Gas Chromatographic Identification and Determination of Barbiturates SIR: The gas chromatographic analysis for barbiturates has been reported by numerous authors (1, 2, 7, 9, 14). It has also been noted that there is a need for special column preparation in order to minimize the tailing associated with these polar compounds (4, 16). With severe tailing, which is the case with unmodified barbiturates, it has been our experience that even with special column preparation quantitation is questionable and sensitivity variable. Because of this problem, most workers have employed spectrophotometry to quantitate and a chromatographic technique for identification (6, 11, 12, 16, 17, 19, 20). However, since many other drugs are known to interfere in spectrophotometric determinations, quantitation' is sometimes impossible. This is a significant problem since multiple ingestions are not rare situations. I n order to resolve these problems, our approach is to minimize the polarity of the barbiturate to be chromatographed. This can be accomplished by methylating the drug to form the 1,3dimethyl - 5,5 - disubstituted barbituric acid derivatives. Stuckey's method is employed for the methylation (18). This derivative, in addition to being less polar, has a shorter retention time than
2
6
the parent compounds on columns coated with SE-30. Figure 1 shows chromatograms of a mixture of 5-ethyl, 5-isoamyl barbituric acid (amobarbital), 5-ethyl, 5-(1-methyl butyl)-barbituric acid (secobarbital), 5-ethy1, &phenylbarbituric acid (phenobarbital), their corresponding methylated derivatives and the methyl ester of palmitic acid (CM) (Sigma Chemical Co.) which is our choice of an internal standard. The ultraviolet spectrum of these derivatives has a maximum a t 2280 A. The position of this maximum is unaffected by changes in pH. This is the evidence upon which the structural assignment of the methylated derivatives is based (18). The molar absorptivity of 6.3 X 10-3 decreases by one half in 20 minutes a t pH 13 (6, 18). EXPERIMENTAL
Apparatus. The instrument used was a Jarrel Ash Model 28-700 equipped with a hydrogen flame detector. The column is a 4-foot l/s-inch copper tubing packed with Anakrom ABS 60-70 mesh coated with 5% Se-30 according to the method prescribed by Horning. This column has been in use for over a year before this data was obtained, The operating conditions used were column temperature 170°, 175', and
IO
14
18
180' C.; nitrogen pressure, 30 p.s.i.; hydrogen pressure, 10 p.s.i., and the amplifier was set a t ampere. The recorder is a Minneapolis Honeywell product with range of 1 mv. for a full scale deflection. Procedure. To a test tube containing 2 ml. of serum, 2 ml. of phosphate solution (saturated solution of sodium dihydrogen phosphate) are added followed by 10 ml. of ether. The tube is stoppered and shaken vigorously for 30 seconds; then centrifuged for 1 minute. Seven milliliters of the ether extract are transferred to another tube in which the ether is removed by heating in water bath with a stream of air. The residue is then dissolved in 2 ml. of a 10% (v./v.) water-methanol saturated with potassium carbonate solution followed by 0.1 ml. of dimethyl sulfate. The reaction is then allowed to proceed for 4 minutes in the bath a t 135' f 2' F. It is important that potassium and not sodium carbonate be used since the former's solubility in the methanol solution maintains the desired alkalinity during the reaction. The methanol is removed also a t 135' F. by a strong steady stream of air. This takes approximately three minutes and it is terminated when only a few droplets of immiscible aqueous and organic liquids remain. One milliliter of l M , pH 6 acetate buffer is added and 1.5 ml. of heptane. Extraction is aided by the use of a vortex mixer followed by centrifugation. One milliliter of the heptane extract is transferred to a small test tube (10 X 75 mm.) from which the heptane is removed similarly to ether and methanol. In this step, excess drying beyond the removal of heptane results in loss of material. The resulting residue is dissolved in 100 pls. of an acetone solution containing 0.5 mg./ml. of the internal standard. Five-microliter injections of this solution are made with a Hamilton syringe using 1 pl. of an inert solvent (heptane) as a back-up liquid to ensure reproducible injections, Following this procedure, calibration data was obtained from samples of normal serums, to which were added increasing amounts of known barbiturates. The results are as indicated in Table I. The total time of analysis is approximately 45 minutes. DISCUSSION
RETENTION T I M E / minutes
Figure 1. Superimposed chromatograms of mixture modified barbiturates A. 8.
of un-
10 p g . of amobarbital and secobarbital 10 pg. of phenobarbital, mixture of methylated derivatives 1. 3 pg of amobarbital 2. 3 pg. of secobarbital 3. 4.5 pg. of phenobarbital 4. 2.5 pg. of internal standard. Column temperature is 175OC.
