ANALYST, JUNE 1990, VOL. 115 757

Catalytic Titration of A/-Penicillamine, A/-Acetylcysteine, Cysteine and 2-Mercaptopropionylglycine*

Pilar Vinas, Manuel Hernandez Cordoba and Concepcion Sanchez-Pedreiot Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30071 Murcia, Spain

S e m i - a u t o m at i c cat a I yt i c tit r a t i o n s of N- pe n i c i I I a m i n e , N- a ce t y I cyst e i n e , cyst e i n e a n d 2 - m e r ca pt o - propionylglycine using silver nitrate as titrant are described. Procedures are based on the inhibitory effect of these thiol-containing drugs on the silver-catalysed phloxin - persulphate reaction. The end-point is determined spectrophotometrically by measuring the absorbance at 537 nm. The proposed methods were applied to the determination of the drugs in pharmaceutical preparations. Keywords: Catalytic titration; N -penicillamine; N -acetylcysteine; c ysteine; 2-mercaptopropion ylglycine

Catalytic titrations have been widely applied to the determina- tion of both organic and inorganic compounds and an extensive review1 has been published.

The silver-catalysed oxidation of phloxin by persulphate in the presence of 2,2'-bipyridyl as activator has been reported previously by Hernandez Cordoba et a1.2 Iodide, bromide and thiocyanate have an inhibitory effect on the catalysed reaction and have been determined by titration with silver nitrate using the catalytic reaction for the indication of the end-point.'

Thiol-containing compounds have a similar inhibitory effect on the silver-catalysed reaction. In this study, semi-automatic spectrophotometric catalytic titrations for the determination of N-penicillamine. 2-mercaptopropionylglycine (drugs fre- quently used as therapeutic agents in Wilson's disease and rheumatoid arthritis and as efficient antidotes in heavy-metal poisoningJ), cysteine and N-acetylcysteine (a well known mucolytic agent4) are described. These drugs are usually determined by chromatographic-5-15 or spectrophotomet- ric1+23 techniques. Catalytic methods for the determination of cysteine have also been published.24-26 Recently, a catalytic titration method for cystine - cysteine mixtures has been reported.27

Experimental Reagents and Solutions

Inorganic chemicals were of analytical-reagent grade and the solutions were prepared with doubly distilled water. Phloxin (dichlorotetrabromofluorescein) solution ( 5 x 10-4 M) was prepared in water from the commercial product (Geigy) without further purification. N-Penicillamine (Fluka, Buchs, Switzerland), 2-mercaptopropionylglycine (Merck, Darm- stadt, FRG) , N-acetylcysteine (Merck) and cysteine (Merck) stock standard solutions (1 x 10-3 M) were prepared in water and kept at approximately 4 "C; working standard solutions were prepared by dilution just before use. Silver nitrate solution (1 x 10-3 M) was prepared daily and kept in the dark. Other reagent stock solutions were 1 M acetate buffer (pH 5 ) and 0.003 M 2,2'-bipyridyl. A 0.15 M potassium persulphate solution was prepared fresh daily.

Apparatus

A Metrohm 616 photometric titrator was coupled to both an Omniscribe recorder and a Radiometer ABU 12 automatic constan t-rate burette.

* Presented at the 3rd International Symposium on Kinetics in Analytical Chemistry, Dubrovnik-Cavtat, Yugoslavia. 25-28 September, 1989.

t To whom correspondence should be addressed.

1 I I 1 I I

1 mM AgN03 (1 division = 1 ml)

Fig. 1. Catalytic spectrophotometric titration of drugs containing thiol groups with 1 X 10-3 M silver nitrate solution. A , Blank titration; B, 2-rnercaptopropionylglycine (161 yg); C, N-penicillamine (148 yg); D, cysteine (121 pg); and E, N-acetylcysteine (166 pg)

0 ' I I I I I I I

1 mM AgN03 (1 division = 2 min)

Fig. 2. Influence of the titrant addition rate on the titration of 121 pg of cysteine with 1 x 10-3 M silver nitrate solution. A , 0.1425; B. 0.285; C, 0.57; and D, 1.14 ml min-1

Procedure

Fill the automatic burette with standard silver nitrate solution of a concentration appropriate to that of the titrand in the sample. Set the wavelength of the photometric titrator at 537 nm and the rate of titrant addition and the chart speed at the optimum values depending on the silver nitrate concentration. Add the reactants to the titration vessel in the following order: 1 ml of 5 x 10-4 M phloxin, 10 ml of 1 M acetate buffer (pH 5 ) , 10 ml of 0.003 M 2,2'-bipyridyl, the sample containing the inhibitory substance and 10 ml of 0.15 M potassium persul- phate solution. Dilute to 50 ml with doubly distilled water. Stir the solution, switch on the recorder and the autoburette simultaneously and record the absorbance - volume graph. Obtain the end-point graphically and calculate the titrand concentration. A blank titration must be performed in the

