THE BARBITURATES

CHAPTER

20

THE BARBITXJRATES J . F . Jackson THE range and production of the barbiturate drugs has increased rapidly since their introduction into therapeutics a t the beginning of this century. One of the factors responsible for this growth was the discovery t h a t the pharmacology of the original barbiturate, 5:5 diethyl barbituric acid, could be modified considerably by altering t h e substituents attached to the pyrimidine nucleus. The substitution of an ethyl group by a phenyl, for example, gives the resulting drug, phenobarbitone, a depressant action on the motor cortex, providing a useful anticonvulsant for the treatment of epilepsy. This depressant action may be further enhanced by attaching a methyl group to one of the nitrogen atoms, forming N-methyl-5-phenyl-5-ethyl barbituric acid, marketed under the trade name "Phemitone." If the short chain alkyl groups are replaced with complex radicals, as in cyclobarbitone, 5-ethyl-5-cyclohexenyl barbituric acid, the hjrpnotic action is more rapid, b u t of shorter duration. B y so changing the chemical configura­ tion of the barbiturate molecule, the time of onset, duration, and intensity of the hj^pnotic action m a y be varied to suit the particular requirements of the physician. Unfortunately, the increasing therapeutic use has been followed by a rise in the number of cases of barbiturate poisoning, which now accounts for more deaths t h a n any other poison except carbon monoxide. I t has been estimated t h a t the number of patients treated for barbiturate poisoning in Great Britain annually exceeds five thousand. This is not reahy surprising when one considers t h a t the patients most likely to have suicidal inclinations are often receiving sedation t r e a t m e n t with barbiturates. I t is evident, therefore, t h a t barbiturate analysis is an important part of the work of the hospital and forensic chemist. The difficulties encountered in this tjrpe of analysis have been enumerated by Curry in a review article on Toxicological Analysis^^^ but they are often not appreciated by those submitting material for examination. A further complication is the number of different barbiturates now used in clinical practice, and it is perhaps fortunate for the toxicologist t h a t of the m a n y hundreds of barbiturates syn­ thesized only about thirty are marketed, and of these, only half are in common use (see Tables 20.3 and 20.4). The modern tendency of pharmaceutical firms to make tablets and capsules containing two or more different barbiturates, and to combine these with other drugs, also presents a problem (see Table 20.1). The identification of barbiturates in biological material has been t h e subject of numerous papers in the past decade. The very number of such references is some indication of the dissatisfaction felt for existing methods. The classical methods of organic chemistry (i.e. determination 494

THE

BARBITURATES

495

T A B L E 20.1

Compound Barbiturate Proprietary Name

Barbiturates Present

Preparations* Proprietary Ñame

Barbiturates Present

Nidar

Butobarbitone Pentobarbitone Phenobarbitone Quinalbarbitone

Butobarbitone Phenobarbitone Quinalbarbitone

Norsed

Amylobarbitone Cyclobarbitone

Evidorm

Cyclobarbitone Hexobarbitone

Tuhial

Amylobarbitone Quhialbarbitone

Fortronal

Butobarbitone Cyclobarbitone Hexobarbitone Phenobarbitone

Plexonal

AUylbarbitone Barbitone Phenobarbitone

Bidormal

Pentobarbitone Butobarbitone

Duodorm

Hexobarbitone Butobarbitone

Ethobral

* T h i s t e r m is restricted t o preparations c o n t a i n i n g m i x t u r e s o f barbiturates. I t d o e s n o t include single barbiturate preparations i n a d m i x t u r e w i t h n o n barbiturate drugs.

of melting-point, elements present, a n d t h e preparation of derivatives) are not generally applicable unless t h e material is pure. Even with suicide cases, when large amounts of barbiturate are sometimes extracted, t h e purity of the residues is often more apparent t h a n real— fractional crystaUization a n d purification processes being necessary before a sharp melting point is obtained. The small residues usually extracted cannot be purified in this way without incurring some loss. I n t h e past, therefore, t h e analyst has h a d t o rely on colour tests a n d micro-crystalline comparisons. Whilst these methods have proved useful, especially as a quick check of extracts from tablets or stomach contents, they sometimes give misleading results when applied t o impure viscera residues. The development of physical methods of analysis such as X-ray diffraction^^^ I.R.<^^ a n d U.V.^*^ spectrophotometry have provided t h e toxicologist with new a n d valuable tools for his work, though t h e high initial expense involved has delayed their application t o clinical toxicology. T h e X-ray diffraction a n d I.R. methods are a valuable check on barbiturate identity b u t t h e occurrence of polymorphism a n d mixed drug poisonings cause complications. T h e U.V. spectrophotometric method, although one of t h e most sensitive means of detecting and estimating barbiturates, is limited because t h e absorption spectra only partiaUy identifies t h e barbiturate. T h e gas chromatography methods<^> probably provide t h e most ef&cient a n d sensitive w a y of separating a mixture of barbiturates b u t require complex equipment

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THE BARBITURATES

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and a high degree of skill. Further more a retention time, hke a n Ry> value, is a non-specific parameter of identification b u t R/ values are obtained with one or more spray reagents which often make t h e identification nearly specific. I t is evident, therefore, t h a t a simple rapid a n d inexpensive technique which offers possibihty of separating a n d identifying barbiturates in impure a n d complex residues, is required. Paper a n d thin layer chromatography fulfil this requirement. Standard solutions. Standard solutions of most barbiturates m a y be prepared in alcohol and a concentration of 10 mg. per ml. is suitable for one w a y paper chromatography when 10 μΐ. are applied t o t h e origin; only 2 t o 4 μΐ. are required for thin layer chromatography (TLC). Providing these standard solutions are stored in well stoppered bottles they wiU keep for a t least three months. The standard a n d test solutions are spotted on t o t h e paper with a capiUary pipette, which is apphed intermittently under a n infra-red lamp. If t h e latter is not available, a n ordinary 60 w a t t electric hght bulb m a y be utilized. I n this case it is useful if t h e lanap is fixed beneath t h e paper. This facUitates t h e control of spot size; t h e paper becomes translucent when wet a n d no further addition of solution is made until t h e area becomes opaque, a m a t t e r of seconds with t h e heat radiated b y the lamp. This technique is n o t required for TLC.

