A Sensitive Spectrophotometric Determination of Ritodrine , Pentazocine , Isoxsuprine Hydrochlorides and Amoxicillin in Pure and Pharmaceutical Samples

A simple, accurate and highly sensitive spectrophotometric method for the determination of ritodrine hydrochloride (RTH), pentazocine hydrochloride (PZH), isoxsuprine hydrochloride (ISH) and amoxicillin (AMX) is described. The method is based on the oxidation of the studied drugs by a known excess of chloramine – T (CAT) in hydrochloric acid medium and subsequent determination of the unreacted oxidant by reacting it with iodide in the same acid medium liberates iodine, which subsequently react with starch to form a stable starch-iodine complex. The reacted oxidant corresponds to the drug content. The coloured complex exhibits a maximum absorption at 590 nm. The apparent molar absorptivity values and Sandell’s sensitivity values are in the range 6.96 x 10 1.43 x 10 L mol cm and 2.45-4.30 ng cm, respectively. The method was successfully applied to the studied drugs in their dosage forms. The results are reproducible within ±1 % and compare favorably with those of official methods of British Pharmacopoeia and the United States Pharmacopoeia.


Introduction
Ritodrine hydrochloride (RTH), chemically 1-(4-hydroxy phenyl)-2-[2-(4-hydroxy phenyl) ethyl amino] propanol, is a β 2 -adrenergic agonist used to arrest preterm delivery in pregnant woman 1,2 .Pentazocine hydrochloride (PZH), ( 2R*,6R*,11R*)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(3methyl-2-butenyl)-2,6-methano-3-benzazocin-8-ol, is an analgesic with antagonist action.Amoxicillin (AMX), 6-(p-hydroxy-α-aminophenyl acetamido) penicillanic acid, is used as an antibacterial drug.Isoxsuprine hydrochloride (ISH), p-hydroxy-N- (1-methyl-2-phenoxyethyl)  norephedrine hydrochloride, is an active peripheral and cerebral vasodilator and it has a direct relaxant effect on the smooth muscular tissue of the blood vessels and uterus.In view of the increased pharmaceutical applications of RTH, PZH, AMX and ISH, their assay and quality control are very important.Spectrophotometric methods have been reported in the literature for the determination of RTH [3][4][5] , PZH [6][7] , AMX 5,8,9 and ISH 3,10,11 .For the determination of studied drugs, other methods include HPLC, [12][13][14] titrimetric 15 and fluorimetric 16,17 are also reported in the literature.Some of these methods required expensive reagents, less sensitive, poor selective, time consuming and tedious experimental procedures.To overcome these limitations in the existing method, there is still a need for a sensitive and cost-effective method for the determination of these drugs that can be adopted for the routine analysis of pharmaceutical samples.In the present investigation, a highly sensitive indirect spectrophotometric determination of cited drugs with chloramine-T-iodine and starch is described, and the proposed method has been employed to the determination of studied drugs in pure and in dosage forms.This new procedure is accurate, highly sensitive, rapid, simple and completely different from the existing methods.

Apparatus
All absorbance measurements were made with an Elico -model SL-171 digital spectrophotometer with 1 cm matched cells.

Reagents
All chemicals used were of analytical reagent grade.Chloramine-T (≅ 0.01 mol L -1 ) was prepared by dissolving 0.28 g of CAT in 100 mL of distilled water and standardized iodometrically.This solution was then diluted subsequently to get 100 µg mL -1 solution.Starch 1%: It was prepared by dissolving 1.0 g of starch in 100 mL of hot distilled water.Solutions of potassium iodide (0.5 %) and hydrochloric acid ( 2.0 mol L -1 ) were used.

Standard procedure
Accurately measured volumes of drug solutions equivalent to 0.2-1.2,0.2-1.6,0.0 -1.5 and 0.5-2.5 µg mL -1 of final solution of RTH, PZH, AMX and ISH, respectively were transferred into a series of 10 mL standard flasks.Then a volume of 0.7mL of 100 µg mL -1 CAT was added to each flask followed by acidification by 1.0 mL of 2.0 mol L -1 hydrochloric acid.After 10 min, 1.5 mL of 0.5 % KI was added to each flask.After 2.0 min 1.0 mL of 1 % starch was added and the contents were diluted to the mark with distilled water and mixed well.The absorbance of the coloured complex was measured at 590 nm against distilled water after 5.0 min.Blank was prepared similarly omitting the drug and its absorbance was measured against distilled water.The decrease in absorbance corresponding to consumed CAT and in turn, to drug concentration, obtained by subtracting the absorbance of a test solution from that of the blank solution.The calibration graph was drawn by plotting the difference in absorbance (absorbance values of test and blank solutions) of the complex against the amount of the drug.The amount of drug was determined from the concurrent calibration graph.

