By Abdulla Mohamed Osman Mohamed Supervised by Prof. Younis M.H.

Younis

Abstract
The main aim of the present investigation is

to perform preliminary steps for the establishment and development of a new analytical technique for the analysis of lactam antibiotics, in general, executed and illustrated by amoxicillin as a model. The method requires a stepwise synthesis of amoxicillin hydroximate ferric ion complex to be an index of determination of drug in capsule dosage form.

 Firstly, amoxicillin hydroxamic acid was prepared via treatment of amoxicillin with hydroxylamine, after establishment of the appropriate reaction conditions that nucleophilic attack, should take place, stereo- selectively, at the carbonyl group of the lactam ring leading to the formation of amoxicillin hydroxamic acid. Secondly, the latter acid was treated with an aqueous solution of ferric chloride; the purple colored amoxicillin hydroximate ferric ion complex was obtained in good yield and moderate stability. The stability of the complex was improved when the reaction was conducted in dimethyl sulphoxide as a solvent. The specificity of the attack at the lactam ring was verified through conducting a control experiment involving reaction of hydroxylamine with acetamide, which formed an unstable colored compound; when treated with ferric chloride aqueous solution. The noticeable low yield and extremely unstability of the complex was evident from the faint purple, transient color.

Thirdly, further evidence for this stereospecific attack was also evident from the disappearance of the carbonyl absorption frequency in the IR-spectrum of the amoxicillin ferric ion complex. Moreover, a number of studies have been carried out, which involved monitoring the stability of the complex towards solvent polarity, pH variations, temperature change resistance and time. Fourthly, UV-spectrophotometric analysis was used for the determination of the stoichiometric ratio of the two reactants by adopting Job`s method of continuous variation and the mole ratio method. It was found that the ratio of amoxicillin hydroxamic acid to that of ferric chloride is 1:6, based on the absorption at max 520 nm. Accordingly, the following chemical formula was suggested: Fe6C16H17N4O6SCl18 and also chemical structures were proposed for amoxicillin hydroximate ferric ion complex.

Introduction
1. Antibiotics
An antibiotic was originally defined as a substance,

produced by one microorganism, which inhibited the growth of other microorganisms. But antibiotic now refers to a substance produced by a microorganism, or to a similar substance (produced wholly or partly by chemical synthesis), which in low concentrations inhibits the growth of other microorganisms.

-Lactam antibiotics
The name lactam is given to cyclic amides is analogous to the name

lactone given to cyclic esters. A -lactam ( beta lactam ) is a four membered heterocyclic ring containing a nitrogen atom and three carbon atoms, with one carbon bearing a carbonyl functionality. It is named as such, because the N-atom is attached to the -carbon atom relative to the carbonyl group. The simplest -lactam is 2-azetidinone. o -lactam ring (azetidinone) which responsible for the antimicrobial activity of all beta-lactam antibiotics Accordingly, -lactam is cyclic amide. Those antibiotics which contain -lactam rings are known as -lactam antibiotics. Such rings are sometimes found to be fused with other heterocyclic rings such as thiazolidine as an example. That fusion leads to creation of chiral centers and forces the molecule to exist in a V-shape. Such a shape enforces the lactam bond to be out of planarity of the ring system and thus inhibits the conjugation and resonance of the N-atom lone pair with the carbonyl group.

Bacteria and the bacterial cell wall

The vast majority of prokaryotes of medical and pharmaceutical

significance are bacteria. Bacteria represent a large and diverse group of microorganisms that can exist as single cells or as cell clusters. Moreover, they are generally able to carry out their life processes of growth, energy generation and reproduction independently of other cells. In these respects they are very different to the cells of animals and plants, which are unable to live alone in nature and can exist only as part of a multicellular organism. They are capable of growing in a range of different environments and can not only cause contamination and spoilage of many pharmaceutical products but also a range of different diseases.1 The bacterial cell wall is an extremely important structure, being essential for the maintenance of the shape and integrity of the bacterial cell. It is also chemically (composed mainly of Peptidoglycan), unlike any structure present in eukaryotic cells and is therefore an obvious target for antibiotics that can attack and kill bacteria without harm to the host.

