The determination of barbiturates and some related drugs by gas chromatography

CLINICA CHIMICA ACTA

THE

195

DETERMINATION

DRUGS

HENRY

BY

OF BARBITURATES

AND

SOME

RELATED

GAS CHROMATOGRAPHY

LEACH

Biochemistry

Dept.,

C 6 A General

Hospiial,

Bangor,

Caerns,

AND P. A. TOSELAND* Biochemistry

Department,

(Revised manuscript

Royal

Infirmary,

Sheffield (U.K.)

received January 16, 1968)

SUMMARY

Details of a technique for the determination of barbiturates and for the identification of some related drugs in blood are described. Results obtained by the comparison of spectrophotometric assay and gas-chromatographic determination of barbiturates indicate that the use of chloroform as extracting solvent at approximately neutral pH gives extracts which do not contain any significant quantity of the more polar barbiturate metabolites. Some evidence is also given that diethylbarbituric acid may be found in blood extracts from cases of barbiturate intoxication when the drug has an ethyl substituent.

The frequency with which barbiturates the necessity to identify individual members

are found in analytical toxicology and of a large group of substances of similar

chemical structure has resulted in a number of studies using gas chromatography1-5. None of the techniques was capable of separating all the common barbiturates on one column and the use of low concentrations of stationary phase led to problems due to peak tailing. In an attempt to overcome this, Cieplinskie added very small amounts of the more polar compounds, dimer and trimer acids, to SE-30 and neopentyl glycol succinate columns and although this resulted in a substantial improvement, only a few barbiturates were reported. The most comprehensive separations were obtained by Parker and Kirk4 and by Brochmann-Hanssen and Svendsen 5 the latter using a number of columns of differing characteristics. These were all low-loaded columns of very high efficiency. Attempts to reproduce these results were erratic and it became apparent that to make columns of a sufficiently high efficiency it was necessary to prepare a number of batches of coated support and columns and select only the best if good results were to be obtained. * Present address:

Department

Clinical Pathology,

Guy’s Hospital,

London,

Clin. Chin?. Acta,

S.E. I. 20 (1968)

195-za3

LEACH

196

This approach was impractical in a busy hospital laboratory and it was decided to explore the possibility of using more heavily loaded columns. Parker and Kirk4 had used 5% SE-30 columns with good results but the resolution of amylobarbitone, pentobarbitone and quinalbarl)itone was not good and, since these three substances were the agents in over 50% of our cases of barbiturate poisoning, it was felt that the main prerequisite of any column was its ability to separate these substances unequivocally. After a number of experiments with Apiezon L, XE-60 and Carbowax columns the final choice was a 10% Apiezon L column. One of our early 10% Apiezon columns has been in use since September 1964 and no significant change in behaviour has been noticed in over z years. In the early stages of this work we became aware by a personal communication of Drost that work on barbiturates using Apiezon columns was in progress by Reith and co-workers in Holland. This work was subsequently pubTABLE I RETENTION TIMES OF SOME BARBITURATES __ ~ ~_._. __--.--

AND

_-

NEUTRAL

DRUGS

Substance

Barbitone (5,5-diethyl) Allobarbitone (5,5-diallyl) Butobarbitone (5-ethyl-5-~~-butyl) Butabarbitone (5ethyl-5-(I-methyl Amylobarbitone (5.ethyl-5-isoamyl)

1.0

0.*Z+

1.47 I .84 I.94 2.14

0.2 I 0.26 0.28 0.31

Nealbarbitone (5-allyl-5-neopentyl) Pentobarbitone (5-ethyl-5-(z-methyl butyl)) ~uinalbarbitone (5.allyl-5-(r-methyl butyl)) Hexobarbitone (1,5-dimethyl-5-(cyclohexen-I-yl)) “Prominal” (Methylphenobarbitone) (g-ethyl-gphenyl-r-methy!)

