Spectrophotometric Determination of Chloramphenicol in Pharmaceutical Preparations

A spectrophotometric method for the determination of trace amounts of chloramphenicol has been proposed. This method depends upon the reduction of the nitro to amino group, condensation with promethazine reagent in the presence of cerium (IV) ions to form a colored dye which exhibits maximum absorption at 606 nm with a high sensitivity (molar absorptivity is 1.292×10 4 l.mol -1 .cm -1 , and Sandell's sensitivity index of 0.025 μg/cm 2 ). Beer' s law is applied within the concentration range of obeyed in 0.4 to 12 ppm with a relative error of -2.73 to + 0.37% and a relative standard deviation of ± 3.84 to ± 0.44%, depending on the concentration level. The method has been successfully applied to the

Reduced chloramphenicol working solution (100μg ml -1 ): 20 ml of reduced CAP solution (500μg ml -1 ) was transferred, the solution was brought to pH 7.0 with sodium carbonate solution, filtered, then the filtrate solution diluted to 100 ml in a standard volumetric flask.

Procedures for pharmaceutical preparations Capsules (BROWN and BURK, (UK) LTD., London)
The contents of 12 capsules (250mg) were weighed and the powder was mixed.The accurately weighed portion of the powder equivalent to one capsule dissolved in 20 ml of distilled water (with warming).The solution was filtered into a 50 ml calibrated flask.To obtain RCAP solution 5 ml of this solution was transferred and react with zinc powder and hydrochloric acid preceded as a procedure described above.

Eye drops (REYERLABS, India)
The contents of five bottles of eye drops (0.5%) was mixed.10 ml of this solution was transferred into a 50 ml calibrated flask and diluted to the mark with distilled water.25 ml of this solution was transferred and proceeded as procedure for RCAP described above.

Eye ointment (HOLEN)
The contents of five bottles of eye ointment (0.1%) was mixed.5 g of this ointment was dissolved in 50 ml of petroleum ether then this solution was extraction using distilling water into 4 portion, each portion contain 50 ml of distilled water.Then filtrated RCAP obtain by using procedure described above.Promethazine.HCI solution (0.1%): The solution was prepared by dissolving 0.1 g of promethazine.HCl from (SDI) in distilled water and completed to 100 ml in volumetric flask.This solution was store in dark bottle; it is stable for at least one week.
Oxidative reagent solution (0.1%):This solution was prepared by dissolving 0.1 g of Ammonium ceric sulphate dihyrate (ACS) from (BDH) in 100 ml of warm distilled water.This solution was used in the same day.Formic acid solution (1N): This solution was prepared by dilution 9.6 ml of concentrated formic acid (25.97 N) (Fulka) with distilled water to the mark in 250 ml volumetric flask.Excipient drugs (1000 ppm): This solution was prepared by dissolving 0.1 g in 100 ml of distilled water.

General procedure and Calibration graph
The aqueous solution of RCAP contains (10-700) µg was transferred to 25 ml calibration flask.A 4 ml of promethazine.HCl reagent (0.1%) solution, 5 ml of formic acid (1N) then 8 ml of oxidative reagent (cerium IV) (0.1%) were added and the volume was made up to the mark with distilled water .The absorbance measured after 15 minute at 606 nm against a blank solution which was prepared in a similar way but without the addition of RCAP .The calibration graph as shown in Fig. (2) was liner over the range of (10-300) µg of RCAP/25 ml (0.4-12 ppm).Higher concentration showed a negative deviation from Beer's law.The apparent molar absorpitivity referred to RCAP 1.292×10 4 l.mol -1 .cm - and Sandel's sensitivity is equal to 0.025 µg/cm.

Results and discussion
The effect of various variables on the color development was tested to establish the optimum conditions for determination of CAP by oxidative coupling with promethaizne.HCl reagent.

Principle of the method
The method involves two steps: Step 1: Oxidation of promethazine.HCl reagent to give a red intermediate.
Step 2: The intermediate couples with reduced CAP to form a blue-green color.

The effect of acids
Effect of different acids has been studied on the intensity of dye; the results are shown in (Table 1).5 ml of formic acid (1N) has been chosen in the next experiments.Because in formic acid, the reaction becomes less sensitive to media environment than others and the formation of the greenish-blue dye is not affected by acid amount.

Choice the reagent
Several different reagents (3.1×10 -3 N) have been studied with 4 ml of each, and also 7 ml of sodium hydroxide (1N) instead of 5 ml acid also have been studied.The results are shown in (Tables 2a, 2b, 2c, 2d).------------- ----------------Thymol From the results it is clear that promethazine give high results in selectivity and color contrast, its solution is easy to prepare and stable therefore it used for next experiments.