Figure 6: The action of transpeptidase (TPase) with its natural substrate (upper) and penicillin G (lower). The OH group on a serine residue at the active site of the TPase attacks the peptide bond between the terminal D-alanyl-D-alanine in the Peptidoglycan precursor, releasing the terminal D-alanine and forming a new peptide cross-link with existing Peptidoglycan in the cell wall. Penicillin G blocks this process by forming a stable penicilloyl-TPase complex

Amoxicillin
Amoxicillin

is amino penicillin(penam -lactam antibiotic) Semi-synthetic product derived from a fermentation product, White or almost white, crystalline powder Slightly soluble in water, very slightly soluble in ethanol (96 per cent), practically insoluble in fatty oils. It dissolves in dilute acids and dilute solutions of alkali hydroxides contains 95.0 per cent to 102.0 per cent (anhydrous amoxicillin).3

 7. Chemical reaction of amoxicillin with

hydroxylamine hydrochloride The reaction of hydroxylamine with the -lactam ring of amoxicillin is known as hydroxylaminolysis which leads to ring opening and the formation of the the corresponding hydroxamate (amoxillohydroxamate).

 Hydroxyl amine hydrochloride. Hydroxylamine is the nucleophile. Hydroxylamine is both an amine and an alcohol, but it always reacts at nitrogen because nitrogen (being less electronegative than oxygen) has a higher-energy (more reactive) lone pair. The more nucleophilic end of NH2OH will attack the more electrophilic carbonyl group. It seems obvious that the more nucleophilic end of NH2OH will be the nitrogen atom depending on the pH of the solution. Normally, hydroxylamine is supplied as the crystalline hydrochloride salt and a base of some kind added to give the nucleophile. Figure 21: State of hydroxylamine changes with pH: the more Nucleophilic atom is marked in black Uses of hydroxylamine Hydroxylamine is used in organic synthesis in the preparation of aldoximes ketoximes hydroxamic acids and heterocycles. It is also being used in in qualitative organic analysis of many different functional groups such as carbonyl compounds, anhydrides , nitryles, amides, acid chlorides, esters , sulphonicacids, and sulphonyl chloride, including synthesis of their crystalline derivative.

 7.3 Hydroxamic acids and their ferric complexes : Hydroxamic acids are chemical compounds produced by the chemical reaction of hydroxylamine with amides ,esters and acid chlorides. These compound are ligands chelate rings are basically didentate chelates (Figure:25), and three are bound to form very stable six coordinate octahedral and chiral Fe (III) colored complexes able to absorb the electromagnetic radiation in the visible range, and hence their concentrations can be determined using UV-Vis spectrophotometers. Figure 22: Hydroxamate and catecholate are bidentate ligands having high ability to chelate ferric ion. In fact, the three chelate units are part of larger single molecules, which means these effectively act as hexa dentate ligands, forming complexes with Fe(III) with very high stability constants. The ligands are highly selective for Fe(III), even over Fe(II). A number of common compounds are known. Ferrichrome is a hydroxamate complex with the hydroxamates as side chains to a peptide ring. Ferrioxamine complexes contain the hydroxamates as part of a simple peptide chain. Hydroxamates release Fe by metal ion reduction, so the ligand can be reused once the reduced Fe(II) is dissociated. Whereas the formation constants are high for Fe(III), they are much lower for Fe(II), so reduction provides one mechanism for iron release, with other chelates then able to compete successfully for the Fe(II). However, simply complex destruction provides another mode of iron release.4

 10. Justifications The assay of amoxicillin as ferric hydroxamate complex is simple using simple techniques such as visible spectrophotometery which is available everywhere in Sudanese laboratories either in universities laboratories industrial laboratories and research laboratories. Such spectrophotometric methods do not require expensive reagents or use of high grade solvents compared to other analytical techniques. From pilots studies and literatures ferric hydroxamate complex is un stable rapidly fades after preparation and this research beside the quantitative analysis of amoxicillin includes stabilization of these complexes. The present research study and the parameters associated with it are expected to solve such problems. e.g. choosing the suitable solvents instead of aqueous media in which the complex is stable. testing whether or not that the hydroxylamine hydrochloride react selectively with the beta lactam ring or amide group side chain or both. To determine mole ratios between ferric chloride and hydroxamic acid to adjust the appropriate molar quantities for achieving proper reaction conditions. Moreover the plan of the practical work would include adjustment pH. If time allows validation parameters for the method will be determined. It is noteworthy to mention that such an study has not been attempted previously in the literature.