2.35 2.55 3.02 4.04 4.79

0.34 0.37 0.43

Rutonal (5-nleth~71-5-phenyl) Phenobarbitone (5.ethyl-5-phenyl) Cyclobarbitone (5-cyclohexen-I-yl-5-ethyl) Medomin (5-(cyclohepten-I-yl)-5-ethyl) 3-Hydroxyamylobarbitone

5.6

0.8

bother

weak

propyl))

6.93 7.’

IO.5

4,4

0.58

0.69

1.0 I .05 1.5 0.63

acids

Megimide Methyl-p-hydroxybenzoic acid Salicylamide p-Nitrophenol Vanillic acid diethylamide

0.62

0.93 1.1 j:&

0.53

New&al o&g fraction Carbromal Ethinamate Methyprylone (Noludar) Phenacetin ~lutethimi~e Caffeine Yhenazone (Antipyrine) Tybamate Methaqualone

-. .__

0.31 0.49 1.25 2.25 4.1 4.38 5.2 5.6 IO.8

Conditions: Apiezon L TOO/,.Carrier gas flow 50 ml/min. Flame Air 300 ml/min. Column temperature: 210’. C&k. C&m. Acta, 20 (1968) 195-203

0.18 0.32 0.59 0.63 0.75 0.8

1.56 ionisation

hydrogen

50 ml/min.

GAS CHROMATOGRAPHY

OF BARBITURATES

197

lished’. Their results were very similar to ours except that we have consistently adequately resolved cyclobarbitone from phenobarbitone whereas they used a permanganate oxidation method to differentiate these two compounds. This could be a peculiarity of the batch of Apiezon we have used but these substances can be readily resolved on 3% XE-60 columns. Retention data for a number of common barbiturates and neutral drugs are given in Table I. It was obvious from considerations of the behaviour of these more heavily loaded columns that retention times for substances of high molecular weight would be somewhat prolonged but it was felt that the good separations, particularly of some of the neutral drugs, outweigl~ed the moderate increase in the time taken for analysis. In any event, gas chromatography using this type of column was still very rapid and in comparison with other methods of chromatography, much more precise. We did consider the possibility of using rather lower loaded columns and running these under temperature-programmed conditions, but the difficulty in obtaining reproducible retention data for the purpose of identification led us to reject this method. In the normal course of analysis, the barbiturates, in particular, would be quantitated and the structure identified by spectrophotometry and gas chromatography would be used only to identify individual members of the barbiturate family. In addition to this we were also concerned with the identification of those drugs which appeared in the same solvent extracts but which had poor ultra-violet spectral characteristics. The possibility did exist, however, that it might be necessary to use only gas chromatography, and therefore the method was investigated to determine whether a satisfactory quantitation could be achieved during a gas chromatography run of extracts from the small amounts of blood which are available in hospital toxicology. MATERIALS

AND METHODS

Two gas chromatographs were used for the bulk of this work. One was a Pye Argon using a %r p ionisation detector; it was fitted with 4’ x 0.25” O.D. (1.2 m x 6 mm) glass columns. The second instrument was a Pye Panchromatograph with a flame ionisation detector. The columns were glass 5’ x 0.25” (1.5 m x 6 mm). Argon was the carrier gas for both instruments and column temperatures were in the range 200” to 225’. Details of the exact conditions are given in the text. Packed columns 25 g go-mo mesh Chromosorb W was washed with acid by suspending the material in several volumes of concentrated hydrochloric acid and stirring gently for 2 11.The acid was decanted and the material washed by decantation using several lots of water until the reaction was neutral. The acid and the water were poured off as soon as the bulk of the material had deposited. The fmer particles remained suspended and were removed by this process. The washed material was filtered by suction and after drying at IIOO was suspended in about 200 ml of a 2% solution of DMCS in toluene in a filter flask. A vacnumwas applied to assist penetration of the solution and after standing for several hours with occasional gentle agitation the suspension was filtered by suction and washed with toluene followed by methanol, It was then dried at 110~. CEilz. Ck+?z. A&z,

20 (1968)

