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Rapid extraction method for barbiturates from blood for gas-liquid chromatographic analysis
MICROCHEMICAL
JOURNAL
Rapid
8, 28-34
Extraction Blood
for
(1964)
Method
for
Gas-Liquid
Barbiturates
from
Chromatographic
Analysis NARESH C. JAIN~ CHARLES R. FONTAN, AND PAUL L. KIRK School
of
Criminology,
University Received
of
California,
March
9, 1961
Berkeley,
California
INTRODUCTION The high incidence of barbiturate poisoning makes desirable a rapid screening method for blood analysis, which could be used with living patients as a confirmation of an overdose of barbiturate, as well as with death cases in which it is necessary to determine the cause of death. A rapid method by which the barbiturate could be identified, and, if necessary, quantitated, with use of small blood samples could serve to eliminate barbiturates as a causal factor, or to focus attention on them for more extended consideration. Gas chromatographic analysis of barbiturate solutions (4, 6, 8) is rapid and reliable for identification of individual barbiturates alone and in mixtures, and with proper calibration of the analytical conditions, for quantitative analysis within the necessary toxicological accuracy. A major fraction of the time necessary for such analyses is involved in the extraction steps conventionally used, in which the pH of the sample must be modified, and is ordinarily combined with multiple extractions from both acidic and basic solution (3, 5). It is necessary to minimize these time-consuming steps if a rapid method is to be devised, especially where a patient is unconscious and treatment may await the results of the analysis. With this point in mind and the fact that it is not desirable to draw large blood samples from living patients, especially from children and those who are in poor physical health, a micromethod which is capable of utilizing blood drawn from the finger tip is ideal. This paper describes a method which is adapted to finger tip blood, and utilizes a very simple extraction procedure suitable for combination with gas-liquid chromatography. The method does not require any adjustment of the blood pH, vigorous shaking of the 28
RAPID
EXTRACTION
OF
BARBITURATES
FROM
BLOOD
29
blood. or any special equipment, and is therefore suitable for emergency use as well as for screening of samples for barbiturate content. The method is fast and capable of detecting barbiturates in submicrogram quantities. .Although fairly impure extracts have been screened for barbiturates and other drugs (6), the method presented here almost completely eliminates the chromatographic background obtained with such extracts and thus facilitates the interpretation of results where the concentration of the drug is very low. By the substitution of pure oxygen for the air which is conventionally used with the fame detector, the chromatographic sensitivity was increased 4 times, permittin g the use of proportionately smaller blood samples for the analyses. Although the spectrophotometric methods that utilize a shift in pH (Bradford and Brackett, Jr., unpublished data; 1, 2, 3) are more specific in identification of barbiturates, they are more laborious than the method proposed here and generally fail to identify the particular barbiturate, the duration of whose action for interpretation or treatment may be very important. Spectrophotometric methods require larger blood samples! 1 ml or more, but in actual practice 5 ml of blood is commonly used. Although 400 nliters of blood was used in accumulating most of the data, only l/SO of the barbiturate present in this sample was actually gas chromatographed. To verify the applicability of the method for readily obtained finger blood specimens: additional tests were made with as little as 50 lditers of blood with essentially the same results. EXPERIMENTAL
.4pparatus. The Hy-Fi gas chromatograph, Aerograph model 600 (Wilkens Instrument and Research Co., Walnut Creek, California), with hydrogen flame ionization detector and the Leeds and Northrup Speedomax H.O-1 m\’ recorder, model S, were used. The chromatographic column (7) was a stainless steel tube of pi inch o.d., 0.093 inch i.d., and 2% feet in length. It was packed with acid-washed lOO- to 120-mesh firebrick which was coated first with 1.5% (w/w) SE 30 and then with 2?; (w/w) Carbowax 20RI. The stationary phase was prepared in two steps. A slurry of firebrick and SE 30 in chloroform was prepared, the solvent being evaporated quickly in a steam bath under vacuum. When dry the material was mixed with a solution of Carbowax 2031 in chloroform and treated in the same manner.