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758 ANALYST, JUNE 1990, VOL. 115

Table 1. Results for thc catalytic titration of N-penicillamine (PA)

AgNo3/M PA takcn/mg PA found*/mg 2 x 10-4 0.0073 0.0064

0.0147 0.0142 0.0221 0.0215 0.0294 0.0288 0.044 1 0.0434 0.0588 0.0580

1 x 10-3 0.0370 0.0355 0.0740 0.0736 0.1110 0.1112 0.1480 0.1487 0.2220 0.2229 0.2960 0.2977

* Means of five replicate determinations.

Standard deviatiodmg 5.76 x 4.02 x 10- 4

4.18 x 10 4.33 x 10-4 4.76 x 10-4 6.02 x 10-4 9.58 x 10 1.83 x 10 1.53 x 10-3 1.25 x 10-3

2.34 x 10-3 2.08 x 10-3

RSD,% 3.0 2.8 1.9 1.5 1 . I 1 .o 2.7 2.5 1.4 0.8 0.9 0.8

Error .YO -4.0 -3.4 -2.7 -2.0 -1.6 -1.4 -4.0 -0.5 +0.2 +0.5 +0.4 +0.6

Table 2. Results for the catalytic titration of N-acetylcysteinc (AC)

AgN03/M AC taken/mg AC found*/mg 2 x 10-4 0.0082 0.008 I

0.0163 0.01 62 0.0245 0.0243 0.0327 0.0328 0.0491 0.0493 0.0654 0.0659

1 x 10-3 0.0415 0.0419 0.0830 0.0840 0.1245 0.1261 0.1660 0.1671 0.2490 0.2477 0.3320 0.3347

* Means of five replicate determinations.

Standard .ieviation/mg 4.61 x 10 J

3.03 x 3.42 x 1.64 x 1.67 x 5.27 x lo--' 7.77 x 10 4

2.03 x 10 3

1.82 x 10-3 1.78 x 1.80 x 10-3 2.71 X 10--3

RSD.% 5.7 1.9 1.4 0.5 0.3 0.8 1.9 2.4 I .4 1.1 0.7 0.8

Error, YO -1.2 -0.6 -0.8 +0.3 +0.4 +0.8 + 1.0 +1.2 +1.3 +0.7 -0.5 +0.8

Table 3. Results for the catalytic titration of cysteine (Cy)

AgNo3/M Cy taken/mg 2 x 10-3 0.0059

0.0118 0.0176 0.0235 0.0353 0.0470

1 x 10-3 0.0303 0.0606 0.0910 0.1213 0.1820 0.2426

* Means of five replicate determinations.

Cy found*/mg 0.0057 0.0120 0.0172 0.0228 0.0349 0.0465 0.0293 0.0597 0.0885 3.1222 0.1809 0.2453

Standard d e v i a t i d m g 1.67 x 1 0 - 4

2.04 x 10 2.97 x 5.35 x 10-4 3.00 x 4.09 x 10-4 1.27 x 10 8.31 x 10 1.83 x 10-i 1.67 x 10-3 1.83 x 2.20 x 10-3

RSD ,YO 2.9 1.7 1.7 2.3 0.9 0.9 4.3 1.4 2.1 1.4 1 .o 0.9

Error, YO -3.4 +1.7 -2.2 -2.9 -1.1 -1.0 -3.3 -1.5 -2.7 +0.7 -0.6 + l . l

absence of inhibitor in order to eliminate possible method- ological errors.

Procedure for the Determination of the Drugs in Pharma- ceuticals

Dissolve one capsule of the drug in water and dilute to 250 ml in a calibrated flask. Filter, dilute if necessary and analyse a suitable aliquot by the general procedure. If an ampoule is to be used, dilute a suitable aliquot in a calibrated flask and proceed as described above.

Results and Discussion The inhibitory effect of organic compounds containing thiol groups on the silver-catalysed phloxin - persulphate reaction

can be applied to the determination of these drugs using catalytic spectrophotometric titrations. When the equivalence point is reached, the first excess of silver catalyses the indicator reaction and a sharp decrease in the absorbance at 537 nm is recorded.

Typical titration curves obtained for N-penicillamine, N-acetylcysteine, cysteine and 2-mercaptopropionylglycine are shown in Fig. 1.