195~203

LEACH

19s

22.5g of the silanised support was treated with 2.5 g of Apiezon L grease dissolved in hcxane. The hexane was evaporated off under vacuum at 60” keeping the flask gently agitated during the evaporation. The dry powder was packed into the glass columns by suction and vibration. These were then conditioned by heating at 2~5” for 48 h with a very slow flow of carrier gas. Extraction and U.V. s$ectro$hotonzetry Two very similar methods of extraction were used in this work. The first employs extraction of 5 ml of blood with 27 ml of chloroform in the presence of I ml of 0.5 M pH 6.5 phosphate buffer. 20 ml of the chloroform is removed after centrifuging and extracted with 5 ml of 0.45 N sodium hydroxides. Because of the smaller total volumes this method is useful when a large centrifuge is not available and it also gives a more concentrated final extract, a useful feature when low barbiturate concentrations are encountered. In the second method 4 ml of blood, I ml pH 7.4 buffer and 30 ml of chloroform are shaken in a roe-ml flask. Solid anhydrous sodium sulphate is added until the water is taken up. The chloroform is decanted and filtered through Whatman No. 43 paper into a 5o-ml centrifuge tube. The blood-sodium sulphate cake is washed with a further 20 ml of chlorofornl and the extracts are pooled and shaken with 8 ml of 0.45 iV sodium hydroxide. The alkali extracts from either method of extraction are then examined by spectrophotometry using Broughton’s method 9. We also make the pH IO extract acid with 2 drops of concentrated hydrochloric acid and run the curve again at this pH of below 2. Concentrations are then calculated as follows: Podmwe extraction E,,, pH 13-E,,, E,,, pH IO-E,,,

pH IO x 10.8 - mg/~oo ml blood 5.94 = mg/roo ml blood

pH 2 x

L140dQ2d~~o~~~to~~ extrnction E,,, pH 13-&~~ pH TOx 16 = mgjroo ml blood E,,, pH IO-E~,, pH 2 x 8.8 = q/100 ml blood These factors are a compromise for the various common barbiturates. For complete accuracy the exact extinction factor should be usedsslo, All the spectrophotometr~ was done on an Optika CF 4R or a Unicam SP 800 recording spectrophotometer using matched I-cm silica cells. Usually the spectrum is recorded between 220 and 420 rnp. All the solutions from the spe~trophotometry and the remainder of the alkali extract are pooled, acidified with HCl and extracted with 2 lots of IO ml of ether. The ether is dried with sodium sulphate and evaporated to dryness. Solid

injection

If the level in blood exceeds 3.0 mgjroo ml the residue is dissolved in I ml methanol or 0.5 ml if below 3.0 mg/roo ml. 50 ~81is transferred to a small capsule of glass or metal packed with glass wool, with a diameter which will allow it to be dropped into the column. After evaporating off the methanol the capsule is dropped into the flash heater zone of the gas chromatographll.

CIh. Cl&a

Acfa, 20 (1968) 19.5-203

GAS CHROMATOGRAPHY

OF RARRITURATES

199

Liquid injection The residue from the ether is transferred to a small tapered glass tube (a “Dreyer’s” tube is ideal) and the transferring solvent evaporated off in a current of air. 50 ~1 of methanol is added to dissolve the residue and 2 ~1 is injected into the instrument with a Hamilton syringe. Immediately before or after the sample has been chromatographed a solution of barbitone or pl~enobarbitone (I mgjml in chloroform) is also run and the relative retention time is calculated. The barbiturate is then provisionally identified from the data in Table I. Calcdatio~z of peak areas After provisionally identifying the barbiturate, aliquots of a standard barbiturate solution are injected until two consecutive peak heights are the same. The peak area of the standard and the test are calculated using the method approved by the Tar Products Test Committee 12, A base line is constructed from the base of one peak to the beginning of the next and the area calculated from peak height x width at half peak height. The identity can then be confirmed by the relation of the peak to that of the standard. The standard solutions contain I mgjml in chloroform. They are stable for over IZ months when kept at -20~. The use of barbitone and phenobarbitone as standards is sufficiently accurate for hospital toxicology. If greater accuracy is required it is better to use a standard barbiturate which has a retention time close to that of the barbiturate which has been identified. This gives peaks of almost identical geometry and this makes peak area calculation simpler. It can also give more certain proof of identity. Table II givesacomparison of some spectrophotometric and gas chromatographic determinations from blood samples and these figures were determined by correcting the spectrop~lotometric values for the molar absorbance of the individual barbituratelo.

