Table I t . Repeatability of Results Monosuifonate content, Date of analysis % mol
February 19 February 25 February 27 March 2 March 29 April April April April
May May July
1 13 16 21 11 12 12
40.8 40.4 39.9 40.7 40.7 39.2 39.1 40.0 40.8 40.0 38.7 39.8
Date of analysis
July July
22 29 4 4 4
November November November November 5 November 9 November 9 November 12 November 16 November 22 November 25 November 26
Monosulfonate content, % mol 39.2 40.5 39.7 39.7 39.4 39.6 39.5 42.1 39.8 39.7 40.5 39.7 38.5
In general, the composition of the eluents is not critical. However, petroleum ether is insufficiently polar to permit complete elution of the monosulfonic acids. I t was found that by adding approximately 5% n-butanol to the petroleum ether, complete separation of the monosulfonic acids was achieved. The retained di- and polysulfonic acids could then be stripped from the column with water. Figure 1 illustrates the excellent separation of monosulfonic acids from a secondary alkane sulfonic acids mixture obtained from the sulfoxidation of n-paraffins. The partial resolution of the polysulfonic acid region into three peaks was achieved by introducing an intermediate elution stage with 2-propanol solvent, between the butanol/petroleum ether and water elution stages. Apparently a major disulfonic acid peak is obtained followed by smaller tri- and tetrasulfonic acid peaks. However, no attempt was made to identify these components, which are indeed taken to be disulfonic acids in calculating the monosulfonate content. The repeatability of the method was checked by analyzing the same sample of sulfonic acids a t intervals over a period of 9 months. Results are shown in Table 11. The standard deviation was &0.8% with 95% confidence limits of 1.5%. Various other tests were made to check the reliability of the method. Thus a standard sample of primary
Table Ill. Sulfated Ash Contents of Neutralized Sulfonic Acid Fractions Obtained from Chromatography Sulfated ash content, % wt
Sample
Theoretical
Found
26.08 37.94 44.72
25.6 39.8
Secondary sodium dodecane sulfonatea (i) (ii) (iii)
Monosulfonate fractionb Disulfonate fractionb (Trisulfonate)
aObtained from t h e sulfoxidation of n-dodecane. tained from previous chromatography r u n s .
...
Fractions were ob-
lauryl monosulfonate (see Experimental) was found to contain 96.3% mol monosulfonate. Similarly the monosulfonate and disulfonate fractions from a few runs were combined, concentrated by distillation, and analyzed to give 96.2% mol and 0% mol monosulfonates, respectively. Another check on the reliability consisted of blending two samples which were previously analyzed; the experimental result (80.6% mol) agreed with the calculated figure (81.9% mol). As a final check on the method, sulfated ash contents were done on the neutralized fractions obtained from a sample of secondary dodecane sulfonate. The results are given in Table I11 and are consistent with good separation of the monosulfonic acid from the di- and polysulfonic acids. The preparation of the sample in the form required for chromatography takes approximately 1 hr. The actual chromatographic separation may be completed in 4 hr. However, one operator can attend simultaneously to several columns. The method described here has been used routinely for the analysis of a wide range of primary and secondary sodium alkane sulfonates with carbon numbers extending from Clo to CZO.
ACKNOWLEDGMENT The authors express their gratitude to Texaco Inc., for permission to publish this paper. Received for review December 11, 1972. Accepted April 2, 1973.
Simultaneous Measurement of Diphenylhydantoin and Phenobarbital in Serum by High Performance Liquid Chromatography James E. Evans Eunice Kennedy Shriver Center for Mental Retardation, lnc., 200 Trapelo Road, Waltham, Mass. 02754
Diphenylhydantoin (DPH) and phenobarbital are the two drugs most often used for the control of epileptic seizures. The value of blood level determinations for dosage of these drugs has been the subject of recent reviews ( I , 2 ) . These determinations are most often made by spectropho(1). S. W. Rose, L. D. Smith, and J. K. Penry, "Blood Level Determinations of Antiepileptic Drugs. Clinical Value and Methods," Washington, D.C., U.S. Department of Health, Education, and Welfare, 1971. (2) F. Buchthal and M . A. Lennox-Buchthal, "Antiepileptic Drugs," D. M. Woodbury, J. K. Penry, and R. P. Schmidt, Ed., Raven Press, New York, N . Y . , 1972, p 193. 2428
tometric or gas-liquid chromatographic methods. Recently, a method was reported (3) for the determination of DPH by converting it to diphenyl ketone and assaying by high performance liquid chromatography (HPLC). The present report describes a method for t,he direct and simultaneous assay of DPH and phenobarbital from small volumes of blood serum by HPLC. (3) V. G. Gauchel, F. D . Gauchel, and L. Birkofer, 2. Klin. Chern. Klin. Biochem., 11,35 (1973)
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
Table I. Comparison of High Speed Liquid Chromatographic and Gas-Liquid Chromatographic Measurement of Diphenylhydantoin in Human Blood Serum pg DPH/rnl Sample 1 2 3 4 5
HPLCa
GLCb
13.2 f 0.26 9 . 8 f 0.25 10.5 f 0.67 10.7 f 0.56 8 . 9 f 0.27
16.2 11.0 12.0 10.8 8.9
C
PATIENT SERUM-
+
SERUM DPH and PHENOBARBITAL
HPLC data are drawn from 5 repeated determinations on each sample and include the standard deviations based on those results. GLC data are from a single determination by t h e method of MacGee ( 4 ) .