Solvents Seven monophasic solvents are described for ascending chromato­ graphy. Four solvents are for conventional paper chromatography, one for high temperature reverse phase chromatography (Street^^^) and two for TLC (see Table 20.2). Paper chromatography solvents. Four solvents are for use with t h e ascending frame technique in t h e Universal T a n k a n d with W h a t m a n No. 1 paper a t room temperature. I n general, all distribute t h e barbiturates in t h e same order; long acting barbiturates nearest t o t h e origin, then t h e intermediate acting, a n d above these, nearest t o t h e top of t h e paper, t h e short a n d ultra-short acting barbiturates. The volume of solvent used is 200 ml. b u t larger amounts t h a n are required for immediate use m a y be prepared, providing t h e y are stored in bottles with weU fitting stoppers. Stock solutions keep for a b o u t a month, b u t a gradual loss of ammonia wiU eventually occur. This wiU be evident when t h e resulting chromatograms are inspected, for t h e distance t h e barbiturate spots travel generaUy varies inversely with the concentration of ammonia. The butyl alcohol a n d amyl alcohol solvents are usuaUy r u n overnight (15-16 hours), b u t a partial dis­ tinction between t h e various types of barbiturates can be achieved overday (7 J hours) especiaUy with amyl alcohol. Increasing t h e time t o 18 hours gives a better separation, b u t no advantage is conferred b y periods longer t h a n this. These solvents m a y be used a second time with only slight variation in values, b u t further use entaUs a sacrifice in t h e quality of separation. The chloroform solvent is much faster a n d separation of t h e long acting, a n d some of the intermediate acting barbiturates, can be achieved in 3 hours. Lengthy equilibration of

498

CHROMATOGRAPHIC T E C H N I Q U E S

atmosphere, solvent a n d paper is not necessary, though t h e addition of a httle concentrated ammonia t o t h e bottom of t h e t a n k is useful if streaking is experienced. Double-zoning occurs sometimes with t h e chloroform solvent. All four solvents are easily removed on completion of the run, 15 minutes drying time in air a t room temperature is usuaUy sufficient. n-Butanol-Water-Ammonia 0-880 (BuWAm). The water a n d con­ centrated ammonia are mixed, forming an approximately I N solution, and this is then shaken vigorously with t h e butanol until a monophasic solvent results. This solvent is t h e most useful when investigating metabolites present in residues as these tend to have very low R , values and often remain too near t h e origin with solvents containing a larger amount of ammonia. The barbiturates are mainly located in t h e t o p half of t h e paper a n d thus are well removed from slow running impuri­ ties. I t is not a good routine solvent however, for t h e degree of separa­ tion of t h e individual barbiturates is poor. n-Butanol-Ammonia 0-880 (BuAm). The increased concentration of ammonia in this solvent, compared t o t h e previous one, decreases t h e distance m a n y of t h e barbiturates travel a n d therefore allows a better separation t o be attained. Amyl Alcohol-Ammonia 0-880 (AAm). This solvent gives better separation of t h e barbituric acid derivatives t h a n t h e other solvents described. Amyl alcohol, as supplied commercially, is a mixture of isomers and so t h e R/ values obtained with this solvent m a y vary slightly unless it is obtained from t h e same source every time. Several of t h e isomeric amyl alcohols m a y be obtained in a pure state b u t t h e extra expense cannot be justified, for consistent values are given with most of t h e ordinary analytical grades; t h e one used here is described for *'milk-testing." Chloroform-Ammonia 0-880 (CAm). I n solvent t r a y : 200 ml. chloroform. I n t a n k b o t t o m : 100 ml. ammonia 0-880. Concentrated ammonia is poured around t h e t a n k bottom a n d t h e chloroform is p u t in t h e solvent tray. The chloroform is thus exposed to ammonia vapour for a t least 3 hours before t h e frame is inserted but more consistent results are obtained if the chloroform is exposed t o ammonia, in this way, overnight. Solvent reaches t h e t o p of the paper in 3 hours a n d evaporates from t h e papers so very quickly t h a t they are ready for examination almost as soon as they are removed from t h e tank. If a spare glass t a n k and t r a y are available, they can be kept ready for immediate use with t h e liquids distributed as above although both should be changed after four t o five days. Phosphate buffer solvent (PB). This buffer is used for high tempera­ ture reverse phase paper chromatography. This utilizes a sewn or clipped cylinder of W h a t m a n No. 1. or 3 paper treated with tributyrin in a simple gas jar tank which is placed in an oven a t 86 t o 96°C. T h e volume of solvent used depends on t h e size of t h e t a n k b u t is usually about 25 to 50 mis. Stock solutions wiU keep for several months b u t t h e ^ H should be checked before use. Equihbration of solvent atmosphere