TABLE

II

COMPARATIVE

DETERMINATIONS

IN

BLOOD

Barbiflwate

SAMPLES

______

--_.___

-- -.-.-.

Awuttnzt determined mg/roo mt ~.________.._~ “. _ I. --__ Spt?CtR?phOtQ?%&p Gas chromatogwp~y

Quinalbarbitone Pentobarbitone Amylobarbitone Pentobarbitone & Quinalbarbitone Pentobarbitone

*.a I.3 I.9 2.2 34

0.8 I.7 2.2 2.8 (Pento 1.6) (Quinal 1.2) 2.9

Butobarbitone Phenobarbitone Butobarbitone Amylobarbitone Amylobarbitone

4.’ 4.’ 4.6 4.9 5.0

3.5 3.7 3-8 4.3 6.1

Plienobarbitone Amylobarbitone Atnylobarbitone Phenobarbitone

5.8 7.6 11.4 13.5

5.2 6.8

Pentobarbitone

& Quinalbarbitone

I.9 ___--

II.7

11.8 2.5

(Pento

1.6)

(Quinal --.

0.9)

* The spectrophotometric results have been corrected on the basis of the absorbance of the individual barbiturates as described by Stevensonlo. C&n. China. Acta, 20

(1~68)

Ig5-203

200

LEACH

Gas chromatography was done using a barbiturate with a similar retention time as a standard as mentioned above, pentobarbitone was used for the short retention time barbiturates, unless the drug was pentobarbitone, for which amylobarbitone was used. ~yclobarbitone was used for phenobarbitone deternlinations. Recently, one of us (ILL.) has been investigating the calculation of peak areas using a Kent Chromalog integrator. Results are promising but mixtures of barbiturates giving peaks close together present some difficulties. For this purpose, an initial injection of the methanol solution of the residue is made to exclude the presence of phenobarbitone. The methanol is evaporated off and the residue redissolved in 50 pl of a o.zo/, solution of phenobarbitone or barbitone in chloroform. Aliquots of this are injected and the concentration of the test solution calculated from the print-out.

With low barbiturate levels from blood samples other peaks appear or1 the chromatogram. These peaks are also found in control blood samples taken from laboratory workers. These peaks have shorter retention times than barbitone and are usually only noticeable using solid injection. With liquid injection most of them occur in the solvent peak. A number of other drugs are extracted by chloroform under neutral or slightly acid conditions. These include some phenolic compounds, the neutral drugs and also a number of bases. The drugs which may be expected to appear in the sodium hydroxide extract together with any barbiturate include megimide, methyl-~-hydro~ybenzoate, salicylanlide, ~-llitropbenol and phenylbutazone. Retention data for these are given in Table I. If neutral drugs are suspected the chloroform remaining from the initial extraction should be evaporated to dryness and the residue dissolved in a small volume of methanol. Aliquots of this are injected. The neutral drugs may include camphor, meprobamate, methyprylone (Noludar), phenacetin, phenazone, glutethimide, caffeine and tybamate. If stomach contents have been extracted, carbromal may also be found. The appearance of bases in extracts which are usually considered to contain only the acidic and neutral drugs is a problem which is often neglected. Many bases will appear in the original chloroform extract and may be included in the “neutral drug” extract. Bradford and Bracketti3 give a number of bases which appear in more than 50:/o yield in the chloroform extract. These include antihistamines, local anaesthetics, narcotics and some classical alkaloids including, for example, strychnine; of this over 70% app ears in the chloroform extract. Apart from problems due to interference the re-extraction of the original blood at an alkaline pH for bases may lead to their being missed. Although the chloroform can be washed with acid to remove bases, some of these are only quantitatively extracted with comparatively strong acid solutions and the possibility of bases appearing in the neutral extract should not be overlooked. An acid wash may remove base interference in the neutralgroup; even if the drug is not extracted, the salt formed may not elute during gas chromatography. Sulphuric acid is better than hydrochloric acid for this purpose. A word of caution is necessary regarding the use of a single column for both basic and acidic substances. This is undesirable and it has been found in practice than non-emerging acids may remain on the column and completely trap bases and vice-versa, and although a reconditioning at 250~ for 48 h may restore

