Fresenius Z Anal Chem (1989) 334:261 -265 Fresenius Zeitschrift fiir

0 Springer-Verlag 1989

Stopped-flow determination of paracetamol and oxyphenbutazone alone and in mixtures by a differential reaction rate method

Manuel Carmona, Manuel Silva, and Dolores Perez-Bendito Department of Analytical Chemistry, Faculty of Sciences, University of Cordoba, E-14004 Cordoba, Spain

Stopped-Flow-Bestimmung von Paraeetamol und Oxyphenbutazon aHein und in Mischungen mit Itilfe einer kinetisch-photometrischen Methode

Summary. A stopped-flow method for the determination of paracetamol and oxyphenbutazone individually and in mixtures with no prior separation is proposed. The method is based on the oxidation of these drugs by the tris(2,2'- bipyridine)-iron(III) complex in an acid medium. The reac- tions are monitored by measuring the rate of appearance of the absorbance of the ferroin complex (2max = 490 rim). The calibration graphs for the individual determination of the two drugs are both linear in the range 5 - 40 gg ml- l, and the precision (R.S.D.) is about 2%. A linear graphical differential reaction rate method is used for the resolution of mixtures of these drugs at the gg mI- t level at paracetamol/ oxyphenbutazone ratios from 6:1 to 1:2. The proposed method is practically free from interferences.

Introduction

ParacetamoI (N-acetyl-4-aminophenol) occupies a pre- dominant place amongst the extensively employed anti- pyretic analgesics. It is frequently formulated in drugs with salicylamide, analgin and oxyphenbutazone. Several meth- ods for the determination of paracetamol and oxy- phenbutazone have been reported, including their assay in drug formulations. Most of these are based on the oxidation of these drugs with oxidising agents using titrimetric or spectrophotometric techniques. Inorganic oxidising agents, such as cerium(IV) sulphate [1-5] , sodium nitrite [6-11], hexacyanoferrate(III) in an acidic medium [12,13], potassium dichromate [14] as well as indophenol formation reaction [15, 16] and organic ones such as iodylbenzoate [17] and iodoxy-benzene [18] have been used with this aim. Although, in general, these substances interfere severely in one or another of the currently available methods for the single determination of each, the resolution of mixtures of these drugs by non-chromatographic techniques has not been reported so far in the literature. A simple and rapid method for the resolution of paracetamol/oxyphenbutazone mixtures should therefore be of great analytical interest.

Offprint requests to: D. Perez-Bendito

This paper describes one such method for the routine kinetic-photometric determination of paracetamol and oxyphenbutazone individually and in mixtures by using the stopped-flow technique for the mixing of sample and re- agents. The method is based on the fast oxidation of these drugs with tris(2,2'-bipyridine)-iron(III) complex in an acidic medium. The resolution of paracetamol and oxyphenbutazone is accomplished by a differential reaction rate method. The reversible transformation of ferroin/ferriin complexes, on which the proposed determination is based, has been scarcely used for indication purposes in kinetic- based analytical methods [19- 22].

Experimental

Reagents

Stock solutions were made from analytical reagent grade chemicals (used without further purification) and distilled water. Paracetamol and oxyphenbutazone solutions (1 mg m1-1) were prepared by dissolving 100.0 mg of the corresponding drug (Aldrich-Chemie) in ethanol:water (2: 3 v/v) and then diluting with this solvent mixture to I00 ml in a volumetric flask. Working solutions were prepared by appropriate dilution of the stock solutions. Solutions of 0.175 tool/1 iron(III) nitrate (Merck) in 0.36 mol/1 sulphuric acid, 0.58 mol/1 2,2'-bipyridine (Aldrich-Chemie) in ethanol:water (3:2 v/v) and 4.5 mol/1 ammonium acetate (Merck) in distilled water were also made.

Apparatus

All photometric measurements were made on a Perkin- Elmer Lambda 5 spectrophotometer. A stopped-flow module designed by the authors [23] was fitted to the spectrophotometer in order to allow the rapid mixing of sample and reagents. Kinetic data (absorbance vs. time) were collected and treated by a Hewlett Packard 98561AE computer and a 16-bit Hewlett Packard analog-to-digital converter, provided with a program written by the authors for application of the initial-rate method and the differential reaction-rate method used for the resolution of mixtures of these drugs. The solutions in the stopped-flow module and in the spectrophotometer cell compartment were kept at a constant temperature by circulating water from a thermo- stated tank. A radiometer PHM62 pH-meter furnished with a combined glass calomel electrode was also used to measure the hydrogen ion activity.