Effect of reagent amount
The effect of reagent amount on sensitivity of method has been studied.A series of solutions contain different volume of promethazine(0.1%)reagent with different amounts of RCAP have been made.The results are shown in (Table 3). 4 ml of the reagent have been chosen from the results obtained because the sensitivity of the reaction is good, the absorbance and correlation coefficient is excellent as well as the absorbance of blank is low.

Choice of oxidative reagent
Number of oxidative reagent (1.5×10 -3 M) in 4 ml has been studied on the absorbance of dye contained; the results are shown in (Table 4).The results shown in (Table 4) show high absorbance for potassium iodate and potassium periodate consider to the blank which give low absorbance, which are not used because they give unstable products.ACS still used for next experiments.

Effect of oxidative reagent amount
Different volumes of oxidative reagent ACS (0.1%) have been studied on the absorbance of solution contain different volumes of RCAP.Table 5 shows 8 ml was the best therefore it was used for the next experiments.

Effect of surfactant
The effect of several types of surfactants on color intensity of the colored product has been investigated.The results indicate that addition of surfactants give no several effect [increasing the intensity or improving the color contrast (∆λ)], therefore it has not been used in the subsequent experiments.

Order of addition
The order of additions of reagent (C, P, O and A) was examined.The results shown in (Table 6) indicated that the order (VII) of addition of reagents was the optimum order due to the high intensity of the formed dye.

Effect of time
The effect of time on the development and stability period of the formed colored product was investigated under optimum experiment conditions described before.The formation of colored product being complete after mixing the component of reaction and the absorbance of the colored species remained constant for, at least 45 minute.Fifteen mintutes have been recommended as a formation colored product for the subsequent experiments.(Table 7).

Final absorption spectrum
After addition of RCAP to solution contain promethazine reagent, ACS (IV) and formic acid under optimum conditions described before, colored product was formed, shows a maximum absorption at 606 nm in contrast to the reagent blank.The maximum wavelength 606 nm has been used in the subsequent experiments.(Fig. 3).

Effect of some excipients on the assay of CAP
To test the efficiency and selectivity of the proposed analytical method, a systematic study of excipients that usually present in dosage showed that there was no interference from excipients up to 1000 µg in the present method as shown in (Table 8).

Accuracy and precision
To check the accuracy and precision of the calibration curve, CAP was determined at three different concentrations.The results (illustrated in Table 9) indicated that the method is satisfactory.

Nature of Dye
Job's method (21) indicates that the colored product has a composition of 1:2 CAP to promethazine reagent (Fig 4).Effect of organic solvent Different organic solvents have been used to examine their effects on the dye.The results are shown in (Table 10) .

Water
According to its good sensitivity and low cost water has been used for dilution.

Application of the method
The proposed method was successfully applied to determine CAP in its pharmaceutical preparations (capsule, eye drop and eye ointment) (Table11) from different sources.This method was compared with the N-NED ( 22) method.The results showed that there was no significant difference between the proposed and N-NED method.

Comparison of methods
Table (12) shows the comparison between some of analytical variables for the present method with that of other spectrophotometric methods.

Table 12 . Comparison of the methods
Present method N-NED (22)  Literature method (23)  Analytical parameters The results indicate that the proposed method is not less efficient than other methods

Conclusion
The determination of chroramphenicol in pharmaceutical preparations has been developed.The method is based on the oxidative-coupling reaction of the drug with promethazine.HCl in the presence of cerium (IV) ion.The blue-green color formed is measured as a function of drug amount.The accuracy of the method has been tested by its application to assay of chloramphenicol in pharmaceutical preparations (capsules, eye drop and eye ointment) and found to be successful.

Fig. 1 .
Fig.1.Structure of chloramphenicol Test and selection of coupling agent in the presence of 5 ml of 1 Test and selection of coupling agent in the presence of 5 ml of 1

Fig 3 .
Fig 3. Absorption spectra of 200µg/25 ml of CAP (treated according to the recommended procedure) against (A) blank (B) distilled water and (C) blank measured against distilled water.

Fig 4 .
Fig 4 .Job's plot for CAP with promethazine solutionHence, the colored product may have the following structure.

Table 4 . Test and selection of oxidative reagent pH Absorbance of Color of Oxidative reagent
The sample became dark turbid blue upon addition of formic acid, while the blank remains yellow. *