30
N. C. JAIN,
C. R. FONTAN,
AND
P. L. KIRK
The operating conditions were: oven temperature, 230” C: injector temperature, 250” C: fow rate of carrier gas (nitrogen), 40 cc per minute; hydrogen flow rate, 52.2 cc per minute; and oxygen flow rate, 171 cc per minute. The air which is customarily used for the hydrogen flame was replaced with oxygen to increase the sensitivity of the instrument. Disposable polyethylene centrifuge cones of about l-ml capacity (Microchemical Specialties Co., Berkeley, California) were used for the extraction. Any small test tube could be employed. Solvents and samples. Acetone and ethyl ether (A.R.) were mixed in equal volume for use in extraction. The sodium salts of amobarbital, barbital, butabarbital, pentobarbital, phenobarbital, and secobarbital were used. Five postmortem blood samples, known from spectrophotometry to contain barbiturates, were also used. Blood samples obtained from 45 normal individuals and containing EDTA (ethylenediamine tetraacetic acid), added as an anticoagulant, were used to determine the gas chromatographic background response. Procedure. Approximately 50 samples were prepared by dissolving weighed quantities of barbiturate in blood, the concentration being 50 ppm (w/v) for sodium phenobarbital and 10 ppm (w/v) for each of the other five drugs. As a matter of convenience 400 yliters of whole blood drawn from patients with no pH adjustment was transferred to a centrifuge cone to which was then added 500 pliters of mixed solvent. The mixture was stirred with a toothpick for about 10 seconds, after which the clear supernatant was transferred with a Pasteur pipette to a S-ml beaker. This process was carried out four times, fresh solvent being used each time and all four supernatants being combined in the beaker. During the extractions the beaker was kept on top of the gas chromatograph, and its contents were evaporated to dryness with the help of an aspirator. The whole procedure took only a few minutes. The resulting residue was taken up to 50 pliters of 95% ethanol, and a l-pliter aliquot was injected into the gas chromatograph. Because it was apparent that considerably smaller blood samples could be used, further tests were made, starting with 0.1 ml samples, and finally using 0.05 ml samples containing seco-, amo-, and pentobarbital. The extracted barbiturate was dissolved in correspondingly smaller volumes of ethanol, down to 10 pliters, and injected into the gas chromatograph in 1-pliter samples. The results were equally good with these smaller samples when the attenuation of the instrument was diminished. For reasons of comparison these smaller samples were the same
RAPID
EXTRACTION
OF
BARBITURATES
FROM
BLOOD
31
bloods usedin larger samples.Disposablepipettes were not found practical for use with these smaller samples,and were replaced by Kirk pipettes. Evaporation of the extract in a conical tube drawn from a s dram shell vial was found to be convenient. RESULTS
If the original concentration of barbiturate in the blood was 10 ppm, 400 Iditers would contain a total amount of 4 ug of the drug. Assuming that the barbiturate was completely extracted in the manner described, and then taken up to 50 uliters of 9556 ethanol, 1 pliter of this ethanolic solution would contain 0.08 I~lgof the barbiturate. However, an injection of 1 yliter of this solution gave a peak height that was ZO-257/c of scale width, indicating that a recovery of 85-90yC’ was obtained. This was calculated from known standards and also checked by the results of the standard classical procedure described earlier. This means that with this percentage of recovery: only about 0.07 Itg of the barbiturate was present in the 1 uliter injected. Results from the five postmortem samplesalso showed that the extraction of barbiturate was 8590% complete. The percentages of barbiturates in these sampleswere determined by a standard extraction method in combination with a spectrophotometer, and the results were compared with the values obtained gas-chromatographically with the simple extraction procedure. A typical chromatogram produced from a blood sample containing 10 ppm of sodium pentobarbital and 10 ppm of sodium secobarbital is shown in Fig. 1. Figure 2 showsa typical chromatogram obtained from a normal blood samplecontaining EDTA. Judged on the basisof retention time and symmetry: none of the 45 bloods tested without barbiturate produced a peak which could be interpreted as representing a barbiturate. The peak registered at 1.1 minutes was produced from all the blood samplestested. DISCUSSION
Most conventional methods (Bradford and Brackett, Jr., unpublished data; 1-3) require pH changes, shaking, and larger blood samples than we have used, which in turn require large volumes of solvent (e.g., 60 ml). As a result, conventional procedures are much more laborious and time consuming. The method described here diminishes all of these difficulties. The avoidance of a pH change is one of the vital factors in the successof this method. When the sample is acidified and extracted, the
32
N.
C. JAIN,
C. R. FONTAN,
Retention FIG. 1. Separation ot’ barbiturates. tion: X 2.
AND
P.
L.
