M. Novotny and K. D. Bartlel lndiano University Bloomington, 47401
Gas-Liquid Chromatography of Derivatized Barbiturates
I
A n undergraduate experiment
Since its introduction in 1952, gas-liquid chromatography (GLC) has hecome one of the most important methods in chemical analysis. Because of its remarkable efficiency in the separation of complex mixtures, and its high sensitivity, GLC has played a prominent role during the last fifteen years in many fields of chemistry, petroleum research, industrial analysis, space-related problems, etc. In addition, there are many indications that the future role of GLC in medicine and life sciences may be far hevond even ontimistic nrevious oredictions. Nevertheless. the teaching bf the basics of themethod and its use at the undereraduate level are often insufficient. Not only future profes&onal chemists, hut biologically-oriented a i d medical-technology students should he taught this powerful technique. In this article, we describe an experiment which has been successfully used by hoth chemistry majors and medical-technology students, and which is representative of determinations carried out daily in routine laboratories. The experiment is relatively simple to perform, and instructive in that it illustrates analytical aspects of GLC while demonstrating an application to one of society's problems-drug overdose. The widespread prescription of barbiturate drugs in modern medicine has led to frequent accidental poisonings, drug abuse, and suicide attempts. The high incidence of overdosage makes the determination of barhiturates in both clinical and toxicological laboratories particularly important. Serum and other biological samples, as well as pharmaceutical preparations must he commonly examined. Because of the similarity of structure of different harbi1 On leave fmm Department of Physical Chemistry, University of Leeds. England. From Supelco, Inc., Bellefonte, Pa.
turates, which are pyrimidine derivatives (I), di-suhstituted a t the 5-position (see the table), ultraviolet spectroscopy does not allow identification without laborious chemical treatments ( I ) . Some barbiturates may he fatal at the 1 mg/100 ml blood level, while others are relatively less damaging a t blood concentrations as high as 8 mg/100 ml and have therapeutic levels near 1 mg/100 ml (see the table). Moreover, the duration of pharmacological action differs widely among the individual drugs (see the table), and their dvnamics must he followed. Thin-laver chromatography can he used for analysis, but does notgive quantitative results of sufficient nrecision (2).
0
0
n
la
0 ~b
Barbiturates are generally prescribed as water-soluble sodium salts (Ih), but on acidification of solutions they are converted to the free acids (la) which may he extracted with organic solvents. The acids can be determined by GLC, hut high temperatures are required, and difficulties in hoth qualitative and quantitative analysis consequent on peak tailing may result (3) unless special column deactivation procedures are used (4). A convenient solution to these prohlems is the use of N,N-dimethyl derivatives (11). These may be prepared by reaction with dimethyl sulfate (5) in sufficient ouantitv for structure (11) to he confirmed by pmr spectroscopy (6). For analytical samples, the methylation may he carried out with tetramethylammonium hydroxide, or, more completely, with trimethylanilinium hydroxide (TMAH), in
Common Barbiturates and their Toxicity and Duration of Action Name
Drue- name
Rx
Barbital
Verona1
CH8CH-
Butabarbital Amoharbital
Butisol Amytal
CHKXCHaCH9-
Pentobarbital
Nemhutal
CHXCHT
Secobarbital Phenobarbital
Second Luminal
C&=CH-CHICHaCH-
Blood level(me/100 mi) . -.
R*
Duration of action
CHaCH?-
8
Long
(CH&CHCH2CH2-
3 3
Intermediate Intermediate
1
Short
1
Short Long
I CH~CH~CHCH~ I
CH~CHEH~CHCH~
I
CH~CHICH~CHCH~ CaHs-
5
Level when conaeioumesa was regained.
Volume 51, Number 5, May 1974
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I
Solvent
+
DM A
Figure 1. Chromatagrams of N.N-dimethyi derivatives of barbiturates on two columns at 140°C. Other conditions as in Experimental. DMA = N.N-dimethyl aniline; 1 = barbital; 2 = 'early phenobarbital' (2-ethyi-2phenylmalondiamide); 3 = butabarbital; 4 = amobarbltal; 5 = pentabarbital: 6 = secabarbitat; and 7 = phenobarbital.
Experimental
Two milliliters of aqueous solutions containing between 1 and 10 mg/100 ml of the barbiturates in the table are acidified with 2 m10.15 M H2SO4.and shaken with 15 ml chloroform for 2 min in a funnel with a PTFE stopcock. After separation, the lower layer is run off into a small beaker and evaporated to -1 ml on a 75'C hot plate in a stream of air. The chloroform solution is then transferred to a small vial, and the evaporation completed. 50 pl of 0.2 M TMAH in methanol8 are added with a 100 pl syringe, and after shaking, the solution is left to stand for 5 min. Five microliters of this solution are then injected over a 20 s period (to facilitate reaction on the GLC column) with a carrier-gas flow of 5 ml/tnin. As soon as the solvent front appears, the flow is increased to 30 ml/min, and any temperathe program is started. The GLC conditions in our laboratory are: Instrument: Hewlett9ackard 7W; Columns: 6 ft x 48 in. o.d. stainless steel packed with 3% w/w (a) SE-30 (methyl silicone elastomer) and (b) OV-17 (methyl phenyl silicone fluid) on 100-120 mesh Gas Chrom Q. The column ends were plugged with silanized glass wool; Detector: flame ionization; Injector and detector temperature: 240'C; Carrier gas: helium; Recorder sensitivity: 1 mVf.s.d. Two milliliters of serum samples containing barbiturates are treated as above, except for centrifuging off protein precipitated by addition of the HISOl (3000 rpm for 5 min) before the chloroform extraction. Alternatively, protein precipitation may he avoided by acidifying the serum with an equal volume of 1M sodium acetate buffered at pH 4.8 (8). Whole blood, urine, or gastric fluid may be treated similar!^. Results and Discussion
Figure 1 shows chromatograms of a mixture of six harhiturates a s dimethvl derivatives on both SE-30 (nonpolar phase) and OV-17 (moderately polar) columns under identical conditions. The widely differing retention properties of the dual-column system allow barbiturates to b e identified positively. For example, a harhiturate pill of uncertain origin and unlabelled composition is shown (Fig. 2) to contain only phenobarbital. Further identification of this compound is afforded by the characteristic 'early phenobarbital' peak 7)1' recently shown (9)to he 2-ethyl-2-phenylmalondiamide resulting from decomposition of phenobarbital in the alkaline TMAH solution. Detector responses (peak area, taken a s proportional to width a t half height x peak height) to the N,N-dimethyl barhituric acids are linear throughout a wide range down to weights chromatographed corresponding to therapeutic levels ( < I mg/100 ml serum), e.g., Figure 3. Quantitative
6-
Peak area Figure 2. Analysis of barbiturate pill under conditions listed in Figure 1 legend. (a) phenobarbital; (b) = 'early phenobarbital.'
the first part of a GLC column (6, 7). Elution of the dimethyl derivatives is then accomnlished a t comnarativelv low temperatures so that few undesirable hiolo&cal art:facts, which may also he extracted from the sample, show in the chromatogram. Dimethyl aniline, a by-p~oductof the reaction, is eluted immediately after the solvent. Determination of barbiturates in biological samples or pharmaceutical preparations by on-column methylation provides valuable experience in clinical laboratory work. The experiment may also be designed to illustrate a number of fundamental chromatographic principles. Small quantities of the drugs (1 mg, sufficient for the experiment) may he purchasedz as solutions without license requirements. Larger amounts may only be obtained with Bureau of Narcotics and Dangerous Drugs, and State drug licenses. 334
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Journal of Chemical Education
.
/cm2 4-
2-
o1
2
4
Wt
IP~
6
Figure 3. Graph of detector response against weight chromatographed for N.N-dimethyl derivatives of (a), secabarblta!: (b). pentobarbital: (c). barbitat. 2pg corresponds to 1 mg/lOO ml in sample.
Figure 5. Programmed temperature chromatagram on 3% SE-30 of derivatired extract of serum containing secobarbital (a), pentobarbitai (b). and barbital ( c ) .
Figure 4. Programmed-temperature chmmatogram of NNdimethyi derivatives of barbiturates (as in Fig. 1) on 3% SE-30.
analysis can thus be carried out by constructing a calibration graph similar to Figure 3, or by simple use of ratios of peak area for the unknown and a single standard solution of the identified barbiturate. Alternatively, to correct for less than 100% recoveries in the extraction, a barbiturate not detected in the qualitative screening test, and of known relative resoonse., mav " be added to the unknown solution before acidification. For low concentrations ( < 1 me1100 ml). or to obtain >90% recoverv. a more elaborate &action and clean-up procedure is &essary. The chloroform layer is washed witb pH 7.2 phosphate buffer, and the barbiturates reextracted witb 0.5 M NaOH. A second acidification and chloroform wash are then carried out. Other chromatoeraohic - . .nrincioles mav be illustrated in the experiment. Temperature programming (Fig. 4) may be compared with isothermal operation (Fig. 1). A temperature-program chromatogram of an extract of a serum containinz barbiturates is shown in Fimre 5. station&-phase polarity effects i r e demonstrated by the much longer retention times ( t ~ on. ) OV-17 than on ~ from SE-30 (Fig. 1). Further, plots of log tR ( t measured the solvent front) against numbers of carbon atoms may be constructed for the barbiturates barbital, hutabarbital, pentobarbital, and secobarbital, and for a homologous series of n-alkanes (CII-CIS) and compared (Fig. 6) for SE-30, OV-17, and a novel polyimide stationary phase,* Poly-I 110. Approximately straight, parallel lines are shown for the different phases. Small deviations are noted for secobarbital, the only barbiturate with an unsaturated substituent group (see the table). The extra polarity of OV-17 as compared with SE-30 is shown in larger t~ for the ~ o l a rbarbiturate derivatives, but shorter alkane t~ values. The polyimide phase, however, shows only slightly shorter barbiturate retention than SE-30, but very much shorter alkane retention. A number of other selective effects of this kind have been noted for this phase (10).
1-0
/
I
18
14
10
No. of C
atoms
Figure 6. Graph of log ( t ~ / m i n )against number of carbon atoms for NWdimethyl derivatives of barbiturates (closed circles: (a) = barbital; (b) = butabarbitai; (c) = pentobarbitai: (d) = secobarbital) and n-alkanes (open circles) on three stationary phases (3% loading)
Acknowledgment
We are grateful to Dr. R. B. Forney, Department of Toxicology, Indiana University, for kindly supplying barbiturate standards. Literature Cited (11 Bmughton. P. M.G., Biorhem. J . 6 3 , m (1956). (21 DeZeouu, Chem.. 40.915(1968). (31 Bmehmsnn-Hsnm, E..J Phorm. Sci.. 51.1017 (1661). (41 A d a m R.F., Purcdi, J. E.. and Ettm, L. S.,Arner Lob., [51,51 (19731 ( 5 ) Martin. H.F.. andDrimi1. Chem.. 38.345119661.
R.A..Anol.
J.L..Annl.
(10) Applier
4
'Methelute,' Pierce Chemical Co., Rockford, Ill. Fmm Applied Science Laboratories, Inc.,State College, Pa.
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