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The Spectrophotometric Determination of Low Levels of Barbiturates in Blood
T h e Spectrophotometric Determination of L o w Levels of Barbiturates in Blood H. M. STONE and C. R. HENWOOD Chemistry Division, Department of Scientific and Industrial Research Lower Hutt. N e w Zealand.
An investigation has been carried out on the determination of low levels of barbiturate in small samples of blood by measurement of ultraviolet spectra. Methods are compared and conditions and precautions described which allow a marked improvement in recovery with very little interference from blood chromogens,reagents, and common anticoagulants and preservatives. Introduction Many methods (Stolman, 1963) have been published describing the determination of barbiturates in blood, by means of their ultraviolet absorption spectra in alkaline solutions. Where blood barbiturate levels are more than 1milligram per 100 millilitres (mg/100 ml), and 10 ml or more of blood is available, very little trouble is experienced with these methods, although high recovery of barbiturate may not always be achieved. In the present investigation it was found that with blood samples of 5 ml or less, and phenobarbitone levels of less than 1 mg/100 ml of blood, considerable interference from reagents and blood chromogens was experienced. Interference from these sources was investigated and it was found that it could completely mask the barbiturate ultraviolet spectral peaks. In addition, depending on choice of solvent and extraction method, the recovery of barbiturate was found to be very variable. The effect of various anticoagulants and ~i.eservativesin the blood on possible interference to ultraviolet spectra of barbiturates, has also been investigated. As a result of this investigation, a method has been adopted which will allow the determination of 10 micrograms of barbiturate in 5 ml of blood (a level of 0.2 mg/100 ml), whereas previously, barbiturate present at this concentration could not be demonstrated adequately. At higher levels recovery was considerably improved. In a particular case, analysis by the modified procedure, of 1 ml of whole blood gave a barbiturate level of 5.5 mg/100 ml, whereas analysis 10 weeks previously by a conventional sodium tungstate procedure (Stewart, 1961) on a 5 ml sample of whole blood showed approximately 2.0 mg/100 ml. Experimental All spectra were run on a Beckman DK2A recording spectrophotometer. Diethyl ether Three common British sources of analytical reagent grade diethyl ether were examined. Ether from one source interfered with the ultraviolet spectra. When twenty-five ml were evaporated, the residue dissolved in 2 ml of 0.45N NaOH, and the spectrum recorded, a strong peak was present at 240 millimicrons (mp) and a weaker peak a t 315 mp. Pre-extraction with N sodium hydroxide solution only partly removed this interference. Each of the other sources of diethyl ether when extracted with sodium hydroxide imparted an intense brown colour to the alkali. This interference could be removed by extracting successively with 0.1N NaOH (twice) very dilute hydrochloric acid (once; this must still be acid