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A rapid high resolution chromatographic method for serum barbiturates: Instant thin-layer chromatography
Clin.
Biochem.
A RAPID
3, 287-293
(1970)
HIGH
RESOLUTION FOR SERUM INSTANT THIN-LAYER
K. ITIABA, Department
CHROMATOGRAPHIC BARBITURATES: CHROMATOGRAPHY
J. C. CRAWHALL,
of Clinical Biochemistry, Royal Victoria Hospital, (Received
May
AND
McGill Montreal,
METHOD
C. M. SIN University Canada
Clinic,
10, 1970)
SUMMARY 1. Barbiturates were extracted from acidified serum with chloroform and diethyl ether. 2. The various barbiturates in the extract were separated by chromatography on fibre glass sheets impregnated with silica gel. 3. The barbiturates were visualized by being sprayed with Rhodamine B and the fluorescence quenching effect observed under ultraviolet light. 4. The R, values of various barbiturates in three different solvent systems are described. 5. Serum barbiturates could be separated in 5 or 20 min into groups having clinical significance. Urinary barbiturates could also be identified in a similar manner but an additional extraction step was required.
THE RAPID IDENTIFICATIONANDCLASSIFICATION of barbituratesarean important function of the Clinical Chemistry laboratory in the management of patients who have taken an over-dosage of drugs. The use of drugs as tranquilizers and sedatives is prevalent in modern urban communities, and many pharmaceutical preparations are combinations of drugs of which only part may be barbiturates. It is this type of preparation which is frequently taken by patients making suicidal attempts. It is also important to know whether patients who show drug overdosage symptoms have taken barbiturate, and whether the serum levels of barbiturate are compatible with the symptoms, or whether additional drugs or organic disease may be involved. A variety of methods are employed in toxicology for determining barbiturate levels. The low specificity of all the chemical methods and the small structural differences that exist have made their identification difficult, unless highly purified preparations are made. In the case of purified preparations, melting Correspondence: Royal Victoria
Dr. Hospital,
K. Itiaba, Montreal
Department of Clinical 112, Quebec, Canada.
Biochemistry,
McGill
University
Clinic,
ITIABA
288
et al.
points, X-ray diffraction, and infra-red absorption methods are suitable. Most of the current methods available to the clinical chemist involve organic solvent extraction of the barbiturates followed by ultra-violet spectrophotometry (1-d). Earlier methods, based on the non-specific colour reaction of barbiturates with cobalt ion in the presence of an amine, have proved unsatisfactory and unreliable (5-r). Differential hydrolysis (8) has been tried but found successful only with single components. Methods involving paper chromatography (9, IO) have been developed but they are time consuming. Gas chromatographic methods (U-14) permit rapid quantitative determinations of barbiturates once the derivatives have been prepared, but this requires specialized techniques and very expensive equipment. This has also been the case with spectrophoto-fluorometric procedures (15) which have not been much used. On the other hand, numerous thin-layer chromatographic methods are available (16~SO), most probably because of the inexpensive equipment involved and the relative rapidity of the procedure. The present report deals with an extremely rapid method for the determination and separation of the three main types of barbiturates (based on their clinical toxicity). The method is designed to provide a rapid, sensitive and reliable indication of the presence and type of barbiturate in serum and other body fluids. MATERIALS
AND
NIETH~DS
Extra&on Procedure A 2-ml sample of serum was brought to pH 4 with the dropwise addition of 6 mol/l HCl and then extracted 3 times with 20 ml chloroform. The pooled extract was decolourized with a spatula (approx. 0.1 mg) of activated charcoal, filtered and evaporated almost to dryness on a steam bath. The residue was dissolved in 4 ml diethyl ether and evaporated to dryness under a stream of nitrogen. The final residue was then dissolved in 50 ~1 of solvent mixture (83yo methanol - 170Jo acetic acid). A preliminary extraction was required with urine and peritoneal dialysate fluid. One hundred milliliters of fluid was added to a 5 X 5 cm piece of cation exchange paper-type SA-2, Reeve Angel Co, Clifton, New Jersey-and agitated for 30 min (91). The ion exchange paper was then extracted with citrate buffer, pH 2.2, and chloroform and the rest of the procedure continued as above for serum. Instant Thin-Layer Chromatography (ITLC) Samples of 2-20 ~1 of the extract were spotted on fibre glass sheets impregnated with silica gel (Gelman Co, Ann Arbor, Michigan). Standards Standards consisted of the sodium salts of phenobarbitone (Phenobarbital), barbitone (Barbital), amylobarbitone (Amytal), pentobarbitone (Nembutal), and quinalbarbitone (Seconal). Stock solutions of 20 mg% were made up fresh daily. Since some instability of barbiturate solutions was observed, these were kept on ice. Working solutions were prepared by l/10 dilution of the stock solutions just before use.