KIRK
Time (minutes) (A) Pentobarbital;
(B) secobarbital. Attenua-
extract becomes contaminated with hemoglobin and probably its decompositionproducts. An additional advantage of selectivity toward the group of compound extracted is obtained where the extraction is made at neutral pH, since the more acidic drugs will not be well extracted. The omission of shaking is also important since it also causeshemolysis and tends to cause emulsions with putrefied blood samples. This procedure did not produce emulsionseither with fresh or putrefied blood. When 10 cc of Hz0 was shaken with 10 cc of the solvent mixture, it was observed that the aqueousphase increased in volume due to part of the solvent mixture going into the water phase. A similar phenomenon was noted when a sample of blood was stirred with the solvent mixture: the blood increasedin volume and the solvent volume decreased,which indicates a partial saturation of the blood with acetone and ether. With subsequent extractions, the acetone and ether, already in the blood from the first extraction, are equilibrated with fresh solvent and carry the dissolved
organic
RAPID
t
, 0
EXTRACTION
, I
Retention
, 2345678 Time
OF
BARBITURATES
,
FROM
(
,
33
BLOOD
, 9
, IO
, II
12
(minutes)
FIG. 2. Normal backaround response of blood.
barbiturates with them, and are removed with the pipette. Depending on the barbiturate, and its pK, a given amount will be present in the blood as the free acid. With each extraction: as the latter is removed by the solvent, the equilibrium shifts to give more free acid which is subsequently removed. The free acid form has a partition coefficient which is favorable to the organic extraction solvent the magnitude of which is large enough to remove 85590% of the drug in 4 extractions. If metabolites of the barbiturates are present, they will usually be in the form of glucuronides or sulfates, or as oxidized compounds. These either will not be extracted or: on the column employed, will have retention times long enough so as not to interfere with the barbituric acid peaks. Where metabolism converts the ingested barbiturate to another similar barbiturate, peaks from both acids will be observed. This was observed. for example, with pentothal, which showed a partial conversion to pentobarbital. Another main feature of this method is the replacement of air which is normally used with flame ionization detectors by pure oxygen. This increases the sensitivity 4 times and makes it possible to use very small
34
N. C. JAIN,
c. R. FONTAN,
AND
p. L. KIRK
quantities of blood, a factor which, by decreasing the large volume of solvent generally used in conventional methods, simplifies very much the quantity and type of glassware used. In addition, in standard methods where a considerable amount of time is wasted in washing glassware and in evaporation of copious quantities of solvents, here very little time is spent in either washing glassware, as most of it is disposable, or in evaporation of solvents, as the quantities used are very small. SUMMARY
A rapid micromethod for the simple extraction and tentative identification of barbiturates present in blood in therapeutic doses by gas-liquid chromatography is described. The extraction is made with a mixture of acetone and ethyl ether in equal volumes at the pH of blood and takes only a few minutes. Most of the glassware used is simple and disposable, thus saving time which is wasted in conventional methods. Air which is normally used in the flame ionization detector is replaced by pure oxygen, thus increasing the sensitivity four times, and making possible the use of very small samples of blood. The method is capable of detecting barbiturates in submicrogram quantities. ACKNOWLEDGMENTS This investigation was supported by a Public Health Service research grant EF 00021 from the Division of Environmental Engineering and Food Protection, Public Health Service, and by a research grant from the Committee on Research, University of California. REFERENCES 1. 2.
3. 4. 5.
6.
7. 8.
CURRY, A. S., Identification of barbiturates. Nature 133, 1052 (1959). GOLDBAUM, L. R., Determination of barbiturates. Ultraviolet spectrophotometric method with differentiation of several barbiturates. Anel. Chem. 24, 1604-1607 (1952). GOLDBAUM, L. R., An ultraviolet spectrophotometric procedure for the identification of barbiturates. J. Pharmacol. Exptl. Therp. 94, 68-75 (1948). KAZYAX, L., AND KNOBLOCK, E. C., Application of gas chromatography to analytical toxicology. Anal. Chem. 35, 1448-1452 (1963). KOPPANYI, TH., DILLE, J. M., MURPHY, W. S., AND KROP, S., Studies on barbiturates. II. Contributions to methods of barbital research. J. Am. Pharm. Assoc. 23, 1074-1079 (1934). PARKFX, K. D., FONTAN, C. R., AND KIRK, P. L., Rapid gas chromatographic method for screening of toxicological extracts for alkaloids, barbiturates, sympathomimetic amines, and tranquilizers. Anal. Chem. 35, 356-359 (1963). PARKER, K. D., FONTAN, C. R., AND KIRK, P. L., Improved gas chromatographic column for barbiturates. Anal. Chem. 35, 418419 (1963). PARKER, K. D., .AND KIRK, P. L., Separation and identification of barbiturates by gas chromatography. Anal. Chem. 33, 1378-1381 (1961).
