Cathodic stripping voltammetry of the peptide felypressin in trace amounts

Analy tica Chimica Acta, 156 (1984) 43-49 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

CATHODIC STRIPPING VOLTAMMETRY FELYPRESSIN IN TRACE AMOUNTS

OF THE PEPTIDE

ULF FORSMAN Astra Liikemedel AB, Research S-l 51 85 Siidertiilje (Sweden)

and Development

Laboratories,

Pharmaceutical

Analysis,

(Received 16th March 1983)

SUMMARY Felypressin, a peptide containing eight amino acids including cystine, is studied by cathodic stripping voltammetry (c.s.v.) at a mercury drop electrode at pH 4.6 in the concentration interval 5 X 1O-g-7 X lob7 M. Excess of copper ions is required to obtain the C.S.V. activity. The stripping peak potential is -0.55 to -0.70 V vs. SCE depending on the excess of copper(I1). The accumulated product is adsorbed both in its oxidized and reduced state. Interference from c.s.v.-active substances which desorb in the reduced state can be eliminated by applying a repetitive cyclic scan and evaluating the second or third scan. Lypressin and somatostatin, two other cystine-containing peptides, are also c.s.v.-active.

Peptides are playing an increasingly important role in pharmacology. In this development, new demands are placed on methods for their determination. The peptide felypressin (I; m.w. 1040) is used in combination with local anaesthetics at a low formulation level (5 X lo-’ M). At present, the

Is-s1

H-Cys-Phe-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH,

(I)

bioassay technique comprising measurement of blood pressure in rats [l] is used for the determination of the peptide at this low concentration. Liquid chromatographic methods have been presented for several peptides closely related to felypressin [2, 31 . A detection limit of 30 ng was reported [2]. The development of highly sensitive analytical methods for peptides is naturally of great interest. Felypressin contains cystine, which suggests that the very sensitive technique of cathodic stripping voltammetry (c.s.v.) should be applicable. Cystine, as well as cysteine, penicillamine and several penicillins have previously been investigated by C.S.V. [4-6] . These substances can accumulate at a mercury electrode in the form of a copper complex when excess of Cu(I1) is present. Detection limits as low as 2 X lo-lo M were reported. These compounds can also be accumulated as mercury complexes, but this approach requires solutions extremely free from Cu(II), because this ion, 0003-2670/84/$03.00

0 1984 Elsevier Science Publishers B.V.

44

even at concentrations much lower than that of the sulphur compound, interferes with the accumulation of the mercury complex. In the present paper, the C.S.V. characteristics of felypressin are reported. An acetate buffer of pH 4.6 was chosen because felypressin is less stable in neutral and alkaline media. EXPERIMENTAL

Equipment, sample and reagents A PAR-174 polarographic analyzer was used. The working electrode was a Metrohm EA-290 hanging mercury drop electrode (HMDE); a drop with a surface area of 0.9 mm2 was used throughout. A platinum auxiliary electrode and a saturated calomel reference electrode were also used. All potentials given refer to the SCE. Felypressin (Sandoz, Switzerland) was obtained in solution with a declared concentration of 25 IU ml-’ (4.13 X lo4 M). Solutions diluted to 4.8 X lo* M were used as standard solutions. The acetate buffer, pH 4.6, was composed of equal volumes of 0.05 M acetic acid and 0.05 M sodium acetate in water. Stock solutions of copper nitrate (Merck) were 1 X 1O-2-l X lo4 M. Appropriate amounts of stock solution was added to the buffer to produce the desired copper(I1) ion concentration. All reagents were of analytical grade. Procedure An aliquot of the solution to be investigated was added to 10 ml of deaerated pH 4.6 buffer solution. Accumulation was done at -0.30 V for 1 min, with stirring unless otherwise stated. A repetitive cyclic scan of 100 mV s-‘, with an initial cathodic scan direction, was then applied. Scan reversal was done manually at the desired potentials. RESULTS

AND DISCUSSION

Stripping voltammetric pattern In Fig. 1, cyclic stripping voltammograms obtained from felypressin both before and after the addition of Cu(I1) ions are shown. In the 5 X lo* M solution, two cathodic peaks, cl and CL and one anodic peak, al, are obtained in the first scan cycle. In the second cycle, the peak c: is lost, and the voltammogram shows only one cathodic, c2, and one anodic, a2, peak. In the 2 X lo-’ M solution, a hump is obtained at potentials more positive than the peaks described above. This hump is present also during the second scan cycle. The peaks cl, c2, a,, a2 all increase in height when Cu(I1) is added. The peak separation between the anodic and cathodic peaks decreases with increasing excess of Cu(I1) and reaches a minimum value of about 20 mV. The accumulated product responsible for the peak couples cl/al and c2/& is adsorbed in both its oxidized and reduced forms, because the anodic

45

c;