GAS CHROMATOGRAPHY

OF BARBITURATES

201

Fig. 1. Representative chromatogram of a number of common barbiturates. zo5’, 50 ml/min carrier, 50 ml/min hydrogen.

the column,

it is better

to use a second

examined. Of the newer non-barbiturate

column

hypnotics,

if post-mortem methaqualone

10%

Apiezon L,

extracts

are to be

is readily

extracted

from acid solution and will appear in the neutral group. Chlordiazepoxide is converted in the body to the lactam which can be extracted with the barbiturates but the retention time is very long on Apiezon L and no interference from this source is to be expected. Diazepam and Oxazepam, two related compounds, also have very long retention times. In two cases of Oxazepam intoxication a peak with a relative retention time to barbitone of 0.2-0.3 was found. This has not been identified and could, of course, be co-incidental. One peak which is occasionally

found in chromatograms

of blood

extracts

corresponds with that of barbitone and there is some, far from conclusive, evidence to suggest that this might be its true identity: I. It does not appear in normal control blood extracts and it has not been detected in the solvents used for extraction. 2. When the barbiturate is a short- or medium-acting

drug, the peak height

increases relative to the main barbiturate when successive analyses are done over a period of a few days. This behaviour suggests the presence of a small quantity of a slowly eliminated barbiturate. 3. When subjected to ion exchange chromatography on Dowex co1umns*4, it appears in the same fraction as diethylbarbituric acid. 4. This peak has only been found when the main barbiturate has an ethyl substituent. It is possible that some dealkylation may take place in the body, but there is also a possibility that traces of diethylbarbituric some commercial barbiturate preparations. The degradation of the main barbiturate unlikely,

for reduction

of the column temperature

acid may appear as an impurity during gas chromatography only produces longer retention Clin.

Chim.

Acta,

20 (1968)

in

seems times 195-203

202

LEACH

and the relative

amounts

of the main barbiturate

and this compound

remain

the

same. Further to establish

work is in progress in an attempt

to identify

this peak conclusively

and

its origin.

Some doubt has recently been expressed15>1B on the validity of blood barbiturate determinations in view of the possibility that the presence of pharmacologically inactive metabolites

might give misleading

spectrophotometry

results. Comparison

and by gas chromatography

good. If metabolites

with an intact

barbiturate

of the results obtained

shows that agreement structure

by

is reasonably

were extracted

under the

conditions described, the levels found by spectrophotometry should be considerably higher than those found by gas chromatography, unless the gas chromatographic behaviour of the metabolite was identical to that of the parent compound. Studies with the major metabolite of amylobarbitone show that the 3-hydroxy compound has a different retention time, on a number of different columns, from the parent barbiturate and it seems reasonable to assume that the hydroxy and acetoxy metabolites of other barbiturates could also be expected to exhibit different behaviour. If any substantial proportion of the barbiturate extracted from the blood were a metabolite, then either a second peak should be found or it would be a substance with a very short or a very long retention time. In either event the amount of barbiturate calculated from the peak area would be proportionately less than the value found by spectrophotometry. The 3-hydroxy derivative of amylobarbitone has been studied17y18 and the solubility

and partition

coefficient

of this substance

prevent its recovery

blood or aqueous solutions when chloroform is used for extraction. The hydroxy derivative of pentobarbitone described by Maynert

from

and Dawsonls

is also much more soluble in water than in the usual solvents, and although it can be recovered from urine saturated with salt when ether is used, it is not extracted by chloroform. The hydroxylated

metabolites,

when treated

with hexamethyldisilazane

form

the corresponding silyl ether. The 3-hydroxy derivative of amylobarbitone has a different retention time from that of the parent compound on both Apiezon L and 3% XE-60 columns, and the silyl derivative also has a different retention time on these columns to both the parent barbiturate and the 3-hydroxy type of behaviour might reasonably be expected from other

compound. A hydroxylated

similar barbi-

turates.