JOURNAL
Rapid
8, 28-34
Extraction Blood
for
(1964)
Method
for
Gas-Liquid
Barbiturates
from
Chromatographic
Analysis NARESH C. JAIN~ CHARLES R. FONTAN, AND PAUL L. KIRK School
of
Criminology,
University Received
of
California,
March
9, 1961
Berkeley,
California
INTRODUCTION The high incidence of barbiturate poisoning makes desirable a rapid screening method for blood analysis, which could be used with living patients as a confirmation of an overdose of barbiturate, as well as with death cases in which it is necessary to determine the cause of death. A rapid method by which the barbiturate could be identified, and, if necessary, quantitated, with use of small blood samples could serve to eliminate barbiturates as a causal factor, or to focus attention on them for more extended consideration. Gas chromatographic analysis of barbiturate solutions (4, 6, 8) is rapid and reliable for identification of individual barbiturates alone and in mixtures, and with proper calibration of the analytical conditions, for quantitative analysis within the necessary toxicological accuracy. A major fraction of the time necessary for such analyses is involved in the extraction steps conventionally used, in which the pH of the sample must be modified, and is ordinarily combined with multiple extractions from both acidic and basic solution (3, 5). It is necessary to minimize these time-consuming steps if a rapid method is to be devised, especially where a patient is unconscious and treatment may await the results of the analysis. With this point in mind and the fact that it is not desirable to draw large blood samples from living patients, especially from children and those who are in poor physical health, a micromethod which is capable of utilizing blood drawn from the finger tip is ideal. This paper describes a method which is adapted to finger tip blood, and utilizes a very simple extraction procedure suitable for combination with gas-liquid chromatography. The method does not require any adjustment of the blood pH, vigorous shaking of the 28
RAPID
EXTRACTION
OF
BARBITURATES
FROM
BLOOD
29
blood. or any special equipment, and is therefore suitable for emergency use as well as for screening of samples for barbiturate content. The method is fast and capable of detecting barbiturates in submicrogram quantities. .Although fairly impure extracts have been screened for barbiturates and other drugs (6), the method presented here almost completely eliminates the chromatographic background obtained with such extracts and thus facilitates the interpretation of results where the concentration of the drug is very low. By the substitution of pure oxygen for the air which is conventionally used with the fame detector, the chromatographic sensitivity was increased 4 times, permittin g the use of proportionately smaller blood samples for the analyses. Although the spectrophotometric methods that utilize a shift in pH (Bradford and Brackett, Jr., unpublished data; 1, 2, 3) are more specific in identification of barbiturates, they are more laborious than the method proposed here and generally fail to identify the particular barbiturate, the duration of whose action for interpretation or treatment may be very important. Spectrophotometric methods require larger blood samples! 1 ml or more, but in actual practice 5 ml of blood is commonly used. Although 400 nliters of blood was used in accumulating most of the data, only l/SO of the barbiturate present in this sample was actually gas chromatographed. To verify the applicability of the method for readily obtained finger blood specimens: additional tests were made with as little as 50 lditers of blood with essentially the same results. EXPERIMENTAL
.4pparatus. The Hy-Fi gas chromatograph, Aerograph model 600 (Wilkens Instrument and Research Co., Walnut Creek, California), with hydrogen flame ionization detector and the Leeds and Northrup Speedomax H.O-1 m\’ recorder, model S, were used. The chromatographic column (7) was a stainless steel tube of pi inch o.d., 0.093 inch i.d., and 2% feet in length. It was packed with acid-washed lOO- to 120-mesh firebrick which was coated first with 1.5% (w/w) SE 30 and then with 2?; (w/w) Carbowax 20RI. The stationary phase was prepared in two steps. A slurry of firebrick and SE 30 in chloroform was prepared, the solvent being evaporated quickly in a steam bath under vacuum. When dry the material was mixed with a solution of Carbowax 2031 in chloroform and treated in the same manner.
