A Simple Kinetic Spectrophotometric Method For The Determination of Certain 4-Quinolones in Drug Formulations

An accurate simple and selective kinetic procedure for the determination of certain 4-quinolones namely, norfloxacin (I), ofloxacin (II), enrofloxacin (III), fleroxacin (IV), ciprofloxacin (V) and pefloxacin (VI) is described. The procedure is based on reacting the studied compounds in acidic media (0.1 M HCL) with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) in presence of cerium (IV) ammonium sulphate as an oxidant at room temperature for a fixed time of 20 min. for (I), (III), 12 min for (II) and 30 min for IV , V and VI, then the absorbance of the reaction product is measured at 630 nm. The concentration of the studied compounds is computed using the corresponding calibration curve equation for the fixed-time method. The absorbance-concentrations plot is rectilinear over the range 20-100 ug.ml-1 for (I), 2 - 20 ug.ml-1 for (II), 10 - 74 ug.ml-1 for (III), 10 - 60 ug.ml-1 for IV, 10 - 50 ug.ml-1 for IV and 8 - 40 ug.ml-1 for VI. The determination of the studied compounds by the fixed-concentration and rate constant methods is feasible with the calibration equation obtained, but the fixed-time method proved to be more applicable. The procedure was applied successfully to commercial tablets and ampoules and the results obtained were compared statistically with the reference methods.


Introduction :
The studied 4-quinolones are antibacterial agents, structurally related to nalidixic acid but with a wider antibacterial spectrum and greater activity'.Various techniques have been utilized for the determination of the studied 4-quinolone compounds either per se or in dosage forms.The most commonly used methods for the determination of the studied compounds were reported in the recently published revied.Also, spectrophotometric3", spectrofluorimetric8~1', condu~timetric'~ and HPLC" methods were reported for their determinations .
Kinetic-based methods of pharmaceutical analysis are not widely applied, although they offer certain advantage of elimintating the additive interferences which are likely affect other methods such as titrimetry or direct spectrophotometric methods, beside, it offers certain selectivity .
The literatures are still poor in analytical procedures based on kinetics especially for pharmaceutical or biological fluids.
Furthermore some specific advantages in the application of kinetic methods can be expectedI4; 1-Possibility of no interference of the coloured and or turbidity background of the samples, and ... A kinetic method is proposed for the determination of studied compounds based on the formation of a coloured reaction product with 3-methyl 2-benzothiazolinone hydrazone HCL (MBTH) in the presence of Ce (IV) as an oxidant and measuring the absorbance at 630 nm in an acidic medium.MBTH has different advantages I5*l6 including : a) good solubility in water , b) oxidative coupling reactions over a wide pH range, c) highly sensitive colour reaction .The proposed method is more sensitive than the titrimetric methods adopted by USP" for the drug in bulk and pharmaceutical preparations.The method is simple, rapid and readily adoptable to both the bulk drug and unit dose determinations.

Apparatus:
The spectrophotometric measurements were made on the UV.Visible1 Spectrophotometer, model UV-1601 Shimadz recording Spectrophotometer (P/N 206 -67001) equipped with kinetic accessory provide with temperature controlled cell(Tcc -240 A) Thermoelectrically temperature.
The purity of these compounds was established by applying the recommended official" or reference method^^^-^^.Pharmaceutical preparations containing the studied compounds were purchased from commercial sources in the local market.

Stock Solutions:
Standard solutions 10.0 mg of the studied compounds were prepared freshly every day in 0.1 M HCL and were successfully diluted to contain the reported concentration range in ug.ml-' (Tab.5) .

General Procedure:
Transfer aliquots 1.0 ml of stock solution of each drug into a series of 1 Om1 calibrated flasks.Add to each flask Iml of 0.3% MBTH and 2.3 f 0.3 ml of 1% Cc (IV), shake well and let to stand for 20 min.for (I), (III), 12 min for (11) and 30 min.for IV,V and VI complete to the mark with distilled water.Measure the absorbance of the resulted solution at 630 nm with time during 40 minutes at ambient temperature (25'C).A blank experiment is prepared simultaneously.Determine the drug concentration during the first 40 minutes of reaction by using the appropriate graphs.Plot log reaction rate versus log concentration of the drugs to get order of the reaction.For calibration curves , carry out the same procedure.Measure the absorbance of the solution at 630 nm.Plot the values of absorbance against the concetration of each drug in ug.ml-' at the specified fixed time.Alternatively, the regression equation is derived (Tab.4).Alternatively the regression equation is derived.Procedure for Spectrophotometric Determination of the Studied Compounds in Dosage Forms.