CHROMATOGRAPHIC
Solvent (i) (ii) (iii)
METHOD
Systems Petroleum ether (BP SO-loo”), ethyl acetate Benzene, acetone (100:2). Carbon tetrachloride, acetone (100:5).
289
(100:14).
Development and Detection The standards and unknowns were spotted onto ITLC sheets (12 X 50 cm or 5 X 10 cm). The chromatograms were developed at room temperature, for 20 min in the case of the larger sheets and for 5 min in the case of the semi-micro sheets. The sheets were dried in a stream of cold air in the fume cupboard and then sprayed with fluorescent Rhodamine B (Gelman Co, Ann Arbor, Michigan). The barbiturates quenched the fluorescence of the Rhodamine B dye so that when the sheets were viewed under short wave-length ultra-violet light (250 nm), with a chromate-vue instrument (Model C-5, Ultra-violet Products, Calif.), the barbiturates appeared as light spots on a dark background. The spots were outlined with pencil and compared with those of the known standards. The Rr and R, values were determined, where: R
and
R
I D
=
distance substance travelled distance solvent front travelled
=
distance substance travelled distance phenobarbital travelled
Quantitation The approximate barbiturate unknown with different amounts
x 100.
concentration was obtained by comparing the of standard solution under ultra-violet light.
RESULTS
AND DISCUSSION
The extraction of barbiturates with chloroform was found to be more satisfactory than either diethyl ether or ethyl acetate which have been used in other methods (15,2,8,%!?). The complete extraction procedure which is common to all the methods in general use took no more than 30 min, and gave a recovery of about 80%. The actual developing time for the chromatograms was 5-10 min for the semi-micro sheets and 20-25 min for the larger sheets. Thus the specificity of the method was greatly increased, and the addition of no more than 10 min made it possible to obtain the benefits of combined solvent extraction and chromatographic separation. The Rr and R, values varied in the different solvents, as may be seen from Table I. Solvent (i) gave the.best separation of phenobarbital. On the other hand, the separation of the short-acting barbiturates (e.g. Seconal) from the intermediate-acting types (e.g. Amytal) was more satisfactory in solvent (ii). Solvent (iii) was of interest as it gave relatively better separation within the long-acting barbiturate group (e.g. phenobarbital and barbital). Figure 1 shows the results obtained for the larger sheets with the use of solvent (i). Comparison of the large and the semi-micro sheets are shown in Fig. 2. Both
290
ITIABA
et al.
TABLE AVERAGE -
Rr AND
R, VALUES
OF THE
I
BARBITURATES
ON INSTANT
Solvent (i) Petroleum ether ethyl acetate, 100: 14 Rf 0.41 0.42 0.56 0.58 0.61
Phenobarbitone Barbitone Amylobarbitone Pentobarbitone Quinalbarbitone
RP 100 103 137 142 149
(ii)
THIN-LAYER
CHROMATOGRAPHY
system:
Benzene acetone,
(iii) 100:2
Rr 0.2s 0.29 0.37 0.37 0.45
RP 100 104 133 133 161
( (
Carbon acetone,
tetrachloride 100:5
Rf 0.28 0.34 0.41 0.40 0.46
RP 100 122 147 143 165
Seconal Amytal
Phenobarbital
2 FIG. 3,4,
5
1. Separation of barbiturates by instant thin-layer chromatography & 5 were the individual barbiturates, 2 the aqueous mixture of these to normal serum.
in solvent (i): Channels and 1 the mixture added
small and large strips were developed consecutively, so that (for example, during emergencies) an immediate result was obtained as to the presence or absence of barbiturates from the semi-micro strips. This was then followed by the result from the larger strips as to the type of barbiturate present. By using different amounts of standard barbiturate solutions and comparing the relative sizes of the spots with the unknown, an approximate indication of the amount of barbiturate
CHROMATOGRAPHIC
METHOD
291
Seconal Amy tal Phenobarbital Seconal
& Amytal
Phenobarbital
Origin
FIG. parison
3.