-Methylated derivatives
. . . . Unmodified barbiturates
It will be noted in Figure 1, under the conditions employed, that amobarbital and secobarbital cannot be resolved in their unmodified form in addition to exhibiting poor sensitivity. Upon methylation, both resolution and increased sensitivity are obtained. This is due to a decrease in polarity as a result of the conversion from secondary to VOL. 38, NO. 2, FEBRUARY 1 9 6 6
345
fable I. Relative Chromatographic Data for the Methylated Derivatives of Amobarbital, Pentobarbital, Secobarbital, and Phenobarbital
RELATIVE RETENTION TIME Retention time ratio of barbiturate t o internal standard Amobarbital Temp. O C. Pentobarbital Secobarbital Phenobarbital 0.27 0.29 0.30
170 175 180
170 175 180
1 0.18 0.19 0.18
2 0.39 0.36 0.38
3 0.62 0.66 0.58
4 1.03 0.98 0.85
tertiary amides. Both identification and quantitation are made relative to an internal standard. Table I lists both the relative retention times and relative responses of varying concentrations which are constants over this temperature range. Our calibration encompasses the four commonly used barbiturates listed in Table I. Of these, amobarbital and pentobarbital are not resolvable by this method. A similar situation exists between phenobarbital and mephobarbital, since upon methylatioli, both yield identical derivatives. This does not present a problem clinically because the latter is tnetabolized to phenobarbital by the body, and it is this form which appears predominantly in serum (3). Identification of other barbiturates can be made from their relative retention times and the use of the graph shown in Figure 2. The use and justification of this relationship between the logarithm of the retention or relative retention times and the cube root of summated boiling point number increments have previously been reported by Martin, Driscoll, and Gudzinowicz (13). I n this instance, all derivatives are identical except for the two substituents in the 5 position, and since the boiling point function is additive, it is valid to restrict the calculation to just these increments. ?he values for these increments may be found in the literature (8, 10, 13). Therefore, from this retention data, an approximation of molecular weight may be calculated for a barbiturate for which there is no previous calibration. By decreasing the polarity of barbituric acid derivatives, quantitation by gas chromatography becomes feasible. By using an internal standard, instru-
346
0.66 0.68 0.70
RELATIVE RESPONSE Peak height ratio of barbiturate to internal standard, cone. mg./100 cc. Amobarbital Pentobarbital Secobarbital
Tzmp.
c.
0.34 0.34 0.34
ANALYTICAL CHEMiSTRY
6 1.33 1.35 1.17
1 0.11 0.13 0.11
2 0.26 0.28 0.28
3 0.44 0.42 0.40
4 0.70 0.65 0.65
5
6 0.89 0.92 0.85
1.5 0.22 0.20 0.19
Phenobarbital 3 4.5 6 9 0.38 0.58 0.87 1.19 0.37 0.57 0.82 1.22 0.33 0.53 0.84 1.10
nature of the solid phase and never in our experience has it been related to the nature of column material. LITERATURE CITED
I .
,
,
2.2
I
2.4
t
2.6
s
8
2.6
3.0
3
ts ( B P N
of C s
R proups I
Figure 2. Graph of relative retention times of methylated derivative vs. the cube root of their summated boiling point increments for the substituents at 5 position 1. 2. 3. 4. 5.
Barbital Butabarbital Amobarbital Secobarbital Phenobarbital
mental and injection variations are monitored. It has been our experience on a number of occasions to complete an emergency determination in 45 minutes, that is the time elapsed from turning on the instrument to reporting the result. I n addition to speed, a great advantage is being able to make accurate determinations in the presence of other drugs such as salicylate, gantrisin, and diphenylhydantoin, which have been present in many samples submitted for analysis in this hospital, as determined by spectrophotometric methods. Studies carried out by us, relative to these and other compounds, indicate that thermal decomposition, whenever it does occur, is always related to the
(1) Brockmann, Hanssen, E., Boerheim, Svendsen. A.. J. Pharm. Sci. 50. 804 (1961). ’ ’ (2) Brockmann, Hansseh, E., Boerheim, Svendsen, A., Zbid., 51, 318 (1962). (3) Butler, T. C., J. Pharmacol. Exptl. Therap. 106, 235-45 (1952). (4) Cieplinski, E. W., ANAL. CHEM.35, 256-7 (1963). (5) Fox, J. J., Shugar, D., Bull. SOC. Chem. Berges. 61,44 (1952). (6) Goldbaum, L. R., ANAL. CHEM.24, 1604 (1961). (7) Gudzinowicz, B. J., Clark, S. S., J. Gas Chromatog. 3, May 1965.
(8)