For tablets :
Weigh and pulverize 10 tablets, weigh accurately an amount of the pulverized tablets equivalent to 20.0 mg of the studied compounds and transfer into 100 ml conical flask.Extract the drug three times each with 30 ml of 0.1 M HCL.Filter the extract into a 100 ml measuring flask.Wash the conical flask with few mls of 0.1M HCL and pass the washings into the same volumetric flask.Complete to volume with 0.1 M HCL.Transfer aliquots of these solutions and continue as previously stated above (Tab.6) .

For ampoules :
Transfer aliquot of peflacin mesylate ampoule equivalent to contain 20.0 mg of VI into 100 ml measuring flask dilute and complete to volume with 0.1 M HCI.Transfer aliquots of this solution within the concentration range cited in table 6 into a series of 10 ml calibrated flask.Proceed as mentioned under general procedure .Calculate the nominal content of the ampoules either from a previously plotted calibration graph or using the regression equation.

Results and Discussion: Kinetic and Optimization of the Reaction Conditions:
The reaction between the studied 4quinolones and MBTH in acidic medium in the presence of Ce (IV) as an oxidant yields a green colour.The oxidation product shows maximum absorbance at 630 nrn (Fig. 1).
. .The reaction was studied under various concentrations of both the reagent and oxidant for varying time at room temperature or under the effect of heat.
It was found that the colour intensity reaches maximum after leaving at room temperature.
Room temperature was preferred as upon heating the reaction mixture, precipitatition takes place .
The influence of cerium (IV) ammonium sulphate on the reaction rate was studied in the range 1-4%.It was found to be 2.3 2 0.3 ml. of 1% solution of Ce (IV) was optimum for development of the colour reaction and maximum formation of the product .
As the intensity of the colour increases with time, (Fig. 2) this was used as a usefull kinetic method for the determination of studied 4-Quinolones either in pure form or in pharmaceutical preparations.The rate of the reaction was also found to be concentration dependent.The reaction rate was followed at room temperature with various concentrations of the studied compounds over the range -100 ug.ml-' for I ,2-30 ug.ml-' for I1 and 10-74 ug.mi! for I11 , 10 -60 ug.mi' for IV, 10 -5ug.mi! for V and 8 -40 ug.rni' for VI, keeping MBTH and Ce (IV) concentrations constant.(Fig. 3).

Log concentral~on
Fig. 3 : Plot of log absorbance versus log concentration of norfloxacin at 630mm.
The graph shown in figure 3 clearly indicates that, the reaction rate of the studied compounds obeys the following equation:-Rate = K-[Drugln (1) Where Kis the pseudo order rate constant and n is the order of the reaction.The rate of the reaction may be estimated by the variable-time-method measurement18 as AAIAt, where A is the absorbance and t is the time in seconds.Taking logarithms of rates and concentrations (tables 1A ,lB, 1C and ID) and figure 5, equation ( 1) is transformed into.

Table :l. A.
Logarithms of rates for different concentrations of norfloxacin at room temperature at 630 nm.(r = 0.997 1) Hence k* = 0.5796 Sec. and the reaction is first order (n= 0.683), with respect to ofloxacin concentration.Hence K-= 20.8 sec.and the reaction is first order (n = 1.22), with respect to pefloxacin concentration

Evaluation of the Kinetic Methods:
The quantitation of the norfloxacin, ofloxacin, enrofloxacin, fleroxacin, ciprofloxacin and pefloxacin, under the optimized experimental conditions outlined above, where MBTH concentration was 40 times the initial concentration of norfloxacin and 200 time the concentration of ofloxacin would result in a pseudo first order reaction with respect to their concentrations.
However, the rate will be directly proportional to the drug concentration in a pseudo first order rate equation as follows: Rate = K-(any of the studied4-quinolones) (3) Where Kis the pseudo-first order constant.Several experiments were then carried out to obtain the drug concentration from the rate data according to equation (3).lntial rate, rate constant.fixed-concentration and fixed time'9320 methods were tried and the most suitable analytical method was selected taking into account the applicability, the sensitivity (i.e. the slope of the calibration graph), the intercept and the correlation coefficient (r).(r = 0.9992) For Enrofloxacin over the range 2.783 x 10" to 2.059 x 1 0-4 M.

Table : 2.D.
Values of Kcalculated from slopes of log A versus t for pefloxacin mesylate K-= -5.663 x 1.397 C ( ~0 .9 9 1 0 ) For pefloxacin over the range 1.205 x to 2.410 x M.

Fixed Absorbance Method :
Reaction rates were recorded for different drug concentrations over the range 9.397~ lo-' to     Values of reciprocal of time taken at fixed absorbance (0.3) for different rates of variable concent l / t = 5 .4 ~1 0 -~+ 6 .5 9 8 C (r = 0.9886) Fixed Time Method: Reaction rates were detcrlnined for different concentrations of the studied 4-quinolones.At a preselected fixed-time, which was accurately determined, the absorbance was measured.Calibration graphs of absorbance versus initial concentrations of the studied compounds at fixed times, with the regression equations assembled in tables 4A, 4B, 4C and 4D.