THE BARBITURATES

499

and paper is not required b u t pre-heating of t h e t a n k a n d solvent before inserting the paper cylinder gives more consistent Revalues and shortens the time required to give useful separations. W h a t m a n No. 1 or 3 papers give similar R^ values. No. 3 is easier to handle when wet and forms a more stable cylinder b u t it requires a longer drying time than W h a t m a n No. 1. The choice of paper is a matter of personal preference. Both types of paper m a y be examined with U.V. (254 τημ) light whilst still wet b u t they should be dried with a hot air fan before using other location reagents. This phosphate buffer solvent gives better separation of the barbitur­ ates than t h e other solvents and in a much shorter time. I t is therefore the recommended solvent for urgent preliminary identification. If t h e chromatograms are required as a permanent record they should be stored in sealed transparent polythene envelopes because they eventu­ ally develop a penetratmg odour of rancid butter. Thin layer chromatography solvents. Silica gel G (Merck) plates are prepared as previously described in Chapter 3. They are activated a t 100°C for 1 hour a n d stored in a desiccator. A 10 cm. r u n is adequate at room temperature. Isopropyl—alcohol : chloroform : 0*880 ammonia (PrCAm), This solvent gives better distribution of barbiturates t h a n a n y other TLC solvents we have tried. I t wiU separate phenobarbitone from barbitone, for example, which few published TLC solvents wiU do. R^ values are fairly consistent and solvent rise is quite rapid; a rise of 10 cms. takes about 50 minutes. Solvent should be prepared fresh on t h e day required however as solvent which has been in t h e tank for a few days is less satisfactory. This m a y be due t o loss of ammonia. This solvent is one of eight studied b y SheUard & Osisiogu<'^ using 12 barbiturates, alone and in mixtures. Their extensive experiments showed it t o be one of the best solvents in their series. Chloroform—^Acetone (CAc), This solvent makes a useful partner to t h e PrCAm solvent because each is capable of separating groups of barbiturates n o t resolved b y t h e other. Butobarbitone a n d methylphenobarbitone for example are not resolved b y PrCAm b u t are easily separated by CAc. Conversely barbitone a n d phenobarbitone, secbutobarbitone a n d heptabarbitone, a n d allobarbitone a n d pentobarbitone are not resolved by CAc b u t can be differentiated b y PrCAm. T h e CAc solvent is slightly faster t h a n PrCAm (10 cms rise takes about 30 minutes). I t is easier t o remove a n d one is not troubled b y ammonia fumes. This is a well tested solvent a n d has been used b y m a n y w o r k e r s . P u b l i s h e d R^ values for barbiturates with CAc v a r y more t h a n one would expect b u t this m a y be due t o different grades of solvent, activation of plates or humidity variations. Except for cyclo­ barbitone a n d methylphenobarbitone our values are within 5 units of S u n s h i n e ' s . A stock solution m a y be prepared, which is useful as this solvent m a y also be used for neutral drugs (see Chapter 22). Comparison of paper and thin layer methods. I n general t h e separa­ tion of barbiturates b y TLC is not as good as t h e PC systems. T h e speed of TLC compared with conventional PC methods is attractive however and m a n y routine toxicology laboratories have converted to

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TLC for preliminary screening of biological samples. Final decision must depend on t h e type, variety, a n d amoimt of work dealt with b y the particular laboratory.

Rf Values Paper Chromatography—R^ Values. As t h e solvent front often advances beyond t h e t o p of t h e paper R/ values cannot be determined and so R^, values have been used, t h u s : ^

_ distance moved b y barbiturate ^ distance moved b y pentothal

Pentothal has been chosen as a standard because it travels farther t h a n most other barbiturates in most solvents a n d is easy t o locate b y a physical method (U.V. light, see later). R^ values have been deter­ mined a t room temperature, 18-20°C, a n d on W h a t m a n N o . 1 paper using t h e ascending F r a m e technique. Reverse Phase Chromatography. T h e quoted R^. values were obtained after 20 minutes a t 86°C using 25 ml. of solvent. T a n k a n d solvent were preheated for about 15 minutes before inserting t h e paper, otherwise no special equilibration was employed. Despite t h e use of different paper (Whatman N o . 1 or 3) and minor fluctuations in t h e temperature, t h e Ry> values were reproducible to better t h a n 3 units. TLC. T h e Ry. values given in t h e tables were obtained after t h e sol­ vent front h a d moved 10 cm. from t h e base line. They are only intended as a guide though in general with freshly pre^pared solvent a n d plates the variation was less t h a n 5 per cent.

Location Reagents Ultra-violet Light. The barbiturate spots m a y be identified on t h e dried papers b y illuminating with a n ultra-violet lamp of m a x i m u m intensity 254 ταμ. (Hanovia chromatolite) after t h e paper has been exposed t o a saturated atmosphere of ammonia. They appear as dark spots on a fiuorescent paper a n d can be marked with a pencil. This method is very sensitive a n d weU defined spots can be obtained with 10 t o 25 μg. of barbiturate. W i t h light of 360 ταμ. maximum intensity (Wood's hght) only t h e thio-barbiturates can be clearly observed. This 3rovides a useful means of locating either t h e thio-barbiturates (360 ταμ. ight) or all barbiturates (254 m ^ . light) without chemical t r e a t m e n t of the paper. A permanent record m a y be obtained b y making contact prints of t h e paper on Ilford Refiex paper No. 50 using t h e ultra-violet lamp as a light source. I t has been noted t h a t cyclobarbitone often leaves a fiuorescent spot a t t h e origin, probably a decomposition product due t o t h e instability of this compound. I n order to view t h e barbiturates on TLC plates it is necessary to incorporate a fluorescent material into t h e silica-gel layer. Although such powders are available commerciaUy we prefer t o use t h e ordinary powder as t h e use of the fluorescent powders m a y preclude t h e observa­ tion of fluorescent substances. Plates should be dried, examined with U.V. light a n d fluorescent areas noted. T h e plates m a y then be sprayed

504

CHROMATOGRAPHIC T E C H N I Q U E S

with a dilute fluorescein solution a n d compounds. Fluorescein Spray Sodium fluorescein Sodium hydroxide Water

re-examined for absorbing 4 mg. 4 g. 100 ml.

This is used as described above to detect barbiturates on TLC plates. I t m a y also be used on paper chromatograms where t h e use of ammonia vapour is inconvenient.