 11. Objectives 11.1 General Objectives Amoxicillin ferric hydroxmate complex synthesis and UVspectrophotometric analysis: (a) To synthesize amoxicillin hydroxamic acid using hydroxylamine. (b) To study the Stoichiometric amounts of amoxicillin hydroxamic acid with ferric chloride in the synthesis of amoxicillin ferric hydroxamate complex. 11.2 Specific Objectives (a) To synthesize amoxicillin hydroxamic acid. (b) To synthesize amoxicillin ferric hydroxamate complex. (c) To evaluate the selectivity of the reaction of hydroxylamine hydrochloride with -lactam ring. (d) Determination of the co ordination number ( Job's method ) or molar ratios of the ferric hydroxamate complex. (e) To study the selection of the proper solvent and pH for the stability of the amoxicillin ferric hydroxamate complex.

 1. Analysis of -lactam compounds using hydroxyl

amine as reagent and ferric chloride as a chromogen The method is based upon the fact that penicillin reacts rapidly with hydroxylamine to give hydroxamic acid which form a purple complex with ferric ion that can be determined colorimetrically. But many esters anhydrides and amides also reacts with hydroxyl amine to produce hydroxamic acids. These colored complexes is unstable in nature its intensity faded with time. Hydroxylamine also react with aldehydes and ketones to give crystalline colored oximes that forms also colored complexes with ferric ion .8

 2. Hydroxamic acids Hydroxamic acids of general structure RCONHOH, having R as an organic residue, have been known since 1869 with the discovery of oxalohydroxamic acid by Lossen H.9 Enormous amount of information has accumulated with respect to their biomedical applications, synthesis, and the determination of the structures of their metal complexes.10 Complexation of metal ions by hydroxamic acids is the starting point of a number of analytical determinations.11 All hydroxamic acids, react with ferric chloride to give rust brown complex salts. These colored complexes form the basis for the sensitive qualitative and quantitative determination of carboxylic acids and their derivatives too. 3. Hydroxamic Acids as Ligands Hydroxamic acids are important bioligands, 6 and are involved in numerous biological processes including metal-ion transport and inhibition of metalloenzymes7,8. 1:1 Metal binding to hydroxamic acids usually occurs in a bidentate fashion with the formation of singly or doubly deprotonated (hydroxamato or hydroximato) Ligand.

 4.3 Hydroxamic Acid Formation from Nitriles and

Amides One drop or 30 mg of the compound dissolved in a minimum amount of propylene glycol is added to 2 mL of a 1 M hydroxylamine hydrochloride solution in propylene glycol. Add 1 mL of 1 M potassium hydroxide and boil the mixture gently for 2 min. Allow the mixture to cool to room temperature and add 0.5-1 mL of a solution of 5% alcoholic ferric chloride. A red to violet color constitutes a positive test. Yellow colors indicate negative tests, and brown colors or precipitates are indeterminate.

 7. Reactivity of hydroxylamine towards

amoxicillin as nucleophile The main cause of deterioration of amoxicillin is the reactivity of the strained lactam ring. Thus the betalactam carbonyl group of penicillin readily undergoes nucleophilic attack by hydroxylamine opening the beta lactam ring to form the corresponding hydroxamic acid.17

 8. Lack of planarity and resonance inhibition in the -lactam ring Inhibited resonance in the beta- lactam rings due to lack of planarity. Reducing the double bond character of C-N bond in comparison to that of planer amide and. the nitrogen of beta lactam becomes basic and hence the C-N bond Beta lactam rings weaker than that amides. If delocalization is completely inhibited in an amide then the rate of the reaction .which in the transition state could occur up to 1013 fold faster than analogues resonance stabilized systems. The strain energy of the four-membered ring is high and therefore a reaction involving opening of the beta-lactam ring could take place faster by a factor of up to 10 20 than the analogues bond fission process in a strain-free amide. The inhibited resonance of amides would : a-increase the C-N bond length and decrease the C-N bond strength. b-decrease the C-O bond length and increase the C-O bond strength. c- decrease the charge positive charge density on nitrogen. d- decrease the negative charge density on oxygen.18

 11. Instability of hydroxylamine in excess of ferric

ion Treatment of hydroxylamine with an excess of ferric ion results in the formation of N2O and equivalent amount of ferrous ion.20 2NH2OH + 4Fe+3 N2O (g) +4Fe+2 +4H+ +H2O

