Gas Chromatographic Determination of Meprobamate, 2-Methyl-2-Propyl-l,3-Propanediol Dicarbamate, in Plasma and Urine J. F. Douglas, T. F. Kelley, N. B. Smith, and J. A. Stockage Wallace Laboratories, Division of Carter- Wallace, Inc., Cranbury, N . J.
A gas chromatographic method for the determination of meprobamate in plasma and urine is described. Meprobamate can be separated from extractables normal to plasma and urine and three other common carbamates with this procedure. The technique is reproducible and accurate in the therapeutic range of 1 to 10 pg/ml of meprobamate. The detectability limit of the method using a 1-ml sample is 0.2 pg/ml. Dibutyl phthalate i s used as an internal standard for quantitation by the relative peak area technique. An alternate and more rapid sample preparation found suitable for routine use in the range of 2.5 to 15 pg/ml is described.
THE QUANTITATIVE DETERMINATION of meprobamate in biological fluids was reviewed in 1964 by Berger and Ludwig (I), who stated that although a variety of methods have been published dealing with the determination of meprobamate, only a few were useful when applied to meprobamate in plasma. Many procedures lacked adequate specificity and were tediously impractical or insensitive in the concentration range encountered in practice. We have found the colorimetric technique of Hoffman and Ludwig (2) to be a reliable procedure for this purpose and have used it routinely for a number of years. The therapeutic use of carbamates structurally related to meprobamate led us to investigate other approaches which would differentiate between meprobamate and other common carbamates and lead to a rapid, sensitive, and quantitative procedure. A paper chromatographic technique was found which separated the various carbamates (3) but quantitation at commonly encountered therapeutic concentrations was difficult, Parker, Fontan, and Kirk (4) discussed the gas chromatographic detection of meprobamate on a 0.05 SE 30 column but did not describe the measurement of the drug in biological fluids. Nujogi, Cordova, and Rieders (5) and Bloomer (6) mentioned a gas chromatographic assay for meprobamate, but details were not described. Although Goldbaum and Domenski (7) discussed a qualitative procedure for the identification of meprobamate in tissues using a 5 % SE 30 column, their technique was designed for detection of relatively large quantities of drug (50 pg) in tissues and quantitative data were not reported. (1) F. M. Berger and B. J. Ludwig in "Psychopharmacological
Agents," M. Gordon, Ed., Academic Press, New York, N. Y., 1964.
(2) A. J. Hoffman and B. J. Ludwig, J . Am. Pharm. Assoc., Sci. Ed., 48, 740 (1959). (3) J. F. Douglas and A. Schlosser, J . Chromatog., 6, 540 (1961). (4) K. D. Parker, C . R. Fontan, and P. L. Kirk, ANAL.CHEM., 34, 757 (1962). (5) S. K. Nujogi, V. F. Cordova, and F. Rieders, Nature, 206, 716 (1965). (6) H. A. Bloomer, Clin. Res., 14, 186 (1966). (7) L. R. Goldbaum and T. J. Domenski, J . Forensic Sci., 11, 233 (1966).
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ANALYTICAL CHEMISTRY
DIBUTYL PHTlWkTE
Figure 1. A . Gas chromatograms of Method I chloroform extracts of human plasma Lower chromatogram is from normal plasma without addition of dibutyl phthalate Upper chromatogram from the same plasma with 2 pg of meprobamate added
This paper describes an improved gas chromatographic method for the determination of meprobamate in plasma, urine, and water. EXPERIMENTAL
Equipment and Reagents. The F & M Model 402 dual column gas chromatograph equipped with a flame ionization detector and a 1 MV Minneapolis-Honeywell recorder was employed. The two chromatographic columns (either could be employed for this procedure) were 4-foot glass tubes packed with 3.8% UC-W98 methyl silicone on 80- to 100-mesh Diataport S. They were commercially obtained from Hewlett-Packard, Avondale, Pa. The instrument settings were as follows:
Temperature Column Injection port Detector block
180" C 275" C 225" C
Gas Flow Rates Hydrogen Helium (carrier gas) Oxygen
20 ml per min 65 ml per min 50 ml per min
The instrument’s sensil.ivity settings were range 10 with an attenuation of 2X. Redistilled chloroform and dibutyl phthalate (Eastman) were used. Method I. A 1.0-mI aliquot of plasma or urine and 10.0 ml of redistilled chloroform are thoroughly mixed and the two phases allowed to separate. Eight milliliters of the organic layer are removed and evaporated to dryness in a water bath. The resulting residue is dissolved in 0.1 ml of chloroform containing I.O pg of dibutyl phthalate as internal standard and a 2.6-pl aliquot of the solution is injected into the gas chromatograph. Method 11. In the alternate, more rapid procedure, 0.2 rnl of redistilled chloroform containing 2.0 pg of dibutyl phthalate is added to 1.0 ml of biological fluid, and the