Cobalt Nitrate-Ammonia. Cobalt nitrate, 1 per cent, in acetone for paper or 5 per cent, in ethanol for TLC. After drying, t h e paper is dipped through t h e reagent, dried again and then exposed to ammonia vapour; a convenient method of doing this is t o place a little strong ammonia solution a t t h e bottom of t h e glass t a n k a n d hang t h e papers inside. The barbiturates give bluish violet spots, best seen b y transmitted light. The colour persists for about 20 minutes b u t returns on re-exposure t o ammonia vapour, even after t h e lapse of several years. When t h e initial violet colour has faded a further identiflcation m a y be m a d e ; thiobarbiturates give a permanent light green, hexobarbitone gives a permanent light brown, and cyclobarbitone gives a faint yeUowish spot. I t is essential t o dry t h e paper thoroughly before exposing t o am­ monia, failure t o do so gives a confusing blue-green background in place of the normal white or faint pink one. The best way to dry paper chromatograms is t o leave them exposed to t h e atmosphere for 10 minutes. T h e use of an oven or hot air fan may produce a pink background which makes it difíicult to observe t h e violet barbiturate spots. The reagent m a y be used for reverse phase chromatograms though the tributyrhi prepared papers have an adverse effect on its sensitivity. The 5 per cent solution of cobalt nitrate in alcohol is more effective on TLC plates a n d will detect about 5 μg. of barbiturate. This reagent is far more diagnostic for barbiturates t h a n t h e more frequently used mercury spray reagents. I t is stable for several months.

Mercuric Sulphate-Diphenyl Carbazone. (a) Mercuric Sulphate. 5 g. mercuric oxide dissolved in 100 ml. water + 20 ml. concentrated sulphuric acid (Stock solu­ tion). (b) Water. (c) Diphenylcarbazone 0-1 per cent in ethanol (D.P.C.). Paper Chromatograms. Immediately before use 1 volume of (a) is mixed with 1 volume of (6) a n d t h e dried chromatogram is dipped through t h e solution. Paper chromatograms must be washed in running water for 15 minutes a n d t h e n placed on a p a d of blotting paper to dry. They are then sprayed with t h e D.P.C. solution (c)

THE BARBITURATES

506

Barbiturates give a bluish violet spot which is fairly permanent if stored away from sunlight. TLC plates. The reagent is mixed as above a n d sprayed on t h e TLC plate. N o washing with water is necessary. White spots are visible on the wet plate if a mercury reacting compound is present [N.B. Other

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F I G . 2 0 . 1 . Chromatogram of t h e c o m m o n barbiturates i n t h e Amyl-alcohol-ammonia solvent. (1) Rutonal, ( 2 ) Phenobarbitone, ( 3 ) B a r b i t o n e , ( 4 ) Allobarbitone, ( 5 ) Cyclobarbitone, ( 6 ) Alurate, (7) Butobarbitone, ( 8 ) Methylphenobarbitone, ( 9 ) Hexobarbitone, ( 1 0 ) Pentobarbitone, ( 1 1 ) Amylobarbitone, ( 1 2 ) Thialbarbitone, ( 1 3 ) Quinalbarbitone, ( 1 4 ) T h i o p e n t o n e . ^^2/

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compounds m a y give this reaction apart from barbiturates—(see Table of Interfering Drugs)]. When t h e white spots have been marked, t h e plate is sprayed with the D.P.C. solution (c). Barbiturates give a bluish violet colour whereas m a n y of t h e other mercury reacting compounds give only a pink colour. Barbiturates containing an aUyl group give a more bluish shade t h a n t h e other barbiturates. Potassium Permanganate: 0-1 per cent in water. This reagent m a y be applied t o paper chromatograms b y t h e dipping or spraying tech­ niques. Reverse-phase tributyrin treated papers a n d TLC plates

506

CHROMATOGRAPHIC

TECHNIQUES

require more reagent t h a n conventional paper chromatograms. T h e unsaturated barbiturates give yellow spots on a pale pink background. These should be marked as soon as they appear. If desired this reagent

PHmumroHi Qúmiumm BUTÜBARBirONE

PÍHTOWAL Μΐπυκ{βζ8) URINE xtr-<.cf MIXTURE PENTOBARBITONE WTOBARBITDNE

FIG. 2 0 . 2 . T h e c h r o m a t o g r a m s h o w s , (a) t h e separation o f barbitu­ rates present in c o m p o u n d commercial preparations, a n d (6) a urine e x t r a c t (;ontaüiing p h e n o b a r b i t o n e a n d a m e t a b o l i t e .

may be applied after t h e cobalt reagent providing t h e chromatogram is freed from ammonia.

N.Q.S. Reagent (a) 0-1 Ν Sodium hydroxide. (b) Sodium l,2-naphthaquinone-4-sulphonate saturated solution in 50 per cent v/v ethanol a n d water. Many of t h e thiazide diuretic drugs are co-extracted with t h e bar­ biturates. This reagent is useful therefore as a method of distinguishing between barbiturate and thiazide drugs. The dried chromatograms are lightly sprayed with (a) a n d then with the NQS reagent (6). The thiazides appear as stable orange spots within 15 minutes, t h e barbiturates do not react; Pillsbury a n d Jackson. ^^^^ Sequential Spraying. Maximum sensitivity is obtained generally when each reagent is applied individually b u t t h e following sequence m a y be useful when this is n o t possible: U.V.-^Cobalt reagent—> Mercury Reagent-^ D.P.C. [mercury reagent {c)]->KMxiO^.