 13. Nucleophilic reactivity of hydroxylamine The amine group in hydroxylamines (HONR2) is more nucleophilc than simple amines. There are two explanations for this effect. In one explanation the existence of lone pair electrons directly adjacent to the nucleophilic site is believed to destabilize the ground state, making the nucleophile more reactive. is called the -effect. The -effect arises when two nucleophilic atoms are adjacent to one another. This dramatically increases the nucleophilicity. For example, although hydroxide is about 16,000 times more basic than hydroperoxide (HOO-), hydroperoxide is approximately 200 times more nucleophilic. Another factor that has a dramatic influence on nucleophilicity is solvation. Solvation forces are stronger for small nucleophilic ions than for larger ones, an effect that is especially true in polar protic solvents where hydrogen bond donation from the solvent to the nucleophile is stabilizing. Because the solvent must be stripped from the nucleophile during the attack on the electrophile, the well solvated smaller ions are poor nucleophiles. Therefore, the more "naked" (less solvated) the nucleophile, the better it becomes. Hence, nucleophiles are consistently more reactive in aprotic solvents of low polarity. However, very often the nucleophile will not be soluble in these solvents because many nucleophiles are negatively charged (they are part of salts). Hence, solvents such as DMF and DMSO are commonly used . These are aprotic but polar enough to solubilize the nucleophile, and high reactivity is found.22

 Experiment (1) Reaction Of Hydroxylamine Hydrochloride With Amoxicillin Amoxicillin(100 mg) was placed in six test tubes and then dissolved in absolute ethanol (2 ml) to form a saturated solution. 1M Hydroxylamine hydrochloride (2ml) was added to five of the test tubes. Five of the test tubes was heated in a water bath maintained at 100 C, the sixth test was left at room temperature (unchanged). Time for heating the test tubes was recorded for each, see table (1). Test tubes were then removed after the elapse of specific time duration for each. Test tube were cooled to room temperature and one drop of aqueous ferric chloride (5%) solution was then added. Development of any coloration was recorded. Experment (2) (control experiment) Reaction Of Hydroxylamine Hydrochloride With Acetamide Acetamide (100 mg) was placed in six test tubes and then dissolved in absolute ethanol (1 ml) to form a saturated solution. 1M Hydroxylamine hydrochloride (1ml) was added to five of the test tubes. Five of the test tubes was heated in a water bath maintained at 100 C, the sixth test was left at room temperature ( unchanged). Time for heating the test tubes was recorded for each, see table (2). Test tubes were then removed after the elapse of specific time duration for each. Test tube were cooled to room temperature and one drop of aqueous ferric chloride (5%) solution was then added. Development of any coloration was recorded. Experiment (3) IR spectra of the Reaction products of hydroxylamine hydrochloride with amoxicillin in different molar ratios (1:1,2:1, and 3:1) An accurate three weights (30.2g) of amoxicillin trihydrate were placed in three 50 ml volumetric flasks. Three volumes of hydroxylamine hydrochloride (0.6706%, 5 ml,10 ml and 15 ml) were added respectively into the volumetric flasks. the mixtures were sonicated for 30 minutes. The sonicated mixtures were dried on betray dishes overnight, at room temperature. Crystals were collected and the IR experiment was conducted. See IR spectra in appendixe. And labeled as: 1 - 1:1 hydroxylamine hydrochloride amoxicillin reaction product. 2 - 2:1 hydroxylamine hydrochloride amoxicillin reaction product. 3 - 3:1 hydroxylamine hydrochloride amoxicillin reaction product. Experiment (4) Determination of max of the ferric hydroxamate complex of amoxicillin in aqueous medium, acidic medium, and after 24 hours Amoxicillin (0.05 g) was placed in 100 ml volumetric flask to distilled water (10 ml) and hydroxylamine hydrochloride (5ml ,0.19%) were added and the mixture was well shaken and was sonicated for 30 minutes. Ferric chloride (5ml, 0.444% ) was added to the sonicated amoxicillin-hydroxylamine hydrochloride mixture. A purple coloration was developed and started to fade out gradually the volume was made up to 100 ml by addition of distilled water. The fading of the purple color was increased by increasing addition of distilled water. The solution was scanned in a UV/Vis-spectrophotometer setup in the range 200-750 nm. Another solution was scanned within the same wave length range after the elapse of 24 hours. Another solution which was prepared in the same manner but only differ in using 0.444% ferric chloride in 0.1M hydrochloric acid. see table (3) Experiment (5) determination of the max of the ferric hydroxamate amoxicillin complex in DMSO and after 24 hours Amoxicillin (0.05 g) was placed in a 100 ml volumetric flask. And DMSO( 10 ml) was added followed by hydroxylamine hydrochloride in DMSO (5ml, 0.19%). the mixture was well shaken and sonicated for 30 minutes. Ferric chloride in DMSO( 5ml,0.444%) was added to the sonicated amoxicillin-hydroxylamine hydrochloride mixture. A stable purple colored complex was obtained the solution completed to 100 ml by DMSO. The solution was scanned in a UV/Vis-spectrophotometer setup in the wavelength range 200-600 nm. Another solution was scanned within the same wavelength range after the elapes of 24 hours. Another 2 dilute solution was prepared by taking 15 ml from the stock and completed to 50 ml by DMSO. One of them was scanned in a UV/Vis-spectrophotometer setup in the wavelength range 200600 nm. And the other was scanned within the same wavelength range after the elapsed of 24 hours. See Table (4) Experiment (6) Reaction of hydroxylamine hydrochloride with amoxicillin in different mole ratios (1:1,2:1 and 3:1) Three separate weights of amoxicillin were prepared as follows : amoxicillin trihydrate (0.2 g, accurately)was placed in 100 ml volumetric flasks, dissolved in three separate volumes of hydroxylamine hydrochloride in DMSO (0.6706 %, 5 ml,10 ml and 15 ml), respectively .The three mixtures were shaken and sonicated for 30 minutes, and then the volume was completed to 100 ml by DMSO. A three 10 ml volumes of these solutions were transferred to three 50 volumetric flasks. Ferric chloride in DMSO (7 ml, 1.614 %) was added to each flask. An instant coloration was obtained the volume was then made to 50 ml by DMSO. Each of the three solutions were scanned in a UV/Vis-spectrophotometer set at a wavelength range 300-750 nm. See table (5) Another scanning in the same wavelength rang were conducted after elapse of 24 hours. See table (5) Experiment (7) Determination of the empirical formula of the ferric hydroxamate amoxicillin complex by jobs method of continuous variation Amoxicillin trihydrate (0.05 g) was placed in 100 ml volumetric flask. hydroxylamine hydrochloride in DMSO (5ml, 0.1656%) was added. the mixture was well shaken and sonicated for 30 minutes. The volume was then completed to 100 ml by DMSO. eleven volumes of this sonicated solution treated with successive volumes ferric chloride in DMSO (0.371232%) as it can noticed in Table (6)