262

Procedures

Individual stopped-flow determination of the drugs. Appro- priate aliquots of the paracetamol or oxyphenbutazone stan- dard solution were diluted to 10 ml With ethanol:water (3:2 v/v) solution in a standard flask in order to obtain the sample solution. The reagent solution was prepared by mixing in a 25-ml volumetric flask 5 ml of 0.175 tool/1 iron(III) solution, 10 ml of 0.58 tool/1 2,2'-bipyridine solution, 6 ml of 4.5 tool/1 ammonium acetate solution and the appropriate volume of concentrated hydrochloric acid to ensure a final pH of 1.25 when the flask was diluted to the mark with distilled water. The drive syringes of the stopped-flow module were filled with these solutions, which are mixed in the same proportion in the mixing/observation chamber of the stopped-flow system. The absorbance was monitored at 490 nm at a data collection rate of 250 ms/point and all measurements were carried out at 50 + 0.1~ The initial rate and the analyte concentration were automatically dis- played on a CRT by the computer.

Determination of paracetamol/oxyphenbutazone mixtures. Of the two 10 ml reservoir syringes of the stopped-flow module, one was filled with a solution containing between 5 and 30ggm1-1 of paracetamol and 5 and 10ggm1-1 of oxyphenbutazone, and the other with the above-mentioned reagent solution. The procedure was then identical to that for the determination of drugs alone except for the following: (a) the temperature was kept constant at 35 4- 0.1 ~ throughout the analysis and (b) the absorbance was recorded at 490 nm between 15 and 20 s after the start of the reaction at a data collection rate of 500 ms/point and again after completion (5 rain).

Results and discussion

As stated in literature [24], the tris(2,2'-bipyridine)-iron(IlI) complex, Fe(bipy)~ +, undergoes a colour change to intense

0.3- A

w 0.2- u z B tl] a: ~: 0.1- c)

m <

0 . 1 -

5 10 15

T IME. s

2% ~b 7% 16o 115 T I M E , s

Fig. 1 A, B. Absorbance vs. time graphs run at 490 nm, A Paracet- amol and B oxyphenbutazone. Drug concentration, 10 pg ml-x in both cases. Temperature 50~ Other experimental conditions as described in Procedure

red, due to the tris(2,2'-bipyridine)-iron(II) complex, Fe(bipy) 2 +, upon reaction with reductants. The reaction is fast when paracetamol or oxyphenbutazone are the re- ductants and can be followed by the stopped-flow technique by monitoring the reaction rate spectrophotometrically at 490 nm (maximum absorption wavelength of the Fe(bipy)~ + formed). Under these conditions paracetamoI is oxidized to N-acetyl-p-quinoneimine [22] and oxyphenbutazone to a similar quinoidal compound. This reducing action of paracetamol and oxyphenbutazone on the ferriin complex is used here for the kinetic determination of both drugs individually and in mixtures.

Figure I shows the absorbance vs. time curves obtained for both drugs with the aid of the data-acquisition system. As can be seen, the reduction is much faster with oxyphenbutazone than it is with paracetamoI. Previous stud- ies carried out on the kinetics of both compounds in this redox reaction showed that the kinetic dependence was similar, so that the forthcoming study of the influence of different variables was carried out on paracetamol only. All concentrations indicated in this study are the initial concentrations in the drive syringes of the stopped-flow module.

Effect of reaction variables

The kinetic procedure for the determination of paracetamol is dependent on factors such as temperature, pH, concentra- tion of oxidant, etc., which must be fixed and studied in order to obtain the most accurate and reproducible results possible.

Increasing temperatures in the range 20 ~ ~ caused corresponding increases in the initial reaction rate. A tem- perature of 50 +_ 0.1 ~ was selected. The Arrhenius plot for both drugs was obtained over the range 30 ~ 50~ The activation energies calculated from the slope were 69.5 and 34.4 kJ mo l - t for paracetamol and oxyphenbutazone, re- spectively. These values show the different reactivity of both drugs in this redox reaction.