THE BARBITURATES

507

The U.V. and cobalt reagent methods are more specific; t h e mercury reagent is more sensitive; t h e D.P.C. reaction assists in confirmation and picks out t h e aUyl barbiturates; and finaUy t h e ΚΜηθ4 indicates the unsaturated barbiturates especiaUy those containing cyclohexenyl or heptenyl groups. The chromatogram should be d r y before proceeding to t h e next reagent. Interfering Compomids. Simple direct solvent extraction of biological fluids, pharmaceutical preparations etc. a t pH 1 t o 7 wiU extract barbiturates plus a n y other acidic a n d neutral solvent soluble com­ pounds present. These non-barbiturate compounds m a y interfere with barbiturate detection or if they react with barbiturate location reagents mislead t h e analyst. The strongly acidic substances m a y be removed b y washing t h e organic solvent extracts with sodium bicarbonate solution. T h e neutral compounds m a y be removed b y making t h e sample alkaline a n d extracting with solvent before proceeding with t h e acid extraction, alternatively t h e neutral compounds m a y be left behind b y extracting the acid solvent fraction with dUute sodium hydroxide which wiU re­ move t h e barbiturates plus other weakly acidic substances. These separations are not always complete however a n d frequently the clinical chemist does not wish t o employ these more complicated extraction techniques. A list of t h e common interfering substances which m a y be co-extracted m a y therefore be useful, (see Table 20.5). Fortunately t h e behaviour of barbiturate spots with short-wave U.V. light (254 ταμ.) a n d their reaction with t h e cobalt nitrate reagent is almost specific. If a spot on a chromatogram is located b y U.V. (254 πιμ.) light as a dark absorbing spot, which appears or intensifies only under alkaline conditions, and if it also gives a positive cobalt nitrate test— it is almost certainly a barbiturate. Other drugs have been reported as giving a positive reaction with cobalt salts b u t this is usuaUy because t h e test has been done in a test tube a n d not on a chromatogram.

Special Techniques 1. Separation of Amylobarbitone, Pentobarbitone, and Quinalbar­

bitone. Most of t h e solvents used for t h e chromatography of barbit­ urates give inadequate separation of these compounds, which are frequently encountered in toxicological examinations, and occur in mixtures of proprietary preparations, such as " T u i n a l " a n d "Nidar." The difficulty in separating Amylobarbitone a n d Pentobarbitone is probably due t o t h e fact t h a t they are isomers (see formulae below). Quinalbarbitone has an unsaturated aUyl substituent a n d m a y be distinguished from Amylobarbitone a n d Pentobarbitone b y using t h e potassium permanganate dip. When r u n as a mixture with either of the other two barbiturates, however, only one large spot is obtained and t h e presence of Amylobarbitone or Pentobarbitone m a y fail t o be detected, unless one notices t h a t only t h e t o p portion of this large spot is reacting with t h e potassium permanganate reagent. Maynert a n d Washburn^^^^ studied t h e reaction of concentrated sulphuric acid with some of t h e barbiturates a n d showed t h a t certain of t h e 5 : 5 disubstituted barbiturates were dealkylated. Brooker^^^^

TABLE

Interfering R/ X 100 P.B. Solvent

Substance

20.5

Compounds

Synonym

D i s t i n c t i o n from Barbiturates

Group

5

Polythiazide

Nephril

N . Q . S . reagent

Β

8

Glutethimide

Doriden

BrJStarch KI*

C

8

Theophylline

Brg/Starch K I *

C

12

Pheneturide

Phenyl ethyl acetyl Urea

Brg/Starch K I *

A

15

Carbromal

Adalin Bromural

BrJStarch KI*

Β

17

Frusemide

Lasix

N.Q.S.

A

20

Methyclothiazide

Enduren

N.Q.S.

Β

21

Teclothiazide

Depict

N.Q.S.

Β

26

Ethinamate

Valmidate

F/HCl*

C

28

Phenylbutazone

Butazolidine

BrJStarch KI*

Β

35

Phenacetin

Acetophenetidin

BrJStarch KI*

C

46

Mephensin Carbamate

Tolseram

F/HCl*

C

55

Methyprylone

Noludar

BrJStarch KI*

C

56

Benzthiazide

Fovane

N.Q.S.

Β

60

Bemegride

Megimide

Bra/Starch K I *

Β

62

Hydrochlorthiazide

Esidrex Hydril

N.Q.S.

A

64

Benzoic Acid

73

Styramate

Sinaxar

F/HCl*

C

75

Chlorothiazide

Saluric Civril

N.Q.S.

A

80

Paracetamol

Panadol

BrJStarch K I

A

85

Salicylic A c i d

Salicylates

FeCla

A

87

Salicylamide

Salamid

BrJStarch KI*

A

92

Hippuric A c i d

BrJStarch K I *

A

A

* F o r details of t h e s e reagents see Chapter 22 (Neutral D r u g s ) Group A. Strong acid group: unlike barbiturates, t h e s e c o m p o u n d s m a y b e e x t r a c t e d from organic s o l v e n t w i t h NaHCOg solution. Group B. Weak acid group: (i.e. B a r b i t u r a t e g:roup): t h e s e drugs c a n n o t b e separated from t h e barbiturates b y simple e x t r a c t i o n m e t h o d s . Group C. Neutral drug group: (see Chapter 22) unlike barbiturates t h e s e drugs will r e m a i n in t h e organic s o l v e n t p h a s e w h e n it is e x t r a c t e d w i t h dilute sodiimi hydroxide. N.B. I f chloroform is u s e d a s t h e e x t r a c t i n g s o l v e n t m a n y other drugs m a y b e CO-extracted w i t h t h e barbiturates; e.g. s u l p h o n a m i d e s , p h e n o t h i a z i n e s a n d s o m e alkaloids.

509

THE BARBITURATES

utilized this reaction to distinguish Amylobarbitone from Pentobar­ bitone. We have extended this acid treatment to t h e above barbitur­ ates, and t h e results obtained after paper chromatography of t h e pro­ ducts are given in the table below. Unambiguous identification of any mixture of these barbiturates is now possible.

Method 1. A miUigram or less of t h e " u n k n o w n " barbiturate is heated with a few drops of concentrated H2SO4 on t h e water b a t h for 1 hour. 2. T h e reaction mixture is carefuUy düuted with a few mis of water and extracted with ether. 3. E t h e r extract dried with knife point of anhydrous Na2S04 a n d evaporated. 4. Residue dissolved in about 0-5 ml. CHCI3 and 25-50μ\. aliquots used for chromatography.