Experiment (8) Determination of imperial formula of amoxicillin hydroxamate ferric ion complex by (mole ratio method) An standard solution of hydroxyl amine hydrochloride (0.66382516 %) in DMSO (100 ml) was prepared. And another standard solution of ferric chloride (1.5494667 %) in DMSO (100 ml) was also being prepared. Amoxicillin (0.2 gm) was dissolved in exactly in 5 ml of the hydroxylamine hydrochloride ( 0.66382516 %). The solution was sonicated for 30 minutes, and then the solution was made up to 100 ml by DMSO. 10 ml of this stock solution of amoxicillin hydroxamate were placed in seven volumetric flasks. To each of these seven volumetric flasks a serial volumes of 1,2,3,4,5,6,7 mls of ferric chloride solution in DMSO (1.5494667 %) were added ( solutions of group (I) ) see table (7). A reddish-brown colouration appeared, instantly, after each addition. Each of the seven solution were scanned, individually, in a UV-spectrophotometer set at the visible region (300-750 nm) for each a single broad band was obtained at a maximum absorbance max520 nm it was then concluded that the appropriate wave length of absorbance for these solutions is max520 nm. See table (7). Another experiment was then performed utilizing the same procedure but the volume of the ferric chloride solution was kept constant (3ml) and that of amoxicillin hydroxamate was varied -5 -3 4,6,8,10,12,14,16 that is a concentration range 3.821126306x10 M to 1.52845052210 M ( solutions of group (II) ). see table (8). It was again concluded that the appropriate wavelength of absorbance is

