.79

Limit of Detection (μg.ml-1)

0.0411

Limit of Quantitation (μg.ml-1)

0.1338

Repeatability:

The absorption was measured at different concentrations of Amoxicillin and was repeated at concentrations of 0.47 mmol. L-1 and 0.12 mmol. L-1 for five occasions.It was noticed to be somewhat convergent and both the standard deviation, the relative standard deviation, and the interval of confidence were calculated. It is noted that the less the concentration of up to 2 ppm and specifically for the evaluation of pharmaceutical compounds (organic) increases the error rate because the quantity of very little material and vice versa as shown in Table 9.

Table 9. Repeatability of AMX complex 

1.259,1.262,1.260

5

0.12

0.806,0.841,0.783

0.810 and 0.826

0.8132 ± 0.0206

2.544

0.0256

Applications:

A validity of the method was checked successfully by determine 200ppm of AMX prepared by dilution. 10mL comprising the same levels of the prepared sample solution were transmitted to centrifugal pipes and each solution followed the suggested CPE procedure for AMX and the drug content was evaluated spectrophotometrically at λmax415  nm . Assay solutions (Capsule) were prepared and the quantity taken from the reduction solution was measured and the quantity of AMX was calculated in all assay kinds. Relative error E percent, standared deviation SD, recovery percentage (Rec%) were also calculated. The outcomes are illustrated as shown in Table (10) below.

Table10.  Results summary representing normal additions to the pharmaceutical assessment of Amoxicillin.

Table10.  Continued

Linearity and range by HPLC:

The linearity of an analytical procedure as the ability to obtain test result which are directly proportional to the concentration (amount) of AMX in the sample at 300 ppm as shown Figure 14 and Table 11. also was studied analyzing 5 concentrations of the reference standard from (25 to700) μg.mL-1 for Amoxicillin solutions to determine in HPLC technique. As shown Figure13 which represent linearity by HPLC. 

Figure 13.Calibration curve of Amoxicillin by HPLC

Figure14. HPLC Chromatogram  of  AMX ( 300 μg/ml).

Table 11. Summary of outcomes representing normal additions to the pharmaceutical assessment of Amoxicillin

Ere%

RSD%

SD

Efficiency of determination

Practically found content of active ingredient (ppm)

Height of peaks(mV) 

Theoretical calculated active material ppm

Confidence interval of the mean

W+t0.05/2 (n-1)SD/ (gm)

Wt.of sample(g) that equivalence to 500 mg of active of ingredient to obtain 0.47 mmolL-1of Amoxicillin in 100 ml

Practically found content of active ingredient (g)

Pharmaceutical drug company and claimed  content of active ingredient

No.of sample

4.58

3.33

0.0187

103%

209.16

481

300

0.011

0.560

0.02

NORMON

Rondade valdecarrizo,tres-contos-madrid (pain)

500 mg

1

1.26

1.38

0.0079

95%

197.55

440

300

0.005

0.577

0.019

Glomox

Glopal pharma

Dubi UAE

2

0.63

2.52

0.0143

97%

198.7

450

300

0.009

0.565

0.02

Neomox

Neopharma

Abu Dubi  UAE

3

2.45

2.92

0.0168

99%

204.90

461

300

0.010

0.575

0.02

Yenimox Yenipharma

Turkey-Istanbol

4

6.08

3.64

0.0199

93%

187.82

430

300

0.012

0.547

0.019

Labmox

India by labmorate

Pharmaceutical(India)

5

Table14.Staticaly data t-test and F-test

CPE

HPLC

d

_d

Sd for method 1

Sd for method 2

t-calculated-d/Sd*

t-tableted

F-test

(Sd1)2/(Sd2)2

F-tableted

0.018

0.019

-0.001

-0.0014

0.00088

0.00054

-0.703

3.182

2.65

6.38

0.019

0.02

-0.001

0.019

0.02

-0.001

0.017

0.02

-0.003

0.018

0.019

-0.001

t_{0.05 (n-1)} n=4(3.182), P-value ˂ 0.05 sig. difference, p-value ˃ 0.05 non sig. difference

Statistical Analysis of Data:

In the treatment of the data, significance tests such asANOVA, pair t-test and f-test were used in this study. At a significance level of α= 0.05 (confidence of 95%) any value <0.05 will reject Null hypothesis in favour of Alternative hypothesis any value of significant ˃0.05 will accept Null hypothesis and reject the alternative hypothesis [31].as shown table14.

CONCLUSION:

A delicate, selective and ecofriendly spectrophotometric technique is created to determine small levels of Amoxicillin using sodium period as a complexing agent and cloud point extraction. The suggested technique was successful, the prospect benefits of the established technique are time-saving, decreasing the quantity of reagents used as well as minimizing analyst effort.

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Accepted on 18.09.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):732-741.