Figure 2 a shows that the initial rate of the reaction is maximum at pH 1.25. From the corresponding log-log plot a partial order of 0.5 was found with respect to the hydrogen ion concentration in the pH range 1.25-2.0. Therefore, a pH of 1.25 was chosen; this was adjusted by adding concen- trated hydrochloric acid to the reagent solution. The initial rate was also affected by the presence of acetate ion in the reaction medium, although practically all of it occurred as acetic acid at the chosen pH. This influence, as is shown in Fig. 2b, was studied in the range 0.1 to 1.5 mol/1 (initial concentrations of acetate ion added as ammonium salt). It is evident from this figure that the acetate ion has a strong effect on this redox reaction up to a concentration of I tool/1. This behavior has also been reported for the 1,10- phenanthroline-iron(III) complex, though in weakly acid solutions [22].

The influence of Fe(bipy)] + on the reaction rate was studied as follows: first, the iron(III) and 2,2'-bipyridine concentrations were determined separately in order to find their optimum ratio in the complex, and then the optimum concentration of this chelate was determined at a fixed 2,2'- bipyridine/iron(III) ratio. Thus, the effect of the iron(III) concentration on the system was studied in the 4.6 x 10-3 to 2.3 x 10 .2 mol/1 range at a constant concentration of 2,2'- bipyridine (3.1 x 10 -2 mol/1). Increasing iron(Ill) concen-

263

%

x

2 o

E

5.0-

3.0"

10-

210 , / / ,

pH

b 8.0 ~ ~ ~o..--

6.0"

4.0-

2.0"

o12 o'.6 ~'.o ,'.~ IN Hz, Ac] ,molll

Fig. 2a, b. Effect of a pH and b acetate ion concentration on the reaction rate. 10 ~tg ml ~ paracetamol. For details see text

trations resulted in increasing initial rates up to l . l x 1 0 - 2 m o l / 1 , roughly corresponding to the stoi- chiometric ratio between this metal ion and the ferriin ligand. Higher concentrations had no effect on the reaction rate. Thus, an iron(III) concentration o f 1.4 x 10 -2 mol/1 was selected for further studies. This iron(III) concentration was fixed in the optimization of that of 2,2'-bipyridine, which was varied from 3.1x~0-2mol/1 to 1 .2x l0- tmol /1 . As with iron(III), the initial rate also increased with in- creasing concentration of this variable up to 7.7 x 10 -2 mol/1, which corresponds to a ligand :metal ratio o f 5.5:~.0. The reaction rate remained unchanged at higher concentrations. This excess of 2,2'-bipyridine over the stoichiometric ratio enhanced the stability o f the tris(2,2'-bipyridine)-iron(III) complex. A concentration of 9.3 x 10-2 mol/1 o f 2,2'-bipyri- dine was chosen for further investigations.

The influence of the ferriin complex concentration was assayed in the range 1 .75 -3 .5 x 10 - 2 mol/1, taking these concentrations as the initial iron(III) added to the reagent solution. As shown in Fig. 3, a zero-order kinetic dependence was found at complex concentrations higher than 3 x 10- 2 moI/1. In order to ensure the optimum yielding the minimum possible relative standard deviation for the initial rate measurements, a 3 .5x 10 - 2 mol/1 tris(2,2'-bi- pyridine)-iron(III) complex concentration was chosen.

Finally, under the experimental conditions used, the re- actions were pseudo first-order with respect to paracetamol and oxyphenbutazone. The pseudo first-order equation pro- posed for both drugs, based on the results of the above experiments, is as follows:

d(Fe(bipy)3 z + )/dt = k~ (drug)

where kr is the corresponding pseudo first-order rate constant. This constant was computed in each case from the

20-

% m

b x 15- o

c I0-

j 2'.0 215 3'.o

[Fe(bipy)~+] • lO'2molll Fig. 3. Effect of the tris(2,2'-bipyridine)-iron(III) complex concen- tration on the oxidation rate of 10 gg ml -a of paracetamol. Ex- perimental conditions as described in Procedure