Table : 4.A.
Regression equations at different fixed times for norfloxacin in the range 6.265x10-' to 0.1325~1 O -~M at constant concentration of MBTM and Ce (IV) at roo111 temperature.

Table :4.B.
Regression equations at different fixed times for ofloxacin in the range 5.534x10-' to 5 .5 3 4 ~ 1 O -~M at constant concentration of MBTH and Ce (IV) at room temperature.

Table :4.C.
Regression equations at different fixed times for enrofloxacin in the range 2.783~10-' to 2 .0 5 9 ~ 1 O -~M at constant concentration of MBTH and Ce (IV) at room temperature.After optimizing the reaction condition, the fixed time method was applied to the determination of the studied compounds in pure form.Statistical analysis2' of the result obtained by the proposed and official or reference methods2226, revealed no significant difference between the performance of the two methods regarding the accuracy and precision.The validity of the method was evaluated by statistical analysis of the regression data2'.The results are shown in table 5.

Application of the Fixed Time Method to the Determination of the Studied Compounds in Dosage forms.
The proposed method was further applied,to the determination of the studied 4-quinolones in pharmaceutical preparations.Common tablet excepients such as talc, n~agnesiunl stearate, lactose and starch did not interfere with the assay.The results are shown in table 6.The reaction between the studied 4-quinolones and MBTH in acidic medium in the presence of Ce (IV) as an oxidant yields a green colour.The oxidation product shows maximum absorbance at 630 nnl (Fig. 1).due to oxidation of MBTH by Ce (IV) accon~panied by a si~llultaneous loss of a proton and two electrons forming an electrophilic intermediate which has been postulated to be the active coupling species.In the second step, an electrophilic reaction between the keto group of the drug and the electrophilic intermediate with the formation of coloured product and elimination of one molecule of water.The mechanism of interaction of the studied 4-quinolones with MBTH was determined via molar ratio method2' and It was found that using equin~olar concentration of the studied drugs and MBTH at constant concentration of Ce (IV), show that the drugs interact via the carbotlyl group with the hydrazone group of MRTH by forming formazan (show max.absorbance at 630 nm) in a molar ratio of 2: 1 (MBTH : drug) (Fig. 5).

cectv) l , ti'
The proposal mechonwn of the reocl~on of norfloxac~n with MET tI In the Presence of ceriurn(lV)ommon~um. sulphale Fig. 5 : Stoichiometry of the reaction between norfloxacin-MBTH (5 x 10" M) via molar ratio method.

Conclusion :
The kinetically-based method proposed in this work for the quantitation of the studied compounds is a direct method and more sensitive compared to USP method.Furthermore, the proposed method doesn't require elaboration of treatment and procedures which are usually associated with the chro~natographic methods.

Table 5 :
Analytical parameters for the determination of the studied con~pounds in pure form using fixed time method.

2 -
Possibility of no interference of other active compounds present in the commerical product if they are resisting the chemical reaction conditions established for the proposed kinetic method.

2 .
506~ 1 o -~ M for I, over the range 3.3204xl0-~ to 5.534x10-~ M for 11, 8.348~10-' to 1.558x10-' M for 111, 7.337 x10e5 to 1.087 x10-~ M for IV, 9.038 x10-~ to 1.506 x10-' M for V and over the range 6.025 x10-~ to 1.205 x 1 0 -~ M for VI.A preselected value of the absorbance was fixed, and the time was measured in seconds.The reciprocal (llt) versus the initial concentration of the studied con~pounds (Tables 3A, 3B73C and 3D), was plotted and the following equation of the calibration graph was obtained.
llt = -7 .0 6 9 ~1 0 -~ + 35.792C (r = 0.9959) Regression equations at different fixed times for pefloxacin in the range 2.4 1 x 10" to 1 .2 0 5 ~ ~o -~M at constant concentration of MBTH and Ce (IV) at room temperature.Application of the Fixed Time Method for the Determination of the StudiedCompounds in pure form.
Sylx = standard deviation of residuals S, = Standard deviation of intercept of regression line.Sb = Standard deviation of slope of regression line.

Table : 3
.C. Values of reciprocal of time taken at fixed absorbance (0.3) for different rates of variable concentrations of enrofloxacin at constant concentration of MBTH and Ce (IV).

Table : 6
Spectrophotonetric determination of the studies compounds in pharnlaceutical preparations by Each result is the average of three separate determinations.Figures in parentheses are the tabulated t and F values respectively at P= (0.05)~'. 1. Product of Eipico.Pharm.Co. Cairo, Egypt.2. Product of Hoechest Orient Pharm.Co. Cairo, Egypt.5.Product of Eipico-Pharm.Co-Cairo, Egypt.6.Product of Amriya for Pharmaceutical Industries.Alex .Egypt.