Separation of barbiturates by instant thin-layer chromatography in solvent of large and semi-micro sheets. Channel 1 wts a mixture of the 3 barbiturates, a mixture of seconal and phenobarbital, and channel 3 seconal.
(i): comchannel 2
present in the unknown sample \\:a~ obtained. The lower limit of sensitivit)of the method was of the order of 5 pg (which corresponds to a serum concentration of about 0.5 mg%). The above points are exemplified in the results shomm in Fig. 3 for an actual case of barbiturate intoxication. Barbiturates lvere sho\vn to be present in this epileptic patient’s serum, peritoneal dialysate, and urine, and approximate serum levels were monitored during the patient’s period in coma (Table II). An additional spot can be observed betlveen the origin and phcnobarbital on this patient’s chromatogram as knell as another extraneous spot occurring close to the solvent front. These were most probably metabolites of the barbiturates (16, 17). The spot between the origin and that of phenobarbital was also observed to have the same RI value as Dilantin, a drug with some structural resemblance to phenobarbital which \\:as kno\vn to have also been taken by this patient. Phenobarbital when added to serum also gave a metabolic derivative that had the same mobility as Dilantin in this system. It can also be seen from Fig. 3 that Dilantin is well separated from the barbiturates and thus when present with phenobarbital did not interfere with this method, Other phenobarbital mixtures such as Amesec (aminophylline, ephedrine and amylobarbitone), Fiorinal (all>.lbarbituric acid, caffeine, acetylsalicylic acid and phenacetin), Phelantin (dilantin, methamphetamine and phenobarbitone), Tederal (theophylline, ephedrine and phenobarbitone) and Atropine-phenobarbital were checked at levels of 2 mg/ml and caused no interference with the identification of the barbiturates. Doriden (a-phenyl-a-ethyl-glutarimide), a compound closely related to barbiturates gave a spot similar to Seconal (Rr values of O.Gl) in the petroleum ether-ethyl acetate solvent system, however it was jvell separated from the barbiturates in benzeneacetone (R, value of 0.84) and carbon tetrachloride-acetone (RI value of O.‘iG) solvent systems.
292
ITIABA
et al.
Phenobarbital Dilantin
Origin
1
2
4
3
6
5
7
FIG. 3. Instant thin-layer chromatography in solvent (i) of extracts of body fluids from a patient under treatment for barbiturate overdosage. Channels 1, 2, 3 represent 2, 5, & 20 al from serum, channel 4 represents 5 pg standard phenobarbital, channels 5 h 6 represent 5 19 20 ~1 from urine, and channel 7 represents 25 pl from peritoneal dialysate extract respectively.
TABLE ESTIMATION OFTHE PATIENT BY MEANS CHROMATOGRAPHY Day 1
2 5 6 7 8 11 12
II
SERUM BARBITURATE OF A OF INSTANT THIN-LAYER (ASGIVENINMETHODS) Cont.