Table I I. Reproducibility of Phenobarbital Measurement from Serum Samplea wg DPH/ml 2 3 4 a n = 5 in all cases.
38.6 f 2.1b 29.8 & 2.1 23.5 f 1.0
RETENTION TIME (min.) Figure 1. High speed liquid chromatography of diphenylhydan-
toin and phenobarbital from human blood serum
Trace ( a ) is from a serum pool obtained from persons not receiving anticonvulsant drugs. Trace ( b ) shows t h e effect of adding 2.0 pg each of DPH and phenobarbital to the serum used for trace (a). Trace (c) resulted when serum from a person receiving both DPH and phenobarbital was analyzed
Standard deviation.
EXPERIMENTAL Apparatus. A Varian Aerograph, Series 4000, constant pressure, gas driven pump is used with helium as the pressurizing gas. The column is a 50-cm X 2-mm i.d. stainless steel tube packed with MicroPak (Varian) 10-km average particle diameter silica gel. Detection a t 254 nm was performed with a Laboratory Data Control Model 1285 UV monitor. Reagents. Ethyl ether was redistilled within 3 weeks of use. All other solvents were reagent grade and used without further purification. The chromatography solvent mixture was methylene chloride/methanol/28% ammonium hydroxide (92:7:1, v / v / v ) . DPH and phenobarbital standards were obtained from Aldrich Chemical Co. Procedure. DPH and phenobarbital are extracted from 50-pl to 200-pl serum samples by mixing them with 1.0 ml of 0.1M NaSP04 and 2.0 ml of ethyl ether in a 3-ml glass-stoppered centrifuge tube. After centrifuging to clear the phases, the upper phases are discarded. The lower phases are acidified (pH 6.8) with 0.1 ml of 2N HC1, mixed with 1.5 ml of ethyl ether, and centrifuged to clear the phases. One-milliliter volumes of the upper phases are transferred, using 1.0-ml volumetric pipets, to 1.0-ml conical vials (Regis Chemical Co.) from which the ether is evaporated to dryness under a stream of nitrogen. Immediately before injection, the samples are dissolved in 50 pl of the chromatography solvent and 20 pl of this solution is injected into the HPLC column. The helium pressure was adjusted to provide a 0.75 ml min-I solvent flow rate. Standard samples of water of the same volume as the serum sample used, containing 10 pg ml-1 of each DPH and phenobarbital, are also analyzed. Drug concentrations are calculated by comparing the peak height response of the sample to that of the standards.
RESULTS AND DISCUSSION Figure 1 demonstrates typical chromatograms obtained from ( a ) 100-p1 samples of pooled serum from unmedicated persons, ( 6 ) serum t.o which DPH and phenobarbital has been added to a concentration of 10 pg ml-1 each, and (c) serum from a patient receiving both drugs. The peaks were identified by injection of DPH and phenobarbital standards separately. An interfering peak eluting just prior to phenobarbital is seen if the ethyl ether is not redistilled.
Table I compares the DPH levels measured by this procedure on 100-p1 serum samples to those obtained by single GLC determinations by the procedure of MacGee ( 4 ) . The precision of this method was measured by five repeated analyses of each of these samples. The mean relative standard deviation for DPH was 3.8% from those data. Samples 2, 3, and 4 from Table I also contained phenobarbital and those data measured from the same chromatograms used for DPH assay are shown in Table II. The mean relative standard deviations for phenobarbital measurement taken from these data are 5.6%. The extraction used with this procedure is designed to provide removal of neutral and basic compounds in as few steps as possible before extraction of DPH and phenobarbital. Many such clean-up procedures now used involve extraction of the drugs into organic solvent from acidified serum, then into a basic buffer, and finally acidification of that buffer and extraction into an organic solvent. The present procedure eliminates the first extraction by doing the basic extraction directly on the serum buffer mixture. This elimination of one extraction and transfer step results in less sample and time loss. The efficiency of this extraction was shown to be 86% when correction was made for aliquoting losses during removal of the last ether phase. This method for the simultaneous determination of DPH and phenobarbital has shown itself to be fast and dependable in 6 months of use for evaluation of more than 300 patient serums. The sensitivity of detection allows the use of small volumes of serum and makes determinations possible with blood drawn into capillary tubes from finger sticks. In this laboratory, one technician can process a group of 24 samples in 4 to 5 hours. Received for review May 24, 1973. Accepted July 16, 1973. This work was supported by the Dreyfus Medical Foundation and by U.S. Public Health Service Grant HD05515. (4) J.
MacGee,AnaL Chern., 4 2 , 4 2 1 (1970)
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