C

w

c,

E 3

G Cl

0

C2

2

-0.3

-0.5

-0.7

-0.9

-0.3 E/V

-0.5

D 0

al.02 -0.7

-0.9

vs. SCE

Fig. 1. Cyclic stripping voltammograms: (A) 5 x 10d M felypressin; (B) Cu(I1) added to A to give a 1 X lo-’ M concentration; (C) 2 X lo-’ M felypressin; (D) Cu(I1) added to C to give a 1.4 X 10d M concentration. Curves 1 and 2 are the first and second scan cycles, respectively.

and cathodic peaks are of equal height and are obtained on repetitive cyclic scans. The loss of the peak c; in the second scan cycle, indicates that the product responsible for this peak desorbs at potentials negative to its peak potential. The stripping procedure for felypressin was mainly based on the peak c2 obtained in the second cycle, where the influence of c; is eliminated. The peak height of c2 increases linearily with sweep rate, u, (r2 = 0.9999) when examined between 20 and 200 mV s-i, in accordance with the behaviour of a film stripping peak. An anodic scan, conducted after the accumulation in presence of Cu(II), yields an anodic peak at + 0.06 V and a cathodic peak at the same potential upon scan reversal. The peaks are situated close to the peak couple of copper amalgam and are therefore obscured when high excesses of Cu(I1) are present. The analytical utility of this peak couple for the determination of felypressin is thus limited, and was not investigated further. Dependence on the presence of copper(H) ions The presence of copper ions seems to be essential for obtaining the stripping peaks. The dependence of the peak height, i,, of peak c2, on the level of copper(I1) ions is shown in Fig. 2. With an increasing excess of Cu(II), a constant value of i, is approached. Simultaneously, the peak width decreases, and the peak potential, E,, is displaced in the negative direction (Fig. 1). The peaks cl, al, a2, follow the same pattern. The E, value of c2 from 5 X lo-* felypressin in the presence of 1 X lo-’ M Cu(I1) was -0.55 V, but -0.65 V in the presence of 1 X lo5 M Cu(I1). The influence of the Cu(I1) concentration on the peak shape follows the characteristics previously reported for cysteine and penicillamine [ 51.

46

20-

IO

30

20 Excess

40

Fig. 2. Peak current of c2 from 5 copper(I1).

00

-0 E/V

of Cu(lI) X

2

-0.4

-0.6

vs SCE

lo-’ M felypressin as a function of the molar excess of

Fig. 3. Peak current of c, as a function of accumulation pressin in the presence of 2 X lo-’ M Cu(I1).

potential for 5

X

lo-’

M fely-

It is well-known that cystine, cysteine, and several other sulphur-containing compounds yield cathodic stripping peaks in the absence of Cu(II), because of the reduction of accumulated mercury compounds [4-81. In the cases of cystine, cysteine and penicillamine, the stripping peak for the mercury compound is well separated from the peak for the copper compound [4-6] . Because the stripping activity is very low when felypressin is in excess over Cu(I1) in the solution, it appears that the peptide does not form, or forms only very slowly, an accumulated mercury compound. The peak c’l probably is not due to a mercury compound of felypressin, because this peak is obtained also in presence of excess of Cu(I1). Accumulation potential The dependence of i, of peak c2 on the accumulation potential, E,,,, is shown in Fig. 3. The peak height rapidly decreases at E,,, more positive than about 0.0 V. This is also the redox potential of the copper amalgam system, which indicates that the stripping peak is only obtained if copper amalgam is formed from excess of Cu(I1) during the accumulation period. This is in accordance with the accumulation of the copper compounds of cysteine, cystine and penicillamine [4, 51. An increase in the Cu(I1) concentration, as stated above, displaces the peak potential in the negative direction. The interval within which i, is independent of E,,, is then simultaneously increased in the negative direction. Peak current as a function of felypressin concentration The peak heights of q, c2, a,, a,, are linearly related to the felypressin concentration as long as the excess of copper(I1) is so large that the height and shape of the stripping peak is unaffected by the Cu(I1) concentration (Fig. 2). (A detailed discussion of the dependence of linearity on the Cu(I1) concentration has been given in connection with the C.S.V.of cysteine and penicillamine [ 51.) In the presence of 5 X 10e6 M Cu(II), 4.8 X 1O-8-2.3 X