(mg%)
5.0 4.0 2.0 1.7 E 0.8 0.6
(17,22,23) have shown that there is a poor correlation between the serum barbiturate level and the degree and duration of coma in a patient. However, information about the gross levels of barbiturate in the plasma are valuable in the therapeutic management of the patient and subsequent studies of plasma barbiturate concentrations demonstrate the effectiveness, or otherwise, of the therapeutic measures. Future studies may show that quantitative measurement of barbiturate after separation from intermediary metaboli tes is better correlated with the patient’s clinical course than the apparent barbiturate concentration based on gross calorimetric measurements. Previous
studies
CHROMATOGRAPHIC
METHOD
293
REFERENCES 1. GOLDBAUM, L. R. Determination of barbiturates. Ultra-violet spectrophotometric method with differentiation of several barbiturates. Anal. Chem. 24, 1604-1607 (1952). 8. WILLIAMS, L. A. & ZAK, B. Determination of barbiturates by automatic diBerentia1 spectrophotometry. Clin. Chim. Acta. 4, 170-174 (1959). 3. WALKER, J. T., FISHER, R. S. & MCHUGH, J. J. The quantitative estimation of barbiturates in blood by ultra-violet spectrophotometry; analytical method. Amer. J. Clin. Path. 8, 451461 (1948). 4. ANNINO, J. S. Clinical Chemistry, Little Brown & Co, Boston, 3rd ed. (1964) p. 340. 6. GETTLER, A. 0. The significance of some toxicologic procedure in the medico-legal autopsy. Amer. J. Clin. Pathol. 13, 169-177 (1943). 6. CURRY, A. S. Rapid method of screening for barbiturates. Brit. Med. J. 2, 1040-1042 (1963). 7. ZAAR, B. & GRONWALL, A. A micro method for determination of barbiturates in serum as mercuric complexes. Stand. J. Clin. Lab. Invest. 13, 225-230 (1961). 8. BROUGHTON, P. M. G. A rapid ultraviolet spectrophotometric method for the detection, estimation and identification of barbiturates in biological material. Biochem. J. 63, 2Oi-213 (1956). D. A. Rapid paper chromatography of barbiturates. Clin. Chem. Acta. 7, 9. PODMORE, 176-179 (1962). 10. CURRY, A. S. Identification of barbiturates. Nature 183, 1052 (1959). C. E. & GILLEN, H. W. Gas chromatography analysis for anti-convulsant 11. PIPPENGER, drugs in biologic fluids. Clin. Chem. 15, 582-590 (1969). K. D. & KIRK, P. L. Separation and identification of barbiturates by gas id. PARKER, chromatography. Anal. Chem. 33, 13781381 (1961). 13. KAZYAK, L. & KNOBLOCK, E. C. Application of gas chromatography to analytical toxicology. Anal. Chem. 35, 14481452 (1963). E. & SVENDSEN, A. B. Separation and identification of 14. BROCHMANN-HANSSEN, barbiturates and some related compounds by means of gas-liquid chromatography. J. Pharm. Sci. 51, 318-321 (1962). S., DUGGAN, D. E., VASTA, B. M. & BRODIE, B. B. A spectrophoto15. UDENFRIEND, fluoremetric study of organic compounds of pharmacological interest. J. Pharmacol. & Exp. Therap. 120, 26-32 (1957). R. L. &PERRY, W. F. Rapid identification of common sedatives and stimulants 16. STREET, by thin layer chromatography. Clin. Biochem. 2, 197-203 (1969). C. E., SCOTT, J. E. & GILLEN, H. W. Thin layer chromatography of 17. PIPPENGER, anticonvulsant drugs. Clin. Chem. 15, 255-260 (1969). 18. SUNSHINE, I., ROSE, E. & LEBEAU, J. Barbiturate detection using thin-layer chromatography. Clin. Chem. 9, 312-316 (1963). J. & ROLLASON, J. G. A rapid method for the partial identification of 19. KELLEHER, barbiturates in blood using thin layer chromatography on microscope slides. Clin. Chem. Acta 10, 92-94 (1964). 0. V. Determination by thin layer chromatography of phenytoin in serum in the 20. OLESEN, presence of barbiturates and other anti-epileptic and various drugs. Acta Pharmacol. Toxicol. 23,414s (1965). V. P., KIM, W. K. & EGLITIS, I. Detection of narcotic drugs and tranquillizers, 21. DOLE, amphetamines and barbiturates in urine. J. Amer. Med. Assoc. 198, 349-352 (1966). G. A. The estimation of barbiturates in blood. Biochem. J. 34, 73-77 (1940). 2.9. LEVVY, w. STEVENSON, G. W. Spectrophotometric determination of blood barbiturate. Anal. Chem. 33, 1374-1378 (1961). I. Chromatographic and Electrophoretic Techniques. Interscience Public. Inc. 94. SMITH, New York, 2nd ed., 1 (1960), pp. 390.