The effect of both chemical and instrumental variables was studied in order to obtain maximum accuracy and greater sharpness of the end-point. The optimum reagent concentra- tions were found to be the same as those for the maximum rate of the catalysed reaction in the absence of an inhibitor. Hence the indication of the end-point is sharper. These values were 1 x 10-5 M phloxin, 0.2 M acetate buffer (pH 5 ) , 6 x 10-4 M 2,2'-bipyridyl and 0.03 M potassium persulphate.

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ANALYST, JUNE 1990, VOL. 115 759

Table 4. Results for the catalytic titration of 2-mercaptopropionylglycine (MP)

AgNo3/M MP taken/mg MP found*/mg 2 x 10-4 0.0083 0.0079

0.0167 0.0163 0.0251 0.0246 0.0334 0.0335 0.0501 0.0502 0.0668 0.0673

1 x 10-3 0.0402 0.0398 0.0804 0.0805 0.1206 0.1214 0.1608 0.1614 0.2412 0.2414 0.3216 0,3209

* Means of five replicate determinations.

Standard deviationtmg 2.51 x 1 0 F 3.74 x 10-J 4.02 x 10-3 4.47 x 10-4 4.67 x 10-5 5.76 x 1.13 x 10-3 1.24 x 10-3 1.71 x 10-3

2.47 x 10-3 1.74 x 10-3

1.17 X lo-’

RSD ,Yo 3.2 2.3 1.6 1.3 0.9 0.8 2.8 1 .5 1.4 0.7 1.0 0.5

Error,% -4.8 -2.4 -2.0 +0.3 +0.2 +0.7 -1.0 +o. 1 +0.7 +0.4 +o. 1 -0.2

Table 5. Determination of the drugs in pharmaceutical preparations

Sample Drug Found value* Certified value Cupripen? . . . . N-Penicillamine/mg per capsule 249.0 250.0 FluimucilS . . . . N-Acetylcysteine/mg per ampoule 301.5 300.0

* Average of three separate determinations ‘r Cupripkn capsules (Lab. Rubio): 250 mg of N-penicillamine per capsule plus excipient. t Fluimucil ampoules (Lab. Zamb6n): 300 mg of N-acetylcysteine in 3 ml of solution at pH 6.5.

The optimum concentration of the silver nitrate titrant solution ranged between 1 x 10-3 and 1 X 10-4 M . Higher concentrations led to copious precipitates which made the localisation of the end-point difficult. When more dilute silver nitrate solution was used, the sharpness of the end-point decreased and the accuracy of the procedure was diminished.

The influence of the rate of addition of the titrant was studied between 0.14 and 1.14 ml min- 1 for both 1 x 10-3 and 2 X M silver nitrate concentrations. Fig. 2 shows the results of the determination of cysteine (121 pg) using a 1 x 10-3 M titrant concentration. Rates of addition higher than 0.57 ml min-1 produced positive errors, whereas the lower rates gave cysteine recoveries of less than 100%. Therefore, in order to obtain optimum results, a rate of addition of 0.57 ml min-1 was selected. A value of 1.14 ml min-1 is recommended when 2 x 10-4 M silver nitrate solution is used as titrant. In this instance, slower rates of addition caused the reaction to take place at a rapid rate before the equivalence point was reached.

Results and values for the precision of five replicate determinations for the semi-automatic spectrophotometric catalytic titration of N-penicillamine, N-acetylcysteine, cysteine and 2-mercaptopropionylglycine with silver nitrate are given in Tables 1 4 .

Table 1 shows that the average error in the determination of N-penicillamine is 2.5% {[relative standard deviation (RSD) 1.9%]} and 1.0% (RSD 1.5%) with the use of 2 x 10-4 and 1 x 10-3 M silver nitrate solution, respectively. Values of 0.7 and 0.9% for the error and 1.8 and 1.4% for the RSD were obtained in the determination of N-acetylcysteine (Table 2), showing no differences between the two titrant concentra- tions. For cysteine, the average error is 2.0% (RSD 1.7%) and 1.6% (RSD 1.8%) for 2 X 10-4 and 1 X 10-3 M silver nitrate solution, respectively (Table 3). For the determination of 2-mercaptopropionylglycine (Table 4) , the best results were obtained with the use of 1 x 10-3 M silver nitrate, the average errors being 1.7% (RSD 1.7%) and 0.4% (RSD 1.3%) for 2 x 10-4 and 1 X 10-3 M titrant concentrations, respectively.

The proposed methods were applied to the determination of the drugs in pharmaceutical preparations with good results, as shown in Table 5. The results agree well with those certified.

This work was supported by a grant from CICYT (Project No. 0053/87).

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Paper 91043341 Received October 2nd, 1989

Accepted November Ist, 1989

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