Table 1. Analytical figures of merit for the determination of paracetamol and oxyphenbutazone by the stopped-flow technique

Characteristics Paracetamol Oxyphenbutazone

Dynamic linear range 5 - 40 ~tg ml- 1 5 - 40 gg ml- t Detection limit a 1.0 ~tg ml- 1 1.5 ~tg ml- 1 Precision (R.S.D.) b 1.8% 2.1% Sampling rate c 60 h- 1 90 h- a

a 3or value (n = 30) for 5 gg drug ml -x (25) b For t0 I-tg drug m1-1 (n = 11) c Each sample was analysed in triplicate

integrated rate equation in terms of the absorbance, which can be written as:

l n ( A ~ - A t ) = In Ao--kc t

where Ao is the initial absorbance, At the absorbance at time t and A~ the final absorbance. The slope of the plot of ln (A | vs. time provides the pseudo first-order rate constant. The average values found w e r e kp = 0.074 s - 1 for paracetamol and/Co = 0.104 s - 1 for oxyphenbutazone. Their ratio w a s ko/k p = 1 .40 .

Individual kinetic determination of paracetamol and ox•phenbutazone

Absorbance vs. time graphs were obtained for solutions containing different amounts of paracetamol or oxyphen- butazone under the optimum conditions. The figures o f merit for the determination o f these drugs by the stopped- flow technique under the recommended procedural conditions are shown in Table 1.

Simultaneous determination of paracetamol and oxyphenbutazone

The pseudo first-order rate constant ratio between oxyphenbutazone and paracetamol found under the optimum experimental conditions was rather small and therefore did not allow the application o f any differential reaction-rate method for the resolution of their mixtures with acceptable accuracy. In order to increase this rate constant ratio without appreciable adverse effect on the sensitivity of the determination, the influence of the reaction

264

variables was also studied. Neither the pH nor the tris(2,2'- bipyridine)-iron(III) complex concentration allowed the rate constant ratio to be enhanced; however, the temperature exerted an appreciable effect as can be seen from Fig. 4. At a working temperature o f 35 ~ the rate constant ratio increased up to 2.43, which corresponds to a kp = 0.015 s- ~ for paracetamol and ko = 0.039 s- t for oxyphenbutazone. Under these conditions, the initial rate for both drugs de- creased by about 50%. This temperature was selected as optimum for the simultaneous determination of paracetamol and oxyphenbutazone.

From the differential reaction-rate methods reported in the literature [26] and from previous studies on their accu- racy in the resolution of mixtures of these drugs, a simplified version of the linear-graph Worthington and Pardue method [27] was used.

Consider the following pseudo first-order reactions:

kA A + R , P

kB

B + R ~ P

where kA and kB are the pseudo first-order rate constants. When these reactions proceed simultaneously and their rate constants are independent of each other (the sum of the concentration of A and B reacting to form a common prod- uct, P, is given), at any time t, we can write

(A)t + (B)t = (P)~ - (P), = (A)o exp ( - k a t ) + (B)o exp (-/Cat) (1)

where (A)o and (B)o are the initial concentrations of A and B; (A)t, (B)t and (P)t are the concentrations o f A, B, and P,

30.

i o • 20-

o

"~ 10- ...

E

2b ~'0 ~3 5'O T e m p e r a t u r e , ~

Fig. 4. Dependence of the initial rate of both reactions on the tem- perature. (3 10 Ixgm1-1 paracetamol and �9 10 ggml -~ oxyphen- butazone

respectively, at time t, and (P)~ is the final concentration of P.

By assuming that kA > kB in Eq. (1) and a time t at which exp (--kAt) ~ 0 and exp ( - - k B t ) r 1 (but is not near zero), Eq. (1) can be written as:

(P), = (A)o + (B)o [ l - exp( -kBt ) ] . (2)

A potential simplification of Eq. (2) relies on the assump- tion that exp (--kBt) ~ 1 - -kBt , which is the case when kB is small, since exp ( - k B t ) can be expanded as a series,

exp ( - kBt) = 1 -- kBt + (kRt)2/2! -- (kBt)3/3 ! + . . .