30
N. C. JAIN,
C. R. FONTAN,
AND
P. L. KIRK
The operating conditions were: oven temperature, 230” C: injector temperature, 250” C: fow rate of carrier gas (nitrogen), 40 cc per minute; hydrogen flow rate, 52.2 cc per minute; and oxygen flow rate, 171 cc per minute. The air which is customarily used for the hydrogen flame was replaced with oxygen to increase the sensitivity of the instrument. Disposable polyethylene centrifuge cones of about l-ml capacity (Microchemical Specialties Co., Berkeley, California) were used for the extraction. Any small test tube could be employed. Solvents and samples. Acetone and ethyl ether (A.R.) were mixed in equal volume for use in extraction. The sodium salts of amobarbital, barbital, butabarbital, pentobarbital, phenobarbital, and secobarbital were used. Five postmortem blood samples, known from spectrophotometry to contain barbiturates, were also used. Blood samples obtained from 45 normal individuals and containing EDTA (ethylenediamine tetraacetic acid), added as an anticoagulant, were used to determine the gas chromatographic background response. Procedure. Approximately 50 samples were prepared by dissolving weighed quantities of barbiturate in blood, the concentration being 50 ppm (w/v) for sodium phenobarbital and 10 ppm (w/v) for each of the other five drugs. As a matter of convenience 400 yliters of whole blood drawn from patients with no pH adjustment was transferred to a centrifuge cone to which was then added 500 pliters of mixed solvent. The mixture was stirred with a toothpick for about 10 seconds, after which the clear supernatant was transferred with a Pasteur pipette to a S-ml beaker. This process was carried out four times, fresh solvent being used each time and all four supernatants being combined in the beaker. During the extractions the beaker was kept on top of the gas chromatograph, and its contents were evaporated to dryness with the help of an aspirator. The whole procedure took only a few minutes. The resulting residue was taken up to 50 pliters of 95% ethanol, and a l-pliter aliquot was injected into the gas chromatograph. Because it was apparent that considerably smaller blood samples could be used, further tests were made, starting with 0.1 ml samples, and finally using 0.05 ml samples containing seco-, amo-, and pentobarbital. The extracted barbiturate was dissolved in correspondingly smaller volumes of ethanol, down to 10 pliters, and injected into the gas chromatograph in 1-pliter samples. The results were equally good with these smaller samples when the attenuation of the instrument was diminished. For reasons of comparison these smaller samples were the same
RAPID
EXTRACTION
OF
BARBITURATES
FROM
BLOOD
31
bloods usedin larger samples.Disposablepipettes were not found practical for use with these smaller samples,and were replaced by Kirk pipettes. Evaporation of the extract in a conical tube drawn from a s dram shell vial was found to be convenient. RESULTS
If the original concentration of barbiturate in the blood was 10 ppm, 400 Iditers would contain a total amount of 4 ug of the drug. Assuming that the barbiturate was completely extracted in the manner described, and then taken up to 50 uliters of 9556 ethanol, 1 pliter of this ethanolic solution would contain 0.08 I~lgof the barbiturate. However, an injection of 1 yliter of this solution gave a peak height that was ZO-257/c of scale width, indicating that a recovery of 85-90yC’ was obtained. This was calculated from known standards and also checked by the results of the standard classical procedure described earlier. This means that with this percentage of recovery: only about 0.07 Itg of the barbiturate was present in the 1 uliter injected. Results from the five postmortem samplesalso showed that the extraction of barbiturate was 8590% complete. The percentages of barbiturates in these sampleswere determined by a standard extraction method in combination with a spectrophotometer, and the results were compared with the values obtained gas-chromatographically with the simple extraction procedure. A typical chromatogram produced from a blood sample containing 10 ppm of sodium pentobarbital and 10 ppm of sodium secobarbital is shown in Fig. 1. Figure 2 showsa typical chromatogram obtained from a normal blood samplecontaining EDTA. Judged on the basisof retention time and symmetry: none of the 45 bloods tested without barbiturate produced a peak which could be interpreted as representing a barbiturate. The peak registered at 1.1 minutes was produced from all the blood samplestested. DISCUSSION
Most conventional methods (Bradford and Brackett, Jr., unpublished data; 1-3) require pH changes, shaking, and larger blood samples than we have used, which in turn require large volumes of solvent (e.g., 60 ml). As a result, conventional procedures are much more laborious and time consuming. The method described here diminishes all of these difficulties. The avoidance of a pH change is one of the vital factors in the successof this method. When the sample is acidified and extracted, the
32
N.
C. JAIN,
C. R. FONTAN,
Retention FIG. 1. Separation ot’ barbiturates. tion: X 2.
AND
P.
L.