Biochem.
A RAPID
3, 287-293
(1970)
HIGH
RESOLUTION FOR SERUM INSTANT THIN-LAYER
K. ITIABA, Department
CHROMATOGRAPHIC BARBITURATES: CHROMATOGRAPHY
J. C. CRAWHALL,
of Clinical Biochemistry, Royal Victoria Hospital, (Received
May
AND
McGill Montreal,
METHOD
C. M. SIN University Canada
Clinic,
10, 1970)
SUMMARY 1. Barbiturates were extracted from acidified serum with chloroform and diethyl ether. 2. The various barbiturates in the extract were separated by chromatography on fibre glass sheets impregnated with silica gel. 3. The barbiturates were visualized by being sprayed with Rhodamine B and the fluorescence quenching effect observed under ultraviolet light. 4. The R, values of various barbiturates in three different solvent systems are described. 5. Serum barbiturates could be separated in 5 or 20 min into groups having clinical significance. Urinary barbiturates could also be identified in a similar manner but an additional extraction step was required.
THE RAPID IDENTIFICATIONANDCLASSIFICATION of barbituratesarean important function of the Clinical Chemistry laboratory in the management of patients who have taken an over-dosage of drugs. The use of drugs as tranquilizers and sedatives is prevalent in modern urban communities, and many pharmaceutical preparations are combinations of drugs of which only part may be barbiturates. It is this type of preparation which is frequently taken by patients making suicidal attempts. It is also important to know whether patients who show drug overdosage symptoms have taken barbiturate, and whether the serum levels of barbiturate are compatible with the symptoms, or whether additional drugs or organic disease may be involved. A variety of methods are employed in toxicology for determining barbiturate levels. The low specificity of all the chemical methods and the small structural differences that exist have made their identification difficult, unless highly purified preparations are made. In the case of purified preparations, melting Correspondence: Royal Victoria
Dr. Hospital,
K. Itiaba, Montreal
Department of Clinical 112, Quebec, Canada.
Biochemistry,
McGill
University
Clinic,
ITIABA
288
et al.
points, X-ray diffraction, and infra-red absorption methods are suitable. Most of the current methods available to the clinical chemist involve organic solvent extraction of the barbiturates followed by ultra-violet spectrophotometry (1-d). Earlier methods, based on the non-specific colour reaction of barbiturates with cobalt ion in the presence of an amine, have proved unsatisfactory and unreliable (5-r). Differential hydrolysis (8) has been tried but found successful only with single components. Methods involving paper chromatography (9, IO) have been developed but they are time consuming. Gas chromatographic methods (U-14) permit rapid quantitative determinations of barbiturates once the derivatives have been prepared, but this requires specialized techniques and very expensive equipment. This has also been the case with spectrophoto-fluorometric procedures (15) which have not been much used. On the other hand, numerous thin-layer chromatographic methods are available (16~SO), most probably because of the inexpensive equipment involved and the relative rapidity of the procedure. The present report deals with an extremely rapid method for the determination and separation of the three main types of barbiturates (based on their clinical toxicity). The method is designed to provide a rapid, sensitive and reliable indication of the presence and type of barbiturate in serum and other body fluids. MATERIALS
AND
NIETH~DS
Extra&on Procedure A 2-ml sample of serum was brought to pH 4 with the dropwise addition of 6 mol/l HCl and then extracted 3 times with 20 ml chloroform. The pooled extract was decolourized with a spatula (approx. 0.1 mg) of activated charcoal, filtered and evaporated almost to dryness on a steam bath. The residue was dissolved in 4 ml diethyl ether and evaporated to dryness under a stream of nitrogen. The final residue was then dissolved in 50 ~1 of solvent mixture (83yo methanol - 170Jo acetic acid). A preliminary extraction was required with urine and peritoneal dialysate fluid. One hundred milliliters of fluid was added to a 5 X 5 cm piece of cation exchange paper-type SA-2, Reeve Angel Co, Clifton, New Jersey-and agitated for 30 min (91). The ion exchange paper was then extracted with citrate buffer, pH 2.2, and chloroform and the rest of the procedure continued as above for serum. Instant Thin-Layer Chromatography (ITLC) Samples of 2-20 ~1 of the extract were spotted on fibre glass sheets impregnated with silica gel (Gelman Co, Ann Arbor, Michigan). Standards Standards consisted of the sodium salts of phenobarbitone (Phenobarbital), barbitone (Barbital), amylobarbitone (Amytal), pentobarbitone (Nembutal), and quinalbarbitone (Seconal). Stock solutions of 20 mg% were made up fresh daily. Since some instability of barbiturate solutions was observed, these were kept on ice. Working solutions were prepared by l/10 dilution of the stock solutions just before use.