47

lo-’ M felypressin concentrations gave linear calibration graphs for c2, a,, a?; the equation was i,(nA) = (7.18 f 0.37 X 108)C - 3.0 + 1.2 with r2 > 0.9995. Above 2.3 X lo-’ M, i,C’ decreased, and above 5 X lo-’ M, only a minor increase in peak height was obtained, indicating that saturation of the electrode surface was approached. The integrated peak area of c2 from 7 X lo-’ M felypressin was about 20 PC cmm2.Increased sensitivity was achieved by increasing the accumulation time. After a 5-min accumulation in buffer solution containing 1 X lo-’ M Cu(II), 5 X 1O-g-2.O X 10m8M felypressin gave the following calibration graph: i, (nA) = (3.68 X 10g)C - 5.6 with r2 = 0.9998. The relative standard deviation for 10 determinations in the same solution of 1.5 X lo-’ M felypressin in presence of 5 X 10” M Cu(I1) was 2.4%. Origin of felypressin stripping peaks and degradation of felypressin The C.S.V. characteristics of felypressin are quite different from those of most previously investigated sulphur compounds. First, the stripping activity in absence of Cu(I1) is very low, indicating that the formation of an adsorbable mercury compound of felypressin is negligible. Secondly, the adsorbed product obtained in the presence of Cu(I1) is adsorbed both in its oxidized and reduced form. The mechanism discussed for cystine, cysteine and penicillamine [4,5] is therefore not applicable in this case. Peptides not containing cystine (e.g., substance P with 11 amino acids) are c.s.v.-inactive. This fact, combined with the requirement that copper must be present for the stripping activity of felypressin to occur, indicates that complex formation between the cystine function of the peptide and copper is vital. It is of major importance for application of the stripping method to elucidate whether degradation products of felypressin will contribute to the stripping peak or not. Because the degradation pattern of this peptide is unknown, oxidized glutathione was chosen for this investigation as a model compound for smaller peptides possibly formed by hydrolysis of felypressin. Oxidized glutathione yields a C.S.V.peak at about -0.4 V when Cu(I1) is present. Figure 4 shows the influence of this peptide on the C.S.V.of felypressin. In the first cathodic scan, the peak from oxidized glutathione interferes with the peak from felypressin. The accumulated product from oxidized glutathione does, however, desorb in its reduced state. The interference is thus diminished in the second scan cycle. Furthermore, by reversing the scan from the anodic to the cathodic direction at a more negative potential, the peak from oxidized glutathione disappears. An accumulation potential of -0.45 V gives only the felypressin peak. It is evident that the stripping method can separate felypressin and oxidized glutathione. Lypressin, which is identical to felypressin, with the exception that one phenylalanine is replaced with tyrosine, gives a C.S.V.response identical to that of felypressin. Somatostatin, which contains 14 amino acids, including cystine, yields the peaks cl, c2, al, a2 but c; is not obtained. No separation is thus possible between these peptides.

I

-03

I

,

-0 5

-a 7

,

-09

E/V vs SCE

Fig. 4. Cyclic stripping voltammograms of 2 X lo-’ M felypressin and 4 X lo1 M oxidized glutathione in a mixture; 5 X 10 d M Cu(I1) was present. Accumulation potential: (A) -0.30 V; (B) -0.45 V. Curves 1, 2 and 3 are the first, second and third scan cycles respectively.

Some preliminary investigations were done on felypressin sample solutions treated at 120°C for 20-80 min to produce degradation. The samples were then examined with the stripping method as well as biologically [l] . Both methods showed a decrease in the felypressin concentration with increased time of degradation. The stripping method thus seems to be selective against the degradation products formed. Extended comparative studies are in progress. Determination in a local anaesthetic formulation The stripping method was applied for the determination of felypressin in a local anaesthetic formulation containing 5 X lo-’ M felypressin, 0.12 M prilocaine hydrochloride and 6.6 X 10e3 M methyl-p-hydroxybenzoate. None of the additional constituents interfered with the stripping procedure. No separation step is thus necessary. At present, the stripping method is being applied in parallel with the biological method for comparison, and a full description will be published later.

49

Conclusions Cathodic stripping voltammetry provides a very sensitive method for the determination of felypressin. Extended studies on other peptides are of interest. So far the method seems to be specific for cystine-containing peptides. This, combined with the capability of the method to separate felypressin and smaller peptides such as oxidized glutathione, opens possibilities for the direct determination of mixtures at low concentrations. The application of the method in pharmaceutical analysis for felypressin in local anaesthetics is promising. REFERENCES 1 2 3 4 5 6 7 8

U.S. Pharmacopeia XIX, U.S. Pharmacopeial Convention, Inc., 1975, pp. 356,534. K. Krummen and R. W. Frei, J. Chromatogr., 132 (1977) 27. M. Abrahamsson and K. GrSningsson, J. Liq. Chromatogr., 3 (1980) 495. U. Forsman, J. Electroanal. Chem., 11 (1980) 325; 122 (1981) 216. U. Forsman, J. Electroanal. Chem., 152 (1983) 241. U. Forsman, Anal. Chim. Acta, 146 (1983) 71. T. M. Florence, J. Electroanal. Chem., 97 (1979) 219. R. A. Grier and R. W. Andrews, Anal. Chim. Acta, 124 (1981) 333.