Silanisation of barbiturate residues from blood, following chloroform extraction and chromatography on both Apiezon L and XE-60 columns, shows no shift of the peak or production of a second peak; this evidence also led us to conclude that metabolites of the barbiturates are not present in significant amounts in extracts prepared by the methods we have described. In choosing the amount of barbiturate to be chromatographed we deliberately took an amount which would allow the instrument to be run well below its maximum sensitivity and the amounts injected are usually in the range 0.5 to 5 pg. McMartin and Streetso, in a comprehensive survey of the gas chromatography of sub-microgram amounts of barbiturates and related drugs, have shown that much smaller amounts Cliw. Chim.

Acta,

20 (1968)

rgs-203

GAS CHROMATOGRAPHY

than these which can frequently microgram time which

OF BARBITURATES

203

can be identified. We chose the method given to reduce the interference occur from the production of multiple, often unidentifiable, peaks which appear, particularly from post-mortem material when working in the subrange. We were also concerned with avoiding the lengthening of retention can occur when very small amounts of drugs are chromatographed.

ACKNOWLEDGEMENTS

We are very grateful to Dr. E. Gerald Evans and Dr. Arthur Jordan, the directors of the Bangor and Sheffield laboratories where this work was done. One of us (H.L.) is indebted to the Research Grants Committee of the Welsh Regional Hospital Board for their help and encouragement and also to his colleague Mr. K. B. Hammond for his expert assistance. REFERENCES K. D. P>IRKER, C. R. FONTAN AND P. L. KIRK, Anal. Chem., 35 (1963) 356. K. D. PARKER, C. R. FONTAN AND P. L. KIRK, Anal. Chem., 35 (1963) 418. B. J. GUDZINOWICZ AND S. J. CLARK, J. Gas Chromato,o., 3 (1965) 147. K. D. PARKER AND P. L. KIRK, Anal. Chews., 33 (1961) 1378. E. BROCHMANN-HANSSEN AND A. B. SVENDSEN, J. Pharm. Sci., 51 (1962) 318. E. W. CIEPLINSKI, Anal. Chem., 35 (1963) 256. J. F. REITH, R. F. VAN DER HEIDE AND R. F. A. ZWAAL, Pharm. We~kblad. 7 (1965) 219. D. A. PODMORE, Clin. Chim. Acta, 7 (1962) 176. P. ht. G. BROUGHTON, Biochem. J.. 63 (1956) 207. G. W. STEVENSON, Anal. Chem., 33 (1961) 1374. D. A. PODMORE, J. Chromatog., 20 (1965) 131. R. P. W. SCOTT AND D. W. GRANT, Analyst, 89 (1964) 179. L. W. BRADFORD AND J. W. BRACKETT, Mikrochim. Acta, 3 (1058) 353. S. L. TOMPSETT, Clin. Chim. Acta, 5 (1960) 415. MM.GEALL, Ho+. Med., I (1966) 51. H. MATTHEW AND A. A. H. Lawson, Quart. J. Med., 35 (1966) 539. E. W. MAYNERT, J. Biol. Chem., 195 (1952) 397. M. S. Moss AND J. V. JACKSON, Proc. gvd Intern. Conf. Forensic ImmunoE., Med., Pathol., Toxicol., London, Apvil, 1963. 19 E. W. M~YNERT AND J. M. Dawso~, J. Biol. Chem., 195 (1952) 389. 20 C. MCMARTIN AND H. V. STREET, J. Chromatog., 23 (1966) 232. I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18

C/in. Chim.

Acta,

20 (1968)

195-203