 -lactam antibiotics are used in a wide ranges in the health and medical field due to their high efficiency in eradication of many infectious diseases, so both quantitative and qualitative analysis of these type of antibiotics is too important. There are many different methods of analysis of -lactam antibiotics but needs high grade solvents, very expensive instruments which needs special well trained analysts. The introduction and establishment of this new analytical procedure adopted in this work is meant to: (1) Overcome all of these drawbacks in the analysis of -lactam antibiotics in general. (2) Synthesis of amoxicillin hydroxamic acid chelates with ferric ion which can be used to decrease high levels of ferric ion in blood circulation. (3) Use of these ferric ion complexs for the estimation of the -lactam rings. The principle on which the method adopted in the present research work is based on is the fact that -lactam rings are susceptible to nucleophilic attack by nucleophyles in general, and in particular amines such as hydroxylamines or even weak nucleophyles such as water and alcohols. It is expected that nucleophilic attack by hydroxylamine would lead to ring opening, thus leading to the formation of the corresponding hydroxamic acid, see fig(24).

Amoxicillin hydroxamic acid

Hydroxamic acid is also expected, as a ligand, to chelate with ferric ions leading to the formation of ferric ion complex of unpredicted stability. It is planned that if the preparation of amoxicillin hydroximate ferric ion complex is successful, then it is feasible and easy to examine its stability via UV-Vis spectrophotometric analysis since such complexes are expected to be colored. It is also expected that such complexes would be of health interest and also a quantitative analytical measure of -lactam rings. The present research study involves a number of phases. The first being the reaction of the -lactam ring of amoxicillin with the hydroxylamine, the aim is not only the preparation of the end product hydroxamic acid but also the determination of the stoichiometric ratio of the amoxicillin hydroxamic acid and ferric ion. The recognition of the most susceptible electrophilic site in the amoxicillin liable for nucleophilic attack by hydroxylamine, determination of the exact amount of ferric ion needed for the formation of the complex and finally examination of the stability of the amoxicillin ferric hydroxamate complex in different solvent systems see table (3) and (4). The reaction of hydroxylamine with amoxicillin was attempted in duplicate : one trial at room temperature and the other in which the reaction mixture is heated. After an elapse of appropriate time few drops of an aqueous solution of ferric chloride were added a purple color developed in both reaction mixture, which indicate the formation of amoxicillin hydroxamate ferric ion complex. It was observed that the purple color started to fade out gradually with time indicating the instability of the complex. Alternatively, it was found that when the reaction was carried in a nonaqueous system, in DMSO solvent, and in excess of ferric chloride 15 drops was added more intense and persistent purple color was produced. This was indicative of more yield and increasingly stable complex was afforded. In an attempt to verify that nucleophilic attack preferably, takes place at the carbonyl group of the -lactam ring thus leading to simultaneous ring opening and that attack does occur at the amide carbonyl of the side chain another experiment was conducted. For this purpose acetamide was used as a reactant (control) instead of amoxicillin, but establishing the same reaction conditions. A very faint and transient of purple coloration was produced, this control reaction demonstrated that nucleophilic attack of hydroxylamine upon the molecule of amoxicillin targeted the carbonyl group of the -lactam ring is predominant leading to synchronous ring opening see table (1) and table (2).

elapsed for the reaction between amoxicillin and hydroxylamine hydrochloride.

elapsed for the reaction between amoxicillin and hydroxylamine hydrochloride.

 IR spectra of the reaction products of hydroxylamine hydrochloride with amoxicillin in different molar ratios (1:1,2:1, and 3:1) was carried out to know the site of reaction. The IR-spectrum of standard amoxicillin has shown strong absorption band at 1776 cm-1. The spectrum of the amoxicillin hydroxamate is void of this band, which is a further confirmation that nucleophilic attack occurs on the carbonyl of the -lactam ring, solely. See appendix. Examination of the stability of amoxicillin hydroximate ferric ion complex towards changes of pH, time dilution of the reaction mixture with water and heating have been monitored via the measurement of absorbance and max of the complex using UV-Visspectrophotometric analysis. The results are summarized in table (3) and table (4). It could be noticed that from table (3) the maximum wavelength of absorption max of amoxicillin hydroxamate ferric ion complex has not undergone any structural changes after an elapse of few minutes from the time of preparation as the initial max 487.5 nm suffers a trivial change to max 484 nm. Alternatively, the complex has suffered a remarkable blue shift to a max of 353 nm when it was measured after the elapse of 24 hrs. On the other hand, the stability of the complex was found to be affected by pH suffered a noticeable red shift to max value of 494 nm. It was then concluded that analytical studies for the complex should be carried out promptly after it's preparation in a medium of appropriate pH. Table (3),table (4).