CO

/

HN

1

Barbiturate

1

\

Amylobarbitone (amytal) Pentobarbitone (Nembutal) Quinalbarbitone (Seconal)

NH

CO

OC

R

\

/

c

/

Solvent System. ΚΜηθ4 X reagent 100 Before

H2SO4

A.Am. R^ X 100 After

H2SO4

\R ,

Ethyl

3-methylbutyl

93

Ethyl

1-methylbutyl

89

Allyl

l-methylbutyl

96

_

N o change. 5

+

Attacked. N o spot.

Results obtahied after H2SO4 treatment m a y be confirmed b y retain­ ing a portion of t h e residue for U.V. spectrophotometric examination. Using 0-5 Ν NH4OH as solvent, dealkylated pentobarbitone gives maximum absorption a t 268 m//. instead of t h e usual peak a t 240 ταμ., whüst quinalbarbitone give weak absorption peaks a t 240, 266, and 315 m//.

We have applied the the foUowing results: UNCHANGED:

(same R^) DEALKYLATED :

(Different R^) ATTACKED :

(No spot located) 34

H2SO4 treatment to other barbiturates with

Barbitone, Amylobarbitone, Butobarbitone, Rutonal, Phenobarbitone a n d Methylphenobarbitone. Pentobarbitone, Cyclobarbitone. AUobarbitone, Alurate, Qumalbarbitone, Thiopen­ tone.

510

CHROMATOGRAPHIC T E C H N I Q U E S

Maynert & Washburn^^^) suggested t h a t : — 1. 5 ' 5 ' disubstituted barbiturates containing two primary alkyl groups and 2. barbiturates containing one primary alkyl group and a phenyl group are not affected by the sulphuric acid treatment. The work of Brooker^i^^ and Jackson^^^^ appears to confirm this finding but t h a t of Curry^^*^ and Bogan^^^ showed some discrepancies in the second p a r t of this statement. They reported destruction of the phenyl contahiing

FIG. 2 0 . 3 . Sulphuric acid t e c h n i q u e . " U n k n o w n " s p o t is n o t a t t a c k e d , therefore identified a s a m y l o b a r b i t o n e .

barbiturates. F u r t h e r studies of this reaction by Street and McMartin, ^^^^ and Brown^24) indicates t h a t for some barbiturates t h e final acid strength and treatment of the reaction mixture is very critical. Never­ theless this technique does provide an additional method of identifica­ tion when no other means are available apart from chromatography. Κ barbiturates with a phenyl group are indicated therefore it is essen­ tial to treat a control sample of the suspected barbiturate simultaneously with the unknown.

Extraction of the Barbiturates The t3φe of material to be analysed and the purpose of t h e enquiry govern t h e method used to extract t h e barbiturate. The ammonium sulphate extraction method of NickoUs^^^^ is preferred for substances

T H E BARBITURATES

511

containing a large percentage of protein or if a general search for all types of drugs is requested. A modified Valov m e t h o d i s generally used if barbiturates are t h e only poison suspected, b u t if the material is protem-free a direct ether extraction, after preliminary maceration and acidification, usuaUy proves effective. Troublesome emulsions are sometimes encountered if urine samples are extracted directly, therefore a simplified ammonium sulphate method is shown below. The small amount of extra labour involved generaUy proves worthwhile.

Extraction of Barbiturates from Body Fluids Urine. 1. 50 ml. urine is acidified with dilute HCl, saturated with solid ammonium sulphate (c:i 100 gm./lOO ml.) heated t o 60°C and filtered. 2. The filtrate is extracted with 3 X ¿ volume of ether. 3. This, together with ether washings of t h e residue on t h e filter paper, is extracted with 2 χ J volume of saturated NaHCOg solution to remove a n y strongly acidic substances. 4. The ether solution is then extracted with 2 X ¿ volume of 0-5N N a O H . 5. The N a O H solution is acidified with dUute HCl a n d extracted with 3 X ^ volume of ether. 6. The ether is evaporated off a n d t h e residue is made u p as a 2 per cent solution in chloroform when 10 a n d 20 μΐ. are used for chroma­ tography. Blood and Cerebrospinal Fluid. Blood is first hsemolysed, b y t h e addition of a n equal volume of distilled water. The hsemolysate or t h e C.S.F. is then treated as for urine. Stomach Washings. The first litre of the stomach wash-out should be saved for detection of barbiturates a n d extracted as for urine. Purification of Viscera Extracts. WhUst t h e apphcation of t h e new physical a n d chromatographic náethods have made possible t h e determination of barbiturates in impure residues, t h e traditional methods of organic chemistry should n o t be neglected. AU methods have their inherent defects which can only be eliminated b y utUizing aU t h e avaUable analytical resources. There must be no shadow of doubt in t h e mind of the expert witness t h a t his analysis is correct a n d mixed melting point tests still provide one of the most satisfactory ways of obtaining a final confirmation of the analjrtical residts in medico-legal work. The use of paper chromatography as a means of purifying viscera extracts seems t o have been overlooked. The foUowing method, how­ ever, has been found useful for obtaining pure crystallme material for micro-melting point determinations. The impure extract is dissolved in chloroform a n d placed directly on the starting line either as a series of spots or as a streak. B y using aU the papers held by t h e frame, quite a large amount of extract m a y be purified. The ascending method is used with t h e butanol-IN ammonia solvent system a n d overnight runs are recommended for good separa­ tion. After drying, t h e papers are exposed t o 25a τημ. ultra-violet radiation t o detect t h e barbiturate. The portion of t h e paper which

512

CHROMATOGRAPHIC T E C H N I Q U E S

shows absorption is then cut out and eluted with alcohol. (If ultra­ violet light is not available a small vertical strip is cut from t h e paper, and the barbiturate level located with the cobalt-nitrate reagent.) Evaporation of the alcohol generally yields a pure residue for melting point determinations. This purification process is also useful as a preliminary t r e a t m e n t before utilizing X-ray diffraction, U.V. and I.R. spectrophotometry and chromatography with standard solutions.

