is added to hydrolysis-susceptible esters, complete hydrolysis takes place. In this case no water-free titration is necessary since all POH-groups in aqueous solution can be titrated with lye. The described method is also suitable to determine hydrolysis-susceptible samples with a high degree of accuracy. Figures 3 and 4 show conductometric titration curves. The method allows one to examine, besides phosphorous acid and its monoesters, other acids and esters also for the presence of POH groups.
LITERATURE CITED (1) J. Jander-Ch. Lafrenz, “Wasserahnllche Losungsrnlttel”, Verlag Chernle GrnbH, Weinheirn/Bergstr., 1968. (2) 8 . Kratochvll, Anal. Chern., 48, 355R (1976). (3) H. J. Lucas, F. W. Mltchell, Jr., and C. N. Scully, J . Am. Chem. Soc., 72, 5491 (1950).
RECEIVEDfor review January 10,1977. Accepted June 6,1977. Part of the author’s thesis submitted for his diploma, D.-65 Mainz, West Germany, 1970.
Reaction-Rate Method for the Determination of Hydrocortisone R. M. Oteiza, D. L. Krottinger, M. S, McCracken, and H. V. Maimstadt* School of Chemical Sciences, Universiv of Illinois at Urbana-Champaign, Urbana, Iliinois 6 180 1
A reactlon-rate method for the determination of hydrocortisone Is described. The method is based upon a modification of the widely accepted blue tetrazolium reaction. An analysis tlme of only 30 s Is required. Reiatlve standard deviatlons of about 1% or less are obtained, and the analytical worklng curves are Ilnear. Analysls of pharmaceutical skln preparations by the new rate method gave results which correlate well wlth the time-consuming standard equilibrium method.
Table I. Reaction-Rate Result.for Different Measurement Timesu Measurement time, s Rate, AmA/sb 1.0 5.0
10.0 15.0 30.0 45.0
26.5 25.6 25.2 24.8 23.7 23.8
RSD, % 3.7 1.0 0.8 0.4 0.5 1.0
Analysis of 2.5 mg/dL standard with 15-s delay time. Average of 5 determinations on a single sample. The quantitative determination of corticosteroids by various spectrophotometric methods has been previously discussed (1). One of these is based on the reduction of blue tetrazolium in an alcoholic solution of a strong base by the a-keto1 group on the CI7side chain of the corticosteroid to form a chromagen which has an absorbance maximum a t 525 nm. This absorbance, measured 90 min after mixing the sample with blue tetrazolium and the base, is then compared to that of a standard and blank solution to obtain quantitative information concerning the steroid concentration in the sample (2,3).This is the basis for the official method of the National Formulary ( 4 ) and the United States Pharmacopeia (5). Graham et al. have studied the blue tetrazolium procedure and have noted a first-order dependence of the corticosteroid concentration on the rate of the reaction (6). By employing the time-saving advantage of reaction-rate methods (71, we have developed a new procedure which decreases the analysis time considerably. Results obtained by the reaction-rate procedure are compared with the official method of the USP for pharmaceutical skin preparations.
EXPERIMENTAL Apparatus. The apparatus used for the reaction-rate method was the automated system described by Malmstadt et al. (8). This system provides for automatic aliquoting and mixing of sample and reagent and delivery of the mixed solution into the measurement cuvet (2-cm pathlength, 60-wL volume) by means of a stopped-flow unit incorporated in a modular spectrophotometer. A ratio-recording spectrophotometer (Model 721, GCA/ McPherson, Acton, Mass. 01720) was used for the equilibrium measurements. Reagents. A single 10 mg/dL hydrocortisone stock solution was prepared weekly by dissolving 10 mg of hydrocortisone (Sigma Chemical Co., St. Louis, Mo. 63178) in 100 mL of 95% ethanol. A 0.5% blue tetrazolium (Sigma Chemical Company) solution was prepared by dissolving 0.5 g of blue tetrazolium in 100 mL of absolute methanol. A 5 % solution of tetramethylammonium 1586
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
hydroxide was prepared by dissolving 5 g of tetramethylammonium hydroxide pentahydrate (Sigma Chemical Company) in 50 mL of USP, reagent quality, absolute ethanol (US. Industrial Chemicals Company, Tuscola, Ill. 61953). Different base concentrations were prepared from the 5% solution by appropriate dilution with absolute ethanol. The standard hydrocortisone solutions were prepared daily by adding 2 mL of the blue tetrazolium solution to an appropriate volume of the stock hydrocortisone solution and diluting to 10 mL with 95% ethanol. Sample Preparation. Samples were prepared from the pharmaceutical preparations-creams, gels, and ointments-by the column chromatographic procedure of Graham et al. (9) in which the corticosteroid is trapped in the column while interferences are removed by n-heptane. The corticosteroid is then removed from the column with chloroform. The eluate obtained from the column is carefully evaporated to dryness. The residue from the chloroform eluate is then dissolved in 95% ethanol and diluted to 25 mL. A 5-mL aliquot is then added to 2 mL of the blue tetrazolium solution and diluted to 10 mL with 95% ethanol. This 10-mL solution will be referred to in subsequent discussions as the sample. Approximately 30 minutes are routinely required for the sample preparation which provides an interference-free sample for analysis. Equilibrium Procedure. The equilibrium procedure was the official procedure given in the USP XIX (5) with the absorbance measured 90 min after mixing the standard with the two reagents. Reaction-Rate Procedure. One hundred p L each of a tetramethylammonium hydroxide solution and the appropriate standard or sample are sampled by the automatic syringes of the stopped-flow module (8). The syringes in the module then drive the solutions through the mixer and transfer the mixed solution to the observation cell. The change in absorbance is automatically monitored at 525 nm during the measurement time and used to construct a rate curve, working curve, or provide quantitative concentration information for the pharmaceutical skin preparations. For the results presented, the solutions and spectrophotometer were at ambient temperature in a temperature-controlled lab-
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Table 11. Results Used for Hydrocortisone ReactionRate Working Curvea Hydrocortisone concn, mg/dL
Rate, AmA/sb
RSD, %
1.06 1.59 2.12 2.65 3.18
9.5 13.8 18.3 23.1 27.7
1.2 0.4 0.4 0.5 0.4
Working curve: Slope = 8.62, intercept = 0.2, r = Average of 5 determinations on a single sample. a
0.9998.