 max Determination of amoxicillin hydroxamate ferric ion complex in aqueous and acidic media.

hydroxamate ferric ion complex in aprotic solvent DMSO

 To overcome the instability of the amoxicillin hydroxamate ferric ion complex the solvent must be changed to a protic one, which is DMSO. So another experiment was carried out to determine the stability and the max of the amoxicillin hydroxamate ferric ion complex in DMSO and max after 24 hours and the result was found that after addition of ferric chloride to the sonicated amoxicillin-hydroxylamine hydrochloride mixture in DMSO, a reddish brown color was obtained. This solution is stable and does not undergo any fading in color compared to that of the aqueous one. max was found 520 4 nm. see table(4). Further analysis and studies were established by the reaction of hydroxylamine hydrochloride with amoxicillin in different mole ratios (1:1,2:1 and 3:1) to confirm there is no reaction in the amide side chain with excess hydroxylamine hydrochloride. A colored complex was obtained after addition of ferric chloride in DMSO to amoxicillin hydroxamic acid in DMSO with max 519.5 nm. UV-scanning has shown a very broad single band with maximum absorbance at 519.5 nm. When the number of moles of hydroxyl amine hydrochloride added was increased, the concentration of amoxicillin hydroxamate ferric ion complex was decreased by 15.6 %. See table (5) and graph (1). This was indicative by the UV-absorbance.

of the experiment and after 22 hours

hydroxylamine hydrochloride increased the absorbance of the complex decreased.

 After 22 hrs it could be noticed from table (5) that there is slight red shift in max and decrease in absorbance. This is an additional verification that the hydroxylamine hydrochloride reacted with only one carbonyl of one amide either that in -lactam ring or that in the side chain.the former was most probable to happen. The details of this experment is as follows: Assume that 2 moles of hydroxyl amine hydrochloride react with both -lactam amide group and the amide group in the side chain. A 2 different hydroxamic acids will produced. Both of these two different hydroxamic acids form a colored complex when have been reacted with ferric chloride. And if we assume, again, that these two complexes have the same color ( same max) the intensity of absorbance will increase to double which is totally opposite to the obtained data. Another assumption is that if the two complexes having different color (different max ) the single broad band will split into two bands with constant max value in the case of increase in the numbers of moles of hydroxylamine hydrochloride. Hence the hydroxylamine hydrochloride reacted with only one carbonyl of one amide group in amoxicillin even in excess either that in the side chain or of that in the -lactam ring which can be another confirmation.

for the determination of mole ratio between ferric ion and amoxicillin hydroxamic acid in the complex.

The highest absorbance of the complex that formed between amoxicillin hydroxamic acid ( 7 ml, 1.19410197x10-3 M ) and ferric ion ( 3 ml, 0.022887299 M) has been at max 529 nm is 0.29463.

cross represent the stoichiometric point 2.5 ml ferric chloride and 7.5 ml hydroxamic acid. Amoxicillin

 Taking the linear regression of the graph (2) the point at

the cross is the stoichiometric point at which the volume of amoxicillin hydroxamic acid is ( 7.5 ml, 1.19410197x10-3 M ) and ferric ion is ( 2.5 ml, 0.022887299 M) at max 529 nm The net volume is 10 ml and the concentration of amoxicillin hydroxamic acid and ferric ion in this solution is 8.955764775x10-4 M, 5.72182475x10-3 M, respectively. Based on the formula mentioned above 5.72182475x10-3 /8.955764775x10-4 = 6.388985081 Hence the mole ratio of amoxicillin hydroxamic acid to ferric ion is 1:6

Another method for the determination of the empirical formula of amoxicillin hydroxamate ferric ion complex is by (mole ratio method) which is performed using two groups of solutions in one the volume of amoxicillin hydroxamic acid solution is kept constant and the volume of ferric chloride is varied, while in the other group of solutions volume of ferric chloride solution is kept constant and the volume of amoxicillin hydroxamic acid is varied see table (7) and table (8).

underlined absorbance represents the inflection point.