Procedure for pharmaceutical formulations
In a 100 mL standard flask, an accurately weighed amount (from the mixed and powdered contents of 20 tablets or mixed contents of 10 capsules), equivalent to 50 mg of the respective drug, was dissolved in 5.0 mL of methanol and completed to volume with distilled water and filtered.Appropriate aliquots of the drug solution were taken and the standard procedure was followed for analyzing the drug content.
To analyze the injection solution and syrup, the requisite amount was transferred to a 100 mL standard flask and dissolved in 5.0 mL of methanol and completed to volume with distilled water.The drug content in the diluted solution was determined as described above, and the results of the analysis are given in Table 1.

Results and Discussion
Preliminary experiment was performed to fix the linear range (Beer's law curve) for chloramine-T (CAT) in the optimum experimental conditions, with the use of iodine-starch reagent.Under experimental conditions, the concentration range of CAT was found to be 0 -7.0 µg mL -1 .In the present work, known but excessive CAT was utilized to oxidize the studied drugs in 2.0 mol L -1 hydrochloric acid medium and the unreacted CAT was determined by reacting it with iodine -starch reagent in the same acidic system.The coloured complex shows a maximum absorption at 590 nm.This formed the basis for the determination of studied drugs in microgram quantities.The studied drugs, when added in increasing amounts to fixed amount of chloramine-T, drugs consume CAT and there is a concomitant decrease in the absorbance of the coloured complex on increasing the concentration of drugs (Figure 1).

Effects of acid and reagents
Hydrochloric acid was the medium of choice for oxidation of the drugs by CAT as well as the latter's determination with iodine-starch reagent.A 1.0 mL of 2.0 mol L -1 concentration of HCl was found optimum for the oxidation of the drugs within 10 min, and hence the same concentration was employed for the determination of CAT with iodine-starch reagent.The volume of 1.5 mL of 0.5% KI and 1.0 mL of 1.0 % starch solution in a total volume of 10 mL of reaction mixture were found suitable for the analysis.

Reaction time and stability of colour
The time taken for complete oxidation of the drugs is not critical.Any delay up to 45 min in the determination of unreacted CAT had no effect on the absorbance.The colour of the formed iodine -starch complex was stable for a period of more than 90 min for the studied drugs.

Effect of excipients
In pharmaceutical analysis, it is important to test the accuracy of the method, so recovery experiments were performed using a synthetic mixture of each drug (RTH, PZH, ISH and AMX) with several excipients such as talc, stearic acid, gum acacia, dextrose, sodium alginate etc. by the proposed method and recoveries obtained were in the range 99.8-101.2%.The results suggested that the usual tablet diluents and excipients were found not to interfere with the analysis by the proposed method.

Analytical data
The Beer's law limit, molar absorptivity, Sandell's sensitivity, correlation coefficient, detection and quantitation limits obtained by least square treatment of the results are given in Table 2.

Applications
The proposed method was applied to the quantitative determination of studied drugs in pharmaceutical formulations and the results (Table 1) compare favorably with the official methods of the United States Pharmacopoeia 18 and British Pharmacopoeia. 19A statistical analysis of the results by f-and t-tests at 95 % confidence level showed no significant difference in the accuracy between the proposed method and official methods (Table 1).To ascertain the ruggedness of the method, four replicate determinations at two different concentration levels of the drugs were carried out.The within-day RSD values were less than 1 %.The values of between-day RSD for different concentrations of drugs, obtained from four determinations carried out over a period of 4 days, are given in Table 3, and indicate that the proposed method has reasonable ruggedness.

Conclusions
The method developed is simple, selective and offer the advantages of high sensitivity and a wide range of determination without the need for heating or extraction.The colour developed is stable for a sufficient interval of time and the method unaffected by slight variations in the experimental conditions such as acidity and other reagents.The proposed method was compared with other reported spectrophotometric methods and found to be superior (Table 4).The proposed method can serve as an alternative method for the determination of the studied drugs in pure and in dosage forms.

Figure 1 .
Figure 1.Absorption spectra of the CAT -iodine -starch complex with ISH (a) Blank (without ISH) (b) 0.5 µg mL -1 (c) 1.5 µg mL -1 (d) 2.5 µg mL -1 measured against water.The general reaction scheme of the method for studied drugs with CAT is represented as follows: Studied Drugs + CAT H + Oxidized Drugs + Reduced CAT + Unreacted CAT Unreacted CAT + KI H + Liberated Iodine Liberated Iodine + Starch H + Starch-Iodine blue coloured complex

Table 1 .
Results of assay of RTH, PZH, AMX and ISH in dosage forms a Average of five determinations, b Tabulated value 2.78 , c Tabulated value 6.39

Table 2 .
Optical characteristics and precision data

Table 3 .
Between-day precision of the determination of cited drugs by the proposed method

Table 4 .
Comparison with other reported methods