MER
lAMATE
MEBUTAMATE
i
MEPR 3AMATE
Figure 2. Gas chromatogram showing separation of four carbamates 100 ng meprobamate, 30 ng carisoprodol, 150 ng mebutamate, and 75 ng tybamate
J
\
mixture is thoroughly shaken and centrifuged. The aqueous layer is removed by aspiration and 2.6 pl of the chloroform extract is injected into the gas chromatograph. RESULTS AND DISCUSSION
Figure 1. B. Gas chromatogram of a Method I chloroform extract of plasma obtained from an individual treated with meprobamate
Previous studies of the partition coefficient of meprobamate between water and chloroform (8) have shown that the drug is substantially quantitatively extracted from aqueous media with a 10-fold volume of this organic solvent. Inter( 8 ) J. F. Douglas, B. J. Ludwig, and A. Schlosser, J. Pharmacol. Exptl. Therap., 138,21 (1962).
3
5~
pg/ml Plasma
pg/ml Urine
rg/ml H 2 0
Figure 3. Relationship between relative peak area and meprobamate concentration in three fluids (Method I) Averages of quintuplicate determinations are given with their standard errors VOL 39, NO. 0, JULY 1967
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2.c
1.8
1.6 1.4
;
1.2
3
1.c
2 c
2
.8
K
.6 .4 .2
2.5
5
10
15
2.5
5
pg/rnl Plrama
10
15
2.5
pg/ml Urine
5
10 pl/ml
15
HIO
Figure 4. Relationship between relative peak area and meprobamate concentration in three fluids (Method 11) Averages of quintuplicate determinations are given with their standard errors pretation of the chromatogram obtained following injection of chloroform from a Method I extract in the gas chromatograph shows that meprobamate can be effectively separated from plasma constituents normally occurring in man (Figure 1). Similar results were obtained for dog, rabbit, and monkey plasma. In human urine, however, we sporadically encountered a constituent which gave a blank of less than 2 Mg/ml. This interfering material is presumably dietary in origin since it is only occasionally present in different urine specimens from the same subjects. It can be corrected by using a urine obtained prior to drug intake. The retention time of meprobamate with the column employed, W 98 methyl silicone, is significantly different from the retention times of other biologically active carbamates-carisoprodol, mebutamate, and tybamate (Figure 2)-indicating that not only is this technique specific for meprobamate in plasma, but it also permits the identification of related structures. Meprobamate can be quantitated gas chromatographically when relative peak area is used as an index of concentration since a linear relationship exists between relative peak area and drug concentration in the range of 1-10 pg/ml of plasma, urine, or water (Figure 3). The reproducibility of the procedure is indicated by the standard errors presented in Figure 3. The extraction protocol can be modified to yield a more rapid but less sensitive assay (Method 11). A reproducible
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ANALYTICAL CHEMISTRY
linear response in the range of 25-15 pg/ml is obtained from water, plasma, and urine using this modification (Figure 4). The reduced detectability of this latter procedure is due to two factors: the ratio of final chloroform solution to starting aqueous fluid is approximately one half that of Method I and the amount of meprobamate extracted, 45x while reproducible, is approximately twofold less than the 99 extracted using the first procedure. However, it should be noted that the more rapid procedure has adequate detectability in the range of practical therapeutic concentrations. In addition, quantitative results can be obtained within 15 minutes after receipt of the specimen. The first procedure which requires about 90 minutes is most useful with extremely low concentrations while the extraction modification is the method of choice when therapeutic concentrations of the drug are involved and results are desired quickly. Collection and subsequent infrared analysis of the volatile product corresponding to the meprobamate peak on the gas chromatogram did not indicate any detectable degradation of the drug under the conditions employed. When the collected material was rechromatographed, it did show, however, the presence of a trace amount of degradation product. RECEIVED for review December 23,1966. Accepted April 19, 1967.