oratory maintained at a nominal temperature of 25 "C.
RESULTS AND DISCUSSION As shown in Figure 1,the rate of the reaction is dependent on the base strength. The appropriate base strength can be chosen to provide the degree of sensitivity needed at a minimum cost per analysis. For our system, good sensitivity and precision could be obtained in a short measurement time with the 5% base.
The reaction-rate curves for the five standards with 5% base over a period of 90 s are shown in Figure 2. A delay of 15 s after mixing was employed before the measurement period began. This allows for any nonreproducible behavior near the beginning of the reaction to terminate as shown in the insert of Figure 2. The optimum measurement time was determined by using the 15-s delay and varying the measurement of rate data over various periods from 1 to 45 s. The results are shown in Table I. It can be seen that the best reproducibility, about 0.4%, is obtained with a 15-smeasurement time. Thus, a 15-s delay and a 15-5 measurement time are used for the determinations. The results obtained for the working curve are shown in Table I1 and give a correlation coefficient of 0.9998 and a relative standard deviation of 0.4 to 1.2%. The working curve can be generated in about 8 min for triplicate analyses on each standard. This is more than a factor of 10 less than the time required to prepare a working curve for the equilibrium method where a single determination on a standard, sample, and blank are generally performed (5). Thus, the total analysis time including sample preparation can be reduced from over 2 h using the equilibrium procedure to slightly over 30 min
Table 111. Hydrocortisone Assay Results of Commercial Skin Preparations Assay, % of Declared
Concentration, Preparation 1
2 3 4 5 6
7 8 9
%
Product type
Reaction ratea
Equilibriumb
DifferenceC
0.5 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
cream cream ge 1 gel cream cream cream cream ointment ointment
70.0 58.8 95.6 94.4 93.6 94.3 95.5 95.5 91.6 95.5
71.0 59.3 94.9 95.4 95.3 93.0 95.1 93.8 91.5 94.9
- 1.0 -0.5 + 0.7 - 1.0 - 1.7 + 1.3 t 0.4 + 1.7 + 0.1
10 a Average of 3 determinations on a single sample. rate - % by equilibrium.
Average of 2 determinations on a single sample.
+ 0.6
% of reaction-
ANALYTICAL CHEMISTRY, VOL. 49, NO. 1 1 , SEPTEMBER 1977 * 1587
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Figure 2. Reaction-rate curves for hydrocortisone concentrations of 3.18 (O), 2.65 (+), 2.12 (A),1.59 (X), and 1.06 (0) mgldL using 5% tetramethylammonium hydroxide
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Flgure 3. Reactlon-rate curves for hydrocortisone concentrations of 3.18 (D), 2.65 (+), 2.12 (A),1.59 (X), and 1.06 ( 0 ) mg/dL using 1 % tetramethylammonium hydroxide
with the reaction-rate procedure. Table I11 shows the results obtained on a series of commercial creams, gels, and ointments by the new reaction-rate method and the official method for steroid analysis. Good agreement exists between the two methods. Successive serial dilutions on one of the commercial creams were analyzed by both the reaction-rate and the equilibrium methods. A direct comparison between the two methods 1588
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
yielded a correlation coefficient of 0.9995 and a slope of 1.053. The stopped-flow module used in this study allowed observation of the absorbance of the reaction mixture in less than 1s after mixing. For many laboratories this speed of mixing and transfer of the solution to the measurement cuvette is not possible, but by varying the base concentration this short time is not necessary. Shown in Figure 3 are the reaction-rate curves for the five standards over 2 min with the 1%base.
Table IV. Reaction-Rate Working Curve“ for 1%Baseb Hydrocortisone concn, mg/dL Rate, AmA/sC RSD, o/c 1.06 3.38 0.8 1.59 4.92 0.2 6.78 2.6 2.12 2.65 8.53 2.7 3.18 10.01 0.4 a Working curve: Slope = 3.17, intercept = -0.01, r = Analysis using 30-s delay time and 30-s 0.9993. Average of 4 determinations on a measurement time. single sample. By decreasing the base strength, we were able to slow the reaction so that the reaction-rate curve is linear over a longer period of time. For the case where a manual mixing operation must be performed, it may take 30 s or longer to mix the two solutions and place the cuvette in the spectrophotometer. We show in Table IV the results obtained for a 30-9 delay time and a 30-s measurement time for the series of standards analyzed previously. Good precision and a linear working curve were still obtained, but at twice the previous analysis time. However, this is still a vast improvement over the 90-min equilibrium procedure. It should be emphasized that these results were obtained on an automated spectrophotometric system which incorporates several features to ensure high reliability in its measurements. A beam splitter and reference detector are employed to correct for light source fluctuations which may occur during the measurement time (IO). The stopped-flow module provides precisions better than 0.2% RSD for the aliquoting, mixing, and transfer of solutions to the 2-cm long observation cell. Finally, control of the spectrophotometer,
acquisition of data, and reduction of these data to provide quantitative information are all reproducibly performed by a minicomputer and associated interface electronics. It also should be noted that the sample and standards were run in rapid succession, thus precluding the necessity of thermostating the solutions. If standard and sample information are to be obtained a t significantly different times, precise temperature control of the stopped-flow module can be maintained (8) over long periods. These factors should be considered when comparing results obtained with other instruments.
ACKNOWLEDGMENT The authors thank McKinley Health Center, University of Illinois, and R. D. O’Keefe, Champaign, Ill., for the pharmaceutical skin preparations used in this study.
LITERATURE CITED R. E. Graham, P. A. Wllliams, and C. T. Kenner, J. Pharm. Scl., 59, 1152 (1970). W. Madder and R. Buck, Anal. Chem., 24, 666 (1952). C. Chen, J. Wheeler, and H. Tewell, J. Lab. Gin. Med., 42, 463 (1956). “The National Formulary”, XIV, Mack Publishing Company, Easton, Pa., 1975, p 976. “The United States Pharmacopeia”, XIX, Mack Publlshing Company, Easton, Pa., 1975, p 622. R. E. Graham, E. R. Biehl, C. T. Kenner, G. H. Lwei, and D. L. Midleton, J. Pharm. Scl., 84, 226 (1975). H. V. Malmstadt, E. A. Cordos, and C. J. Delaney, Anal. Chem., 44, (12), 26A (1972). D. L. Krottlnger, M. S.McCracken, and H. V. Malmstadt, Am. Lab., 9 (3),51 (1977). R. E. Graham, P. A. Wllliams, and C. T. Kenner, J. Pharm. Sc;., 59, 1472 (1970). K. R. O’Keefe and H. V. Malmstadt, Anal. Chem., 47, 707 (1975).
RECEIVED for review May 6, 1977. Accepted June 13, 1977. Research partially supported by the NIH through Grant HEW PHS GM 21984-02.
Cyclic and Differential Pulse Voltammetric Behavior of Reactants Confined to the Electrode Surface Alan P. Brown and Fred C. Anson” A. A. Noyes Laboratory, California Institute of Technology, Pasadena, California 9 1125
Experlmental and theoretical cyclic and dlfferentlal pulse voltammograms are compared for reactants Irreversibly attached to the surface of graphlte electrodes. Ouantltatlve agreement between experiment and theory can be Obtained only If account Is taken of possible nonldeal behavior In applying the Nernst equation to the attached reactants. The lntentlonal addttlon of external unwmpensated resistance when recordlng dlfferentlal pulse voltammograms leads to slgnlflcant Increases In the sensltlvtty of thls technique for monltorlng small quantltles of attached reactants. An approximate method Is desctlbed whlch allows the surface concentratlons of attached reactants to be estlmated when the quantltles present are too small to yleld dlscernlble cycllc voltammograms.
Electrochemistry with electroactive reactants attached to electrode surfaces is under active study in a number of laboratories (1-5). In a recent publication (6), we described
the electrochemical behavior of several reactants that were bound to the surface of graphite electrodes by strong, spontaneous adsorption. The experimental data indicated that differential pulse voltammetry could prove to be a more sensitive technique than cyclic voltammetry for examining the electrochemical behavior of such systems. The advantages of the former technique are particularly noteworthy when the quantity of bound reactant is small. In this paper, more detailed experimental results are presented and are compared with theoretical analyses of the expected cyclic and differential pulse voltammetric behavior of reactants irreversibly attached to electrode surfaces. To account for the observed peak heights and wave shapes (e.g., half-peak widths), it was necessary to include activity coefficients which depend on the surface concentrations in the Nernst equation as written for the surface-bound reactants. One unique virtue of the differential pulse voltammetric technique is that enhanced sensitivity can be obtained by the intentional addition of uncompensated resistance to the cell ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
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