CHROMATOGRAPHIC
Solvent (i) (ii) (iii)
METHOD
Systems Petroleum ether (BP SO-loo”), ethyl acetate Benzene, acetone (100:2). Carbon tetrachloride, acetone (100:5).
289
(100:14).
Development and Detection The standards and unknowns were spotted onto ITLC sheets (12 X 50 cm or 5 X 10 cm). The chromatograms were developed at room temperature, for 20 min in the case of the larger sheets and for 5 min in the case of the semi-micro sheets. The sheets were dried in a stream of cold air in the fume cupboard and then sprayed with fluorescent Rhodamine B (Gelman Co, Ann Arbor, Michigan). The barbiturates quenched the fluorescence of the Rhodamine B dye so that when the sheets were viewed under short wave-length ultra-violet light (250 nm), with a chromate-vue instrument (Model C-5, Ultra-violet Products, Calif.), the barbiturates appeared as light spots on a dark background. The spots were outlined with pencil and compared with those of the known standards. The Rr and R, values were determined, where: R
and
R
I D
=
distance substance travelled distance solvent front travelled
=
distance substance travelled distance phenobarbital travelled
Quantitation The approximate barbiturate unknown with different amounts
x 100.
concentration was obtained by comparing the of standard solution under ultra-violet light.
RESULTS
AND DISCUSSION
The extraction of barbiturates with chloroform was found to be more satisfactory than either diethyl ether or ethyl acetate which have been used in other methods (15,2,8,%!?). The complete extraction procedure which is common to all the methods in general use took no more than 30 min, and gave a recovery of about 80%. The actual developing time for the chromatograms was 5-10 min for the semi-micro sheets and 20-25 min for the larger sheets. Thus the specificity of the method was greatly increased, and the addition of no more than 10 min made it possible to obtain the benefits of combined solvent extraction and chromatographic separation. The Rr and R, values varied in the different solvents, as may be seen from Table I. Solvent (i) gave the.best separation of phenobarbital. On the other hand, the separation of the short-acting barbiturates (e.g. Seconal) from the intermediate-acting types (e.g. Amytal) was more satisfactory in solvent (ii). Solvent (iii) was of interest as it gave relatively better separation within the long-acting barbiturate group (e.g. phenobarbital and barbital). Figure 1 shows the results obtained for the larger sheets with the use of solvent (i). Comparison of the large and the semi-micro sheets are shown in Fig. 2. Both
290
ITIABA
et al.
TABLE AVERAGE -
Rr AND
R, VALUES
OF THE
I
BARBITURATES
ON INSTANT
Solvent (i) Petroleum ether ethyl acetate, 100: 14 Rf 0.41 0.42 0.56 0.58 0.61
Phenobarbitone Barbitone Amylobarbitone Pentobarbitone Quinalbarbitone
RP 100 103 137 142 149
(ii)
THIN-LAYER
CHROMATOGRAPHY
system:
Benzene acetone,
(iii) 100:2
Rr 0.2s 0.29 0.37 0.37 0.45
RP 100 104 133 133 161
( (
Carbon acetone,
tetrachloride 100:5
Rf 0.28 0.34 0.41 0.40 0.46
RP 100 122 147 143 165
Seconal Amytal
Phenobarbital
2 FIG. 3,4,
5
1. Separation of barbiturates by instant thin-layer chromatography & 5 were the individual barbiturates, 2 the aqueous mixture of these to normal serum.
in solvent (i): Channels and 1 the mixture added
small and large strips were developed consecutively, so that (for example, during emergencies) an immediate result was obtained as to the presence or absence of barbiturates from the semi-micro strips. This was then followed by the result from the larger strips as to the type of barbiturate present. By using different amounts of standard barbiturate solutions and comparing the relative sizes of the spots with the unknown, an approximate indication of the amount of barbiturate
CHROMATOGRAPHIC
METHOD
291
Seconal Amy tal Phenobarbital Seconal
& Amytal
Phenobarbital
Origin
FIG. parison
3.
Separation of barbiturates by instant thin-layer chromatography in solvent of large and semi-micro sheets. Channel 1 wts a mixture of the 3 barbiturates, a mixture of seconal and phenobarbital, and channel 3 seconal.
(i): comchannel 2
present in the unknown sample \\:a~ obtained. The lower limit of sensitivit)of the method was of the order of 5 pg (which corresponds to a serum concentration of about 0.5 mg%). The above points are exemplified in the results shomm in Fig. 3 for an actual case of barbiturate intoxication. Barbiturates lvere sho\vn to be present in this epileptic patient’s serum, peritoneal dialysate, and urine, and approximate serum levels were monitored during the patient’s period in coma (Table II). An additional spot can be observed betlveen the origin and phcnobarbital on this patient’s chromatogram as knell as another extraneous spot occurring close to the solvent front. These were most probably metabolites of the barbiturates (16, 17). The spot between the origin and that of phenobarbital was also observed to have the same RI value as Dilantin, a drug with some structural resemblance to phenobarbital which \\:as kno\vn to have also been taken by this patient. Phenobarbital when added to serum also gave a metabolic derivative that had the same mobility as Dilantin in this system. It can also be seen from Fig. 3 that Dilantin is well separated from the barbiturates and thus when present with phenobarbital did not interfere with this method, Other phenobarbital mixtures such as Amesec (aminophylline, ephedrine and amylobarbitone), Fiorinal (all>.lbarbituric acid, caffeine, acetylsalicylic acid and phenacetin), Phelantin (dilantin, methamphetamine and phenobarbitone), Tederal (theophylline, ephedrine and phenobarbitone) and Atropine-phenobarbital were checked at levels of 2 mg/ml and caused no interference with the identification of the barbiturates. Doriden (a-phenyl-a-ethyl-glutarimide), a compound closely related to barbiturates gave a spot similar to Seconal (Rr values of O.Gl) in the petroleum ether-ethyl acetate solvent system, however it was jvell separated from the barbiturates in benzeneacetone (R, value of 0.84) and carbon tetrachloride-acetone (RI value of O.‘iG) solvent systems.
292
ITIABA
et al.
Phenobarbital Dilantin
Origin
1
2
4
3
6
5
7
FIG. 3. Instant thin-layer chromatography in solvent (i) of extracts of body fluids from a patient under treatment for barbiturate overdosage. Channels 1, 2, 3 represent 2, 5, & 20 al from serum, channel 4 represents 5 pg standard phenobarbital, channels 5 h 6 represent 5 19 20 ~1 from urine, and channel 7 represents 25 pl from peritoneal dialysate extract respectively.
TABLE ESTIMATION OFTHE PATIENT BY MEANS CHROMATOGRAPHY Day 1
2 5 6 7 8 11 12
II
SERUM BARBITURATE OF A OF INSTANT THIN-LAYER (ASGIVENINMETHODS) Cont.
(mg%)
5.0 4.0 2.0 1.7 E 0.8 0.6
(17,22,23) have shown that there is a poor correlation between the serum barbiturate level and the degree and duration of coma in a patient. However, information about the gross levels of barbiturate in the plasma are valuable in the therapeutic management of the patient and subsequent studies of plasma barbiturate concentrations demonstrate the effectiveness, or otherwise, of the therapeutic measures. Future studies may show that quantitative measurement of barbiturate after separation from intermediary metaboli tes is better correlated with the patient’s clinical course than the apparent barbiturate concentration based on gross calorimetric measurements. Previous
studies
CHROMATOGRAPHIC
METHOD
293
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