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Anal. Chem. 1984, 56, 1222-1226
Normal Pulse Cathodic Stripping Voltammetry of Ethynylestradio1 A. M. Bond* and I. D. Heritage
Division of Chemical and Physical Sciences, Deakin Univeristy, Victoria 3217, Australia
M. H. Briggs Division of Biological and Health Sciences, Deakin University, Victoria 3217, Australia
The cathodic stripping voltammetrlc behavlor of ethynylestradloi was lnvestlgated to provide the basts of a routine method for determlnatlon of this Synthetic steroid hormone. A normal pulse potentlai wave form was determlned to be slgnlflcantly superlor to conventlonal differential pulse and dc stripplng techniques. Microprocessor-based instrumentatlon was utilized to provide greater flexlblilty In the control of potentlai wave form and data acqulsitlon and also to allow complete automation of the technique. Interference from both organic and Inorganic exclplents can be overcome by the method of standard addition and the use of a complexlng agent. The coformulatlon of other ethynyl sterolds however creates overwheimlng Interference and a separatlon step Is recommended. Pharmaceutical levels of ethynylestradiol over the concentratlon range 4 X lo-’ to 6 X IO-’ M can be determined with a precislon of 1.5% and values agree satlsfactorily wlth the nominal content. The limit of detection of ethynylestradiol In standard soiutlons was 5 X lo-‘’ M.
Ethynylestradiol is a pharmaceutically important synthetic steroid hormone that is formulated in dosage regimes that are typically 4 pg to 50 pg per tablet. A sensitive method is therefore required for routine determinations of this compound. Previously these determinations have been performed by GLC and fluorimetry (1) and HPLC (2-4). Recently, we reported a new polarographic response for ethynylestradiol which involves the formation of a sparingly soluble or adsorbed mercury compound (5). This work suggested the possibility of developing an extremely sensitive technique for ethynylestradiol by cathodic stripping voltammetry, CSV, even though other workers had met with virtually no success (6). Indeed CSV has been demonstrated to be a sensitive analytical procedure for a wide range of organic and inorganic compounds that are capable of either forming a sparingly soluble mercury compound or adsorbing onto the electrode (6-12). The reaction of ethynylestradiol with mercury is analogous to the well-documented thiol (R-S-H) reaction (7,8);however in this instance it is the acetylenic (RCECH) functionality of ethynylestradiol which is the “active” part of the compound. Microprocessor-based instrumentation has achieved widespread use in laboratory analyses due to the ease of control available over experimental parameters plus the ability to perform relatively sophisticated data reduction quickly. In this work we have utilized a “home built” microprocessor-based system to provide greater flexibility over the control of the potential wave form generated and data acquisition. The instrumentation is also amenable to automation so as to free the operator from tedious manual control of CSV operations. In this report three different formulations of ethynylestradiol were chosen as a representative group of commercially available pharmaceutical products. They were chosen
on the basis of ethynylestradiol content and coformulation of other steroid hormones in order to determine the nature of possible interferences that could be encountered with the routine application of this technique. A suitable analytical procedure is reported for the determination of ethynylestradiol when interference by coadsorbed interferents is not overwh e1min g.
EXPERIMENTAL SECTION Instrumentation. Initial experiments were performed with an EG&G Princeton applied Research Corp. (PAR) Model 174A polarographicanalyzer, together with a Houston Omnigraph X-Y recorder. The working electrode was a PAR Model 303 static mercury drop electrode used in the HMDE mode. Unless otherwise stated a medium size drop of electrode area 0.016 cm2was used. The PAR 303 system was fitted with a platinum counterlectrode and a Ag/AgC1(3 M KC1) reference electrode. Unless otherwise stated, solutions were degassed with nitrogen for a minimum of 10 min prior to undertaking a stripping experiment. All data were obtained at 20 h 1 “C. Microprocessor-baaedinstrumentation utilized a “home built” 12-bit function generator and data acquisition system that has already been described (13). Machine code programs written for normal pulse cathodic stripping voltammetry were based upon earlier programs that had been written for polarographic techniques (14). In the microprocessor-based experiments the PAR Model 174 instrument was used as a potentiostat and I/E converter only. Chemicals. All chemicals used were of analytical reagent grade unless otherwise stated. Buffer solutions were prepared from distilled-deionized water and distilled AR grade methanol. Stock solutions of ethynylestradiol were prepared by using distilled AR grade methanol and ethynylestradiolsupplied as cited in the acknowledgmentsection. The purity and stability of the ethynylestradiol solutions were confirmed by reverse-phase high-performance liquid chromatography with UV detection at 280 nm. RESULTS AND DISCUSSION The Stripping Process. The electrochemical process involving ethynylestradiol at a mercury electrode has been described before (5) and will not be detailed in this report. The pertinent features, however, for understanding the CSV behavior can be ascertained from a cyclic voltammogram of a 1.6 X M solution of ethynylestradiol in alkaline methanolic solutions (Figure 1). A peak (a) occurs a t -0.33 V on the forward, anodic, scan corresponding to the oxidation of the mercury electrode in the presence of ethynylestradiol. A sharper peak (b) a t -0.37 V on the reverse or cathodic scan is observed corresponding to the reduction or “stripping” of the product of (a) from the electrode. In CSV, a suitable potential is applied to the mercury electrode so as to accumulate the product from the oxidation process onto the electrode. A cathodically scanning potential wave form is then applied to strip this concentrated product from the electrode which results in a CSV current-voltage curve suitable for quantifying the analyte of interest. Figure
0 1984 American Chemical Society 0003-2700/84/0358-1222$01.50/0
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8,JULY 1984
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a
0.1pA
A
Ox
a2
vb 0.5
red
0.4
b2
f J
.05pA
0.3
0.2
-E,Volt
Flgure 1. Cyclic voltammogram of 1.8 X M ethynylestradiol in 50% methanoV0.l M Na,CO,, pH 11.5, at a mercury electrode: scan rate, 200 mV s-’: (a) oxidation process, (b) reductlon, “stripping”
process.
f