Metabolism of Barbiturates If one peruses the standard textbooks on toxicology, it will be found t h a t almost all the analytical techniques are confined to the detection of the drug in the form in which it was ingested. Thus, despite all t h e improvements in analytical science it is still possible, a t least in theory, t o commit t h e perfect murder by administering a poison t h a t is rapidly metabolised b y the body. This is inevitable whilst the processes of detoxification and excretion of drugs are unknown, and whilst the amount of research devoted to producing new drugs far exceeds t h a t available for studying the metabolism of the established ones. However, advances in t h e knowledge of barbiturate metabolism have been made and comprehensive reviews of this topic b y Raventos^^^^ and Stewart & Stolman^^o) emphasize the contribution made by chroma­ tographic methods. Patient research by m a n y workers in this field has revealed the mode of breakdown of m a n y of the barbiturates. I n general, the more complex the barbitm'ate the more complicated is t h e metabolic process, and the greater the percentage of the ingested dose excreted as metabolites. Thus, whilst barbitone, di-ethyl barbituric acid, is excreted almost entirely in its original form, only 0-3 per cent of pentothal, 5-ethyl-5-(l-methyl butyl)-2-thio-barbituric acid, is recovered unchanged. Hydroxylation and oxidation to keto, or carboxy derivatives appears to be the main method of detoxification, coupled with N-demethylation and desulphurization if these radicals are present. R u p t u r e of the pyrimidine ring plays only a very minor role and this has important practical implications because most of the methods for detecting barbiturates depend on the reactions of this structure; as it is present in the majority of the metabolites they will still give positive reactions. We have found this to be so with urine residues from phenobarbitone, pentobarbitone, amylobarbitone, and quinalbarbitone poisonings. Spots giving a positive cobalt nitrate reaction with low R^, values have been observed. Cochin & Daly^^^ have noted several recurring spots whilst investigating urine samples from patients on therapeutic doses of barbiturates by the TLC method. Some of these unusual compounds have been identified. Thus the hydroxy metabolites of p h e n o b a r b i t o n e , p e n t o b a r b i t o n e and amylobarbitone<22) y^^y^ ajl ]3een isolated from h u m a n urkie samples. To ensure extraction of these metabolites most of the routine extraction methods need to be modified. Chloroform, ether and methylene dichloride are not suitable solvents since hydroxylation significantly modifies the solubility of these compounds in water and organic solvents. The partition coefficients of hydroxy-amylobarbitone for

T H E BARBITURATES

513

chloroform-water, ether-water a n d methylene dichloride-water are 0*02, 0-20 and 0-20 respectively. If t h e water phase is saturated with ammonium sulphate t h e coefficient for t h e system ether-water a n d ammonium sulphate is 4-0. The extraction method given above for body fluids can be easily modified, therefore, t o give good recovery of barbiturate metabolites, b y increasing t h e volumes of ether a n d saturating all water phases with ammonium sulphate. A n alternative scheme has been suggested b y Moss^^a) ^j^q fomxd t h a t t h e partition coefficient for hydroxyamylobarbitone using t h e system cyclohexanolwater was 5-0.

Extraction Method for Barbiturate MetaboUtes Body fluid or tissue homogenate. Adjust urine ρΉ. 1 or blood a n d viscera to ρΉ. 6. E x t r a c t with equal volume of cyclohexanol.

\ I cyclohexanol

I B o d y fluid, e t c . D i s c a r d .

w a s h w i t h 1/lOth v o l . 5 % N a H C O » — d i s c a r d w a s h . E x t r a c t c y c l o h e x a n o l w i t h 1/lOth v o l u m e N / 2 N a O H . cyclohexanol.

Discard

Acidify a n d e x t r a c t w i t h 10 χ v o l u m e ether, 3 t i m e s . E v a p o r a t e t o s m a l l v o l u m e , a d d a n h y d r o u s N a 2 S 0 4 a n d charcoal. Filter. Evaporate to dryness. Residue—barbiturate metabolites.

AU future research work on barbiturate tissue levels, excretion rates and distribution studies should include some form of chromatography in order to establish whether t h e results refer t o unchanged barbitur­ ates, metabolized barbiturates or a mixture of both, a n d a lot of t h e earlier work concerned with t h e quantitative aspects of barbiturates in biological material must now be re-evaluated. Clinical Applications. The diagnosis of barbiturate intoxication can often be made from t h e circumstances a n d t h e clinical condition of t h e patient, on admission t o hospital. Chromatographic analysis m a y be utihzed t o confirm t h e diagnosis a n d t o obtain t h e initial identification (i.e. fast, medium, or slow acting barbiturate). If a n ultra-violet spectrophotometer is not avaUable, t h e approximate level of barbiturate in t h e blood m a y be estimated b y visual comparison of t h e intensity of the located spot with known standards. The doctor can t h u s assess the value of various methods of treatment, a n d check t h e progress of the patient. The accuracy of this method, compared with t h e quanti­ tative result given b y t h e ultra-violet spectrophotometer, is about ± 5 per cent. The real value of the paper chromatographic analysis of barbiturates is evident when a case history is n o t avaUable a n d when t h e clinical picture is confused or comphcated b y other factors (e.g. poisoning due