0.16- ~ o O O

(Oxyphen butazone)o

0.12- . I o

o 7 ~ ~176 ~./~o o~

On / / ~176176 0 . 0 8 - / / o

m .171 < o

o o o 0.04 - o

o <, o

o o

; 1'0 1; 2; 2'~ T i m e , s

Fig. S. (P)t expressed in terms of absorbance vs. time plot for a synthetic mixture containing 5.0 gg ml - 1 of each drug

Table 3. Tolerated limits for various species in the simultaneous determination of paracetamol and oxyphenbutazone, 5.0 gg ml- 1 each

Species added Tolerated concentration (gg ml- 1)

Salicylic acid, acetylsalicylic acid, caffeine, phenacetin, glucose, lactose, sucrose, talc, starch, glycerol

Salicylamide

Ascorbic acid

500

400

4

Table 2. Analysis of synthetic mixtures of paracetamol and oxyphenbutazone by the stopped-flow technique

Amount taken (p,g ml- 1) Paracetamol Oxyphenbutazone

Paracetamol Oxyphen- Found Error Found butazone (gg ml- 1) (%) (gg ml- 1)

Error (%)

5.0 5.0 4.9 --2.0 5.0 10.0 5.0 9.9 --1.0 5.1 20.0 5.0 19.7 --1.5 5.3 30.0 5.0 30.2 0.7 5.3

5.0 10.0 5.3 6.0 9.8

0.0 2.0 6.0 6,0

-2 .0

265

and, under these conditions, only the first two terms in the series are significant. This simplification reduces Eq. (2) to:

(P)t = (A)o + (B)okBt (3)

so that the plot of (P), as a function of time becomes a straight line with a slope and intercept which allow the calculation of (B)o (if kB is known) and (A)o, respectively.

In the application of this method to the mixture dealt with in this work, (A)o and (B)o are the initial concentrations of oxyphenbutazone and paracetamol, respectively, in the mixture (kg = ko and ka = kp, and therefore ko > kp). As stated above, this method requires that exp (-kpt) ~- 1 -kpt to be applicable. Taking into account the reported values of this rate constant, this simplification was evaluated in terms of measurement times. First, at short times, the simplifica- tion was more accurate; however, the contribution of the fast component (oxyphenbutazone) was also more appreciable (exp (-kot) r 0). With longer times, it was evident that the fast component had reacted fully, but the errors involved in the simplification were larger (i. e. for a reaction time of 25 s the error was about - 1 0 % ) . Therefore, the time interval over which the differential reaction-rate method can be applied must be selected taking into account these two opposing effects. A measurement time interval between 15 and 20 s after the start of the reaction was chosen. On the other hand, as stated above, the concentration of the slow component (paracetamol) can be determined from the slope of the plot. In this work, more accurate results were achieved if this concentration was obtained from the (P)~o value, taking into account that (P)oo = (A)o + (B)o.

The proposed differential reaction-rate method was applied to the determination of various synthetic mixtures containing oxyphenbutazone and paracetamol in different ratios. The results obtained are summarized in Table 2. Figure 5 shows by way of example a typical plot cor- responding to a mixture containing 5.0 txg m1-1 of each drug. Mixtures of these compounds can be determined in oxyphenbutazone/paracetamol ratios from 1:1 to 1:6 and paracetamol/oxyphenbutazone ratios from 1:1 to 1:2. These results are satisfactory and agree with the relative content of these drugs in pharmaceuticals.

The precision of the method, expressed as the relative standard deviation for a mixture containing 5.0 gg ml - 1 of oxyphenbutazone and 10.0 l-tg ml - ~ of paracetamol was 4.2% and 1.6%, respectively (P = 0.05, n = 11). Finally, the selectivity of this method was also tested. The species assayed in this study (other drugs, excipients and diluents), are

commonly associated with paracetamol and oxyphen- butazone in pharmaceutical samples. As can be seen from the tolerated amounts of foreign species listed in Table 3, the method offers good selectivity in the simultaneous deter- ruination of these analgesics.

Acknowledgement. The authors gratefully acknowledge financial support from the C.A.I.C.Y.T. (Project No. 0979/84).

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Received December 19, 1988; revised February 17, 1989