KIRK
Time (minutes) (A) Pentobarbital;
(B) secobarbital. Attenua-
extract becomes contaminated with hemoglobin and probably its decompositionproducts. An additional advantage of selectivity toward the group of compound extracted is obtained where the extraction is made at neutral pH, since the more acidic drugs will not be well extracted. The omission of shaking is also important since it also causeshemolysis and tends to cause emulsions with putrefied blood samples. This procedure did not produce emulsionseither with fresh or putrefied blood. When 10 cc of Hz0 was shaken with 10 cc of the solvent mixture, it was observed that the aqueousphase increased in volume due to part of the solvent mixture going into the water phase. A similar phenomenon was noted when a sample of blood was stirred with the solvent mixture: the blood increasedin volume and the solvent volume decreased,which indicates a partial saturation of the blood with acetone and ether. With subsequent extractions, the acetone and ether, already in the blood from the first extraction, are equilibrated with fresh solvent and carry the dissolved
organic
RAPID
t
, 0
EXTRACTION
, I
Retention
, 2345678 Time
OF
BARBITURATES
,
FROM
(
,
33
BLOOD
, 9
, IO
, II
12
(minutes)
FIG. 2. Normal backaround response of blood.
barbiturates with them, and are removed with the pipette. Depending on the barbiturate, and its pK, a given amount will be present in the blood as the free acid. With each extraction: as the latter is removed by the solvent, the equilibrium shifts to give more free acid which is subsequently removed. The free acid form has a partition coefficient which is favorable to the organic extraction solvent the magnitude of which is large enough to remove 85590% of the drug in 4 extractions. If metabolites of the barbiturates are present, they will usually be in the form of glucuronides or sulfates, or as oxidized compounds. These either will not be extracted or: on the column employed, will have retention times long enough so as not to interfere with the barbituric acid peaks. Where metabolism converts the ingested barbiturate to another similar barbiturate, peaks from both acids will be observed. This was observed. for example, with pentothal, which showed a partial conversion to pentobarbital. Another main feature of this method is the replacement of air which is normally used with flame ionization detectors by pure oxygen. This increases the sensitivity 4 times and makes it possible to use very small
34
N. C. JAIN,
c. R. FONTAN,
AND
p. L. KIRK
quantities of blood, a factor which, by decreasing the large volume of solvent generally used in conventional methods, simplifies very much the quantity and type of glassware used. In addition, in standard methods where a considerable amount of time is wasted in washing glassware and in evaporation of copious quantities of solvents, here very little time is spent in either washing glassware, as most of it is disposable, or in evaporation of solvents, as the quantities used are very small. SUMMARY
A rapid micromethod for the simple extraction and tentative identification of barbiturates present in blood in therapeutic doses by gas-liquid chromatography is described. The extraction is made with a mixture of acetone and ethyl ether in equal volumes at the pH of blood and takes only a few minutes. Most of the glassware used is simple and disposable, thus saving time which is wasted in conventional methods. Air which is normally used in the flame ionization detector is replaced by pure oxygen, thus increasing the sensitivity four times, and making possible the use of very small samples of blood. The method is capable of detecting barbiturates in submicrogram quantities. ACKNOWLEDGMENTS This investigation was supported by a Public Health Service research grant EF 00021 from the Division of Environmental Engineering and Food Protection, Public Health Service, and by a research grant from the Committee on Research, University of California. REFERENCES 1. 2.
3. 4. 5.
6.
7. 8.
CURRY, A. S., Identification of barbiturates. Nature 133, 1052 (1959). GOLDBAUM, L. R., Determination of barbiturates. Ultraviolet spectrophotometric method with differentiation of several barbiturates. Anel. Chem. 24, 1604-1607 (1952). GOLDBAUM, L. R., An ultraviolet spectrophotometric procedure for the identification of barbiturates. J. Pharmacol. Exptl. Therp. 94, 68-75 (1948). KAZYAX, L., AND KNOBLOCK, E. C., Application of gas chromatography to analytical toxicology. Anal. Chem. 35, 1448-1452 (1963). KOPPANYI, TH., DILLE, J. M., MURPHY, W. S., AND KROP, S., Studies on barbiturates. II. Contributions to methods of barbital research. J. Am. Pharm. Assoc. 23, 1074-1079 (1934). PARKFX, K. D., FONTAN, C. R., AND KIRK, P. L., Rapid gas chromatographic method for screening of toxicological extracts for alkaloids, barbiturates, sympathomimetic amines, and tranquilizers. Anal. Chem. 35, 356-359 (1963). PARKER, K. D., FONTAN, C. R., AND KIRK, P. L., Improved gas chromatographic column for barbiturates. Anal. Chem. 35, 418419 (1963). PARKER, K. D., .AND KIRK, P. L., Separation and identification of barbiturates by gas chromatography. Anal. Chem. 33, 1378-1381 (1961).