514

CHROMATOGRAPHIC T E C H N I Q U E S

to a mixture of drugs, or modification of t h e normal disease symptoms by barbiturate). Forensic AppUcations. Apart from t h e obvious cases such as murder, infanticide, a n d suicide, which necessitate testing for barbiturates in post mortem specimens, t h e use of these hypnotics m a y be a factor in m a n y other crimes. Analysis of food, blood, a n d urine samples, m a y be required t o determine whether a race-horse or a greyhound has been doped. Robbery a n d sexual offences have been a t t e m p t e d after administration of barbiturate as a **knock-out" drug and this m a y entail examination of the dregs in a cup or a stain on a sheet. The analysis of tablets a n d capsules found a t t h e scene of a crime is often required b y the police a n d t h e investigation of suspected contraventions of t h e Dangerous Drugs Act, nearly always entails this type of analysis. Paper chromatography is especially helpful in this work for it provides one of the best methods of separating a n d identifying t h e ingredients of a compound tablet. Detection of normal medicinal doses of barbiturate in t h e urine a n d blood of drivers m a y assume importance in offences under t h e Road Traffic Act, whilst t h e presence of barbiturate in t h e victims of road accidents m a y provide confirmation of witnesses, who declare t h a t t h e injured person staggered in front of t h e vehicle. Although t h e U.V. spectrophotometer provides t h e most sensitive means of detecting small doses, it is useful t o confirm these findings and to check t h e identity of t h e barbiturate with a chromatogram. The foUowing case reports iUustrate some of these applications. Case No, 1. A m a n was found unconscious in a motor car. On admission t o hospital he was given a stomach washout b u t he remained in a coma. A tentative diagnosis of barbiturate poisoning was made, b u t t h e hospital authorities were not willing t o give evidence if proceed­ ings were instituted b y t h e police, on a charge of being under t h e influence of drugs whilst in charge of a vehicle. The stomach washout was submitted t o t h e pohce laboratory for analysis. The total volume was 2J htres a n d as no food residues were present, 1 litre of the washout was directly extracted as previously described yielding a final residue of 69 mg.; this residue, in t h e barbiturate group, was a colourless gum containing some crystalline material. An aliquot of t h e residue was submitted t o paper chromatography. One barbiturate was found with an Rj, value similar t o t h a t of amylobarbitone a n d pentobarbitone. N o reaction was obtained with t h e potassium permanganate dip, indicating a saturated barbiturate. From t h e reaction with cobalt nitrate it was estimated t h a t only 50 per cent of t h e residue was barbiturate. Examination of the residue remaining in t h e original ether after alkah extraction, i.e. t h e neutral drug group, revealed t h e presence of carbromal a n d this was confirmed b y a n X-ray diffraction pattern. Additional separations on t h e original residue finally yielded 24 mg. of carbromal a n d 44 mg. of barbiturate. F u r t h e r paper chromatograms were r u n and t h e portion of the paper a t t h e pentobarbitone level was eluted with alcohol. Comparison of the residue obtained, with a n authentic sample of pentobarbitone confirmed its identity. The total amount of carbro­ mal in t h e stomach washout was 5-2 grains (340 mg.) a n d t h e total amount of pentobarbitone was 1^ grains (98 mg.). On recovering

T H E BARBITURATES

515

consciousness, the patient admitted taking 20 Carbrital capsules; Carbrital is a proprietary preparation containing 4 grains carbromal and 1^ grains pentobarbitone sodium. Case No. 2. A m a n was arrested for driving a motor vehicle whilst under t h e influence of drugs. The pohce constable stated t h a t the accused was unable to walk straight; t h a t his speech was slow and slurred; and t h a t his breath h a d a faint smell of alcohol. H e was medically examined one hour later and a urine sample was obtained. The doctor certified t h a t he could detect no medical cause for t h e man's condition. Examination of the urine sample gave negative tests for alcohol, methyl pentynol, paraldehyde, acetone, sugar, a n d albumen b u t a residue was obtained in t h e barbiturate group. This was submitted t o paper chromatography, and gave the p a t t e r n shown in Fig. 20.2. This is typical of phenobarbitone poisoning for in addition to the pheno­ barbitone spot the slower running spot of p-hydroxy phenobarbitone m a y be seen. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Curry, A . S. J. Pharm. Pharmacol, 1955, 7, 969. T s o - Y u c h H v a n g . Acta Pharm. Internat., 1951, 2, 4 4 3 . Maehly, A . C. Analyst. 1962, 87, 116. B r o u g h t o n , P . M. G., H i g g i n s , G., a n d O'Brien, J . R. P . Lancet, 1956, 270, 180. Parker, K . D . , a n d Kirk, P . L . Anal. Chem., 1961, 33, 1378. Street, H . V . Acta pharmacol. et toxical., 1962, 19, 312. SheUard, E . J . , a n d Osisiogu. I.U. Laboratory Practice, 1964, 13, 516. Cochm, J., a n d D a l y , J . W . J. Pharmacol and Exp. Therap., 1963, 139, 154. B o g a n , J . , R e n t o u l , E . , a n d S m i t h , H . J. Forens. Sei. Soc, 1964, 4, 147. Sunshine, I . Amer. J. Clin. Path., 1963, 40, 576. P i l s b u r y , V . B . , a n d J a c k s o n , J . V . J. Pharm. Pharmac, 1966, 18, 7 1 3 . Maynert, E . W . , a n d W a s h b u r n , E . J . J. Am. Chem. Soc, 1953, 75, 1700. Brooker, Ε . G. Analyst., 1957, 82, 448. Curry, A . S. Nature, 1959, 183, 1052. Street, H . V . , a n d McMartin, C. Clin. Chim. Acta, 1964, 9, 3 0 1 . J a c k s o n , J . V . Chromatographic and Electrophoretic Techniques ( S m i t h , I. E d . V o l 1, H e i n e m a n n , L o n d o n , 1960) NickoUs, L. C. Science in Criminal Investigation, B u t t e r w o r t h , 1956, p . 382. V a l o v , P . Ind. Eng. Chem., (Anal.), 1946, 18, 456. R a v e n t o s , J . J. Pharm. Pharmacol, 1954, 6, 217. Stewart, C. P . , a n d S t o l m a n , A . Toxicology, Mechanisms and Analytical Methods, Vol. 1. A c a d e m i c P r e s s , L o n d o n , 1960. Curry, A . S. J. Pharm. Pharmacol, 1955, 7, 1072. J a c k s o n , J . V . a n d Moss, M. S. Nature, 1961, 192, 5 5 3 . Moss, M. S. Proc Assoc Clin. Biochem., 1965, 218 B r o w n , T. L. J. Pharm. Sei., 1963, 52, 274.