Summary:-

This study includes analytical methods for the determination of the drug amoxicillin trihydrate (Amox.) in some pharmaceutical preparations using Cobalt ion (Co(II)) as complexing metal. The best conditions for complexation were: the reaction time was 20 minutes, pH=1.5 and the best temperature of reaction was 70  ̊C. Benzyl alcohol was the best solvent for extraction the complex.


Introduction
Amoxicillin is one of the important derivatives of semisynthetic penicillin; it is active against Gram positive and to less extent Gram negative bacteria. Its nomenclature according to penicillins is 6-[D(-)-α-Amino-phydroxyphenyl acetamido] penicillanic acid or αamino-p-hydroxy benzyl penicillin (1) ,while its systematic (IUPAC) name is 7-[2-Amino-2-(4-hydroxypHenyl)-acetyl]amino-3,3,dimethyl-6-oxo-2-thia-5-azabicyclo [3,2,0] heptane-4-carboxylic acid, the chemical structure of the drug is (1) The formula structure of Amoxicillin as trihydrate (drug) is C 16 H 19 N 3 O 5 S.3H 2 O, its molecular weight = 419.45 gm.mole -1 . It is off white or almost white crystalline powder, slightly soluble in water and alcohol such as methanol and ethanol (2) . It has UV max. (ethanol): 230,274 nm and in (0.1N HCl): 229,272 nm (1) .Imran et al. prepared complexes of amoxicillin with Zn(II), Cu(II), Ni(II)and Ag(I), they identify these complexes by (C, H, N)elemental analysis and IR Spectra. These complexes have increased the biological activity of the drug (3) . Jian et al. determined amoxicillin in tablets, they used a quick and simple method which is (second differential derivative) at λ max . 282 nm and the standard deviation was less than 1.2% and the standard recovery for the drug was 97-100.5 % (4) .Denis et al. determined amoxicillin and clavulanic acid in blood plasma by HPLC supplied by UV detection, and they found the linearity was (0.62 -20 µg.ml -) while the detection limit for amoxicillin was 0.312 µg.ml -(5) .
Ashry et al. detected phenolic antibiotic like amoxicillin by its reaction with benzocaine in the presence of triethylamine at λ max . = 455 nm the linearity was (2 -16 µg.ml -1 ) while the detection limit was 0.0034 µg.ml -1 (6) .Co(II) forms blue-colored complex in the organic phase with Cyanex 923, a sensitive analytical reagent, the λ max . of the complex was 635 nm and the concentration that obeyed Beer's law is (58.9-589.0 µg. ml -) (7) .Zayed et al. in a new study, prepared different complexes of amoxicillin with Zn (II), Ni (II), Co (II) and Cu (II), these complexes were studied using elemental analysis, IR and mass spectra. The molar ratio of complexes were found to be Metal:Drug =1:1,1:2, and the stability constant K f of these chelates was(10 7 -10 14 ) (8) .In a recent study, Alekseev et al. prepared mixed complexes of β-lactam antibiotics (Ampicillin, Amoxicillin and Cephalexin) in solutions containing Co(II) and glycine anions(Gly). These complexes had been investigated using pH-metric titration at 20 o C in alkaline medium as mixed ligands complex [Co Gly Ampicillin], [Co Gly Amoxicillin], and [Co Gly Cephalexin] (9) . The experimental work showed that the reaction did not proceed at room temperature; heating was needed, media must be acidic for this reason. We studied the effect of pH, temperature, reaction time, extraction time, and suitable solvent for the extraction process.

4.
Spectral study: a) Amoxicillin spectrum: transfer 1 ml from stock solution of amoxicillin to 5 ml volumetric flask then dilute with distilled water the absorbance is measured at (200-1100 nm) using acidic water as blank. Table (

Results and Discussion
Amoxicillin spectrum, illustrated at    Temperature effect: the reaction of the metal and Amoxicillin proceeded slowly, to increase the reaction velocity we intend to increase the temperature of the reaction from 40 to100 ˚C, then the complex is extracted and measured the absorbance using UV Spectrophotometer. Table (3) gives the absorbance of the complex at different temperatures at pH 1.5 and Figure (5) shows the λ max for the complex is at 70 ˚C.

3.
Reaction time: the complex formation increased as well as the absorbance when the reaction time is increased, before the extraction process takes place, we used different times (5-30 min.), Table ( (7) showed that the increase in the aqueous phase to 6 ml with 4 ml of organic phase will decrease the absorbance of the complex.

7.
Times of extraction: the first extraction process is enough to extract the major concentration of the complex because the second extraction process for the complex that remained in aqueous phase gives a very weak absorbance less than 0.1.

Calculation of the ligand (Amoxicillin) to metal (Cobalt) ratio in the complex [Amox.-Co (II)]:
Molar ratio: to detect the ratio of complexation i.e. the molar ratio of Ligand (L) to the Metal (M) by taking different volumes of the Ligand (V L ) with constant volume of Metal (V m ) at the same concentration for each (1.2 X 10 -3 M) at the optimum conditions for complexation, then drawing the relation between absorbance and V L /V m as in Figure  ( Co(II) -2Amox.
Imran (3) and his partners defined the Amoxicillin complex form with transition elements in M:L is 1:2 ratio, and this was identical with proportion of metal with the ligand (drug) that we have reached by molar ratio.

Calibration curve of the UV spectrum:
It was carried out through taking different concentrations for the complex (ppm) and measuring the absorbance, as shown in Table (9). Figure (10) shows the calibration curve of [Amox.-Co(II)] complex that obeys Beer's law for the concentrations (50-500 µg. ml -) at 375 nm.

Determination of the drug concentration in different pharmaceutical preparations:
This was done by taking an average weight of one capsule from six capsules that have been mixed previously then the absorbance of the active ingredient (Amoxicillin) was measured from calibration curve (Figure 10), after complexation process takes place at the optimum condition mentioned above. Note: we carried out the same procedure for 500 mg. amoxicillin capsules of different trademarks (SDI, Julphar and Ajanta). Table  (10) shows the results of these preparations.

Determination of the [Amox.-Co(II)] by Flame Atomic Absorption Spectrophotometer (FAAS):
The complex was prepared under the optimum conditions of pH, temperature, proper solvent etc.. and we used the FAAS to detect the amoxicillin concentration by indirect measurement the absorbance of the Co(II) in the complex as shown in Figure (11), also we can measure the concentration of the amoxicillin in these pharmaceutical preparations using the calibration curve of indirect (FAAS).

Conclusion
The developments of new analytical methods to determine the amount of amoxicillin, such methods are UV-Visible and flame atomic absorption spectrophotometrics, are very sensitive and precise. Amoxicillin forms chelated complex with Cobalt ions at 70 o C and pH: 1.5 and the molar ratio for complex formation, Amoxicillin:Cobalt, is (1:2). The results of analysis of amoxicillin capsules of different trademarks show the amount of amoxicillin in SDI and Julphar capsules are almost as presented by the package, on the other hand the Ajanta capsules show some differences.