A large determinant error was noted in the analysis of hydroquinone and p-phenylenediamine (6-10 Z) and aromatic nitro-compounds (20%) when water was used as the solvent. This effect was probably due to adsorption of the sample on the graphite which was possible because the particular sampling technique used involved the passage of a large volume of sample solution through the cell before the electrolysis. This
problem could probably be minimized by a change in the sampling procedure. However, for the compounds studied, this adsorption effect was negligible when acetonitrile was used as the solvent. RECEIVED for review May 12, 1969. Accepted June 17, 1969.
Gas Chromatographic Method for Determination of Diphenylhydantoin Blood Level Khalid Sabih and Khawla Sabih Clinical Pharmacology & Toxicology Center, University of Kansas Medical Center, Kansas City, Kan. 66103 DIPHENYLHYDANTOIN (5,s-diphenylhydantoin) is a drug widely used in the treatment of grand mal seizures. The rate of this drug has been shown to vary appreciably from one individual to another (1). Several spectrophotometric, colorimetric, and ultraviolet methods (2-5) have been developed to determine concentrations of the drug in biological material. However, some of these methods are nonspecific, time consuming, and require many selective extractions to avoid interference of other drugs-e.g., barbiturates. Recently, a gas chromatographic method for the determination of diphenylhydantoin in blood has been described (6). This method involved the conversion of diphenylhydantoin to its methyl ether by treatment, in the absence of a catalyst, with freshly prepared diazomethane. The application of this method in our laboratories for the determination of blood concentration of diphenylhydantoin in patients was not successful because of the consistent presence of several interfering peaks. This report describes a gas chromatographic method for determining diphenylhydantoin in blood which does not require derivative formation. EXPERIMENTAL
Reagents. Diphenylhydantoin was from Parke, Davis and Co. (Detroit, Mich.). Chloroform and acetone were redistilled analytical reagents. Diazomethane was prepared according to DeBaer and Backer (7), using Diazald (Aldrich Chemical Co. Inc., Milwaukee, Wis.). Apparatus. An F & M Model 5755B gas chromatograph equipped with a flame ionization detector was used for chromatography. The column was 4.5 feet, l/8" stainless steel tubing packed with Gas Chrom Q 80-100 mesh coated with 5 DC-200. The operating temperatures used were column, 220 "C; detector, 330 "C; and injection port, 340 "C. Helium was used as the carrier gas with a flow rate of 80 ml/ min. Similar results were obtained with a 4-foot, U-shaped glass column in a Barber-Colman Model 5000 gas chromatograph. (1) E. W. Loeser, Jr., Neurology, 11, 424 (1961). (2) 0. Svensmark and P. Kristensen, J . Lab. Clin. Med., 61, 501 (1963). (3) J. W. Huisman, C h .Chin?.Acta, 13, 323 (1966). (4) J. Wallace, J. Biggs, and E. V. Dahl, ANAL.CHEM., 37, 410 (1965). (5) J. E. Wallace, ibid., 40, 978 (1968). (6) D. H. Sandberg, G. L. Resnick, and C . 2. Bacallao, ibid., 40, 736 (1968). (7) Th. J. DeBaer and H. J. Backer, "Organic Synthesis," Vol. IV, John Wiley and Sons, Inc. New York, N. Y.,1963, p 250. 1452
ANALYTICAL CHEMISTRY
I
I
0
2
Time (min) Figure 1. Typical chromatogram of diphenylhydantoin Conditions: 5 % DC-200 on Gas Chrom Q. Column temperature 230 "C, helium flow rate 80 ml/min. Chart speed 0.25 in./min. Attenuation 8, range 10*
Procedure. NONBIOLOGICAL SYSTEM.A stock solution of 5 mg diphenylhydantoin in 10 ml acetone was prepared. Aliquots containing 5, 7.5, 10, 12.5, 15, 20, 25, and 30 pg were placed in separate flasks, the solvent removed under a stream of dry nitrogen, and 100 pl of acetone or chloroform added to each residue. One to five p1 samples were injected onto the column. Figure 1 shows a typical chromatogram obtained with diphenylhydantoin which has a retention time of 2.2 minutes. BIOLOGICAL SYSTEM.Diphenylhydantoin was added to plasma samples in amounts of 10, 15, 20, 25, and 30 pg/mI. Two ml of the resulting mixtures were each extracted with 8 ml of chloroform by shaking for 5 minutes with a mechanical shaker and then centrifuged for 15 minutes at 2000 RPM. The aqueous layer was aspirated and 4 ml chloroform extract from each sample evaporated to dryness under reduced pressure or under a stream of dry nitrogen. To each residue was added 100 pl of chloroform or acetone and 1-5 p1 samples utilized for analysis under the conditions described above. Plasma from patients receiving diphenylhydantoin was analyzed in a similar manner except the plasma was acidified
100
Table I. Recovery of Diphenylhydantoin from Plasma DPH added, pg/ml Recovered Recovery, 5
10 15 20 25 30
3 M
.I
5i
4.8 9.9 14.4 20.0 23.8 29.1
98 99 96 100 95 96
'Lm
& Table 11. Comparison of Two Methods for Measurement of Diphenylhydantoin in Plasma Dill's method, Sample Fdml GC, p d m l
0
i
I
0
2
Time (min) Figure 2. Chromatogram of A . glutethimide, B. phenobarbital, C. diphenylhydantoin Conditions: see Figure 1
with 0.5-1 ml 0.5N HC1 before extraction. The concentration of diphenylhydantoin in plasma samples was determined by comparing peak heights to that obtained with a series of standards chromatographed on the same column. This gas chromatographic method was compared to the colorimetric method developed by Dill and his co-workers (8). The latter or modifications of it is the method most frequently used for quantitative assays of diphenylhydantoin reported in the literature.
RESULTS Utilizing conditions cited in the method, the retention time was 2.2 minutes. The retention times for diphenylhydantoin and that extracted from plasma were identical. Chromatograms of extracts of control plasma did not contain any peaks with the retention time of diphenylhydantoin. Addition of increasing amounts of diphenylhydantoin to extracts of plasma containing the drug produced an increase in the peak height having the retention time of the standard. The relationship between integrator response and quantity of the drug is linear at optimum instrument sensitivity when 0.02 Fg and higher amounts were injected. Phenobarbital, which is also frequently used in treatment of epilepsy, did not interfere with the analysis (Figure 2). Glutethimide and valmid, which can be analyzed on the same column but at lower column temperature and flow rate, also did not interfere. The extraction procedure for diphenylhydantoin from plasma was adequate and gave 95-100% recovery of added drug. The recovery of diphenylhydantoin was dependent upon the volume and polarity of the extracting medium. Best results were obtained (Table I) when 5 ml chloroform was used per ml plasma. Therapeutic plasma levels of diphenylhydantoin were detected at optimum sensitivity of the instrument, but as little as 0.02 pg was measurable. Lower amounts of diphenylhydantoin were also detectable at higher sensitivity. The temperature chosen provided optimal recorder response with a relatively short retention time. When column temperature (8) W. A. Dill, A. Kazenko, L. M. Wolf, and A. J. Glazko, J. Pharmacol, Exp. Ther., 118, 270 (1956).
1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19
12.5 16.4 19.8 17.5 21.3 23.2 29.6 23.0 6.7 6.7 22.3 36.4 9.0 33.9 16.4 31.1 64 62.3 67.1
8.0 16.5 13.5 16.6 20.0 21.4 30.0 22.7 6.1 6.0 22.0 36.0 8.7 33.5 14.2 28.0 61 .O 62.0 66.0
was reduced there was a marked decrease in recorder response accompanied by peak broadening and tailing. Reduction of helium flow rate produced similar effects. T o test the reproducibility of the gas chromatographic method, we injected the same volume of a known concentration of diphenylhydantoin several times. This gave the same peak height in each case with a standard deviation of 1.5%. The same results were obtained when this procedure was repeated using plasma samples. This gas chromatographic method was also compared with the method described by Dill and his co-workers (8). Plasma samples from patients receiving diphenylhydantoin and phenobarbital were analyzed using both the colorimetric (8) and the gas chromatographic methods. The results are shown in Table 11. An all glass U-shaped column packed with Gas Chrom Q coated with 2 XE-60 was also tried using a Barber-Colman Model 5000 gas chromatograph. However, the diphenylhydantoin peak showed a great deal of peak tailing at the different temperature and flow rate. A 3 SE-30 glass column was also tried. This column gave a fairly symmetrical peak, but required higher temperature operation and the column required an overnight reconditioning at 300 "C prior to each analysis. An all glass U-shaped column packed with 5 % DC-200 on Gas Chrom Q did not show any significant advantage over the stainless steel column. DISCUSSION
The GLC method described above is accurate, sensitive, and specific for the measurement of diphenylhydantoin concentration in blood and can be used in clinical laboratories. It has VOL. 41, NO. 11, SEPTEMBER 1969
1453
the advantage of measuring diphenylhydantoin directly without performing any chemical reaction on it. Thus, the use of hazardous chemicals, like diazomethane and BFrHF, is avoided. This method requires less than one hour for analysis of several samples, while other methods (2-4, 8) require several hours. Furthermore, this method, because of its direct nature, gives more accurate quantitative measurement of diphenylhydantoin over the other gas chromatographic method (6). The previous method requires the formation of the methyl derivative of diphenylhydantoin by reaction with diazomethane. This reaction gives the N-methyl derivative (9) of diphenylhydantoin in 25-38 yield (IO) which cannot be used as an accurate measurement of diphenylhydantoin in blood. This nonquantitative yield can be attributed to the lack of tautomerization of diphenylhydantoin to its enolic form which was shown by Carington and his co-workers (9) and more recently by Dudley and Bius (11). In addition, diazomethane is known to polymerize rapidly in the light to
produce high molecular weight polymers (I2-14), which in turn may interfere with the analysis of diphenylhydantoin and prevent an accurate qualitative and quantitative measurement. This gas chromatographic method has the advantage of simplicity and specificity over the colorimetric methods which require elaborate extraction or separation procedures to avoid interference by drugs frequently used with diphenylhydantoin such as phenobarbital. The absence of such interference in this method should make it useful in studying blood concentration of diphenylhydantoin in patients placed on multiple drug therapy. ACKNOWLEDGMENT
The authors thank Parke, Davis and Co., Detroit, Mich., for the diphenylhydantoin used in this study. RECEIVED for review April 21, 1969. Accepted June 11, 1969. Work supported by funds from the Public Health Service, Grant GM15956. ~~
(9) H. C. Carrington, C. H. Vasey, and W. S. Waring, J. Chem. SOC.,1953, 3105. (10) F. Arndt, Rev. Fuc. Sci. Univ. Istanbul, 9A, 19 (1944). (11) K. H. Dudley and D. L. Bius, J. Org. Chem., 34, 1133 (1969).
(12) K. R. Kopecky, G. S. Hammond, and P. A. Leermakers, J. Amer. Chem. SOC.,83, 2397 (1961). (13) C. H. Bawn and T. B. Rhodes, Trans. Furaduy Soc., 50, 934 (1954). (14) H. Meermein, Angew. Chem., A60, 78 (1968).
Substoichiometric Radioisotope Dilution Analysis of Tungsten as a Major Constituent in Molybdenum Containing MateriaIs Using To1uene3,4-Dithio1 L. T. McClendon and J. R. DeVoe Analytical Chemistry Diuision, National Bureau of Standards, Washington, D. C. 20234
THERE ARE SEVERAL reagents which produce colored complexes with tungsten ( I ) , thus providing the basis for numerous spectrophotometric methods for the determination of tungsten in various materials. However, these reagents also produce colored complexes with molybdenum, and because most tungsten containing materials also contain molybdenum, an interference problem exists with these photometric methods. Colorimetric methods which result in the formation of stoichiometric complexes can be adapted to radioisotope dilution techniques. The addition of excess complexing reagent to provide quantitative colorimetric analysis can often result in the formation of interfering metal complexes whose partition coefficient is close to that of the desired complex. The use of radioisotope ’dilution analysis with substoichiometric amounts of a complexing reagent, reduces the problem of interference by eliminating the need for quantitative separation. In addition, the radioisotope dilution technique is insensitive to problems of color instability (inherent or as the result of interferences). The technique involves the isolation of equal amounts of analyzed element before and after dilution of the radioisotope (by the analyzed element) (2). Measurement of specific activity of the radioisotope before and after dilution is facilitated by the substoichiometric technique, and (1) E. B. Sandell, “Colorimetric Determination of Trace Metals,” 3rd Ed., Interscience, New York, 1959. (2) J. Ruzicka and J. Stary, Tulunra, 8, 228 (1961).
1454
ANALYTICAL CHEMISTRY
the total amount of element is calculated using the standard substoichiometric radioisotope dilution equation,
x=
Y($
- 1)
where Y is the total amount of element in the radioisotope (known very accurately), and A I is the radioactivity obtained in the extract before dilution with nonactive isotope and A z is the activity obtained in the extract after dilution with nonactive isotope. The colored complexes formed by toluene-3,4-dithiol, often referred to as “dithiol,” were first reported by Hammence (3). Several workers (4-8) have developed photometric methods for determining tungsten and molybdenum in various materials over a span of years. Of course, the success of these methods is very dependent upon the control of the problems (mentioned above) that are inherent in photometric methods. Thus, arising from the need of several independent methods (3) J. H. Hammence, Analyst, 65, 152 (1940). (4) B. Bagshawe and R. J. Truman, ibid., 72, 189 (1947). (5) P. Greenberg, ANAL.CHEM., 29, 896 (1957). (6) S. H. Allen and M. B. Hamilton, Anal. Chim. Acta, 7 , 483 (1952). (7) C. S. Piper and R. S. Beckwith, J. SOC. Chem. Ind., 67, 374 (1948). (8) E. W. Hobart and E. P. Hurley, Anal. Chim. Acta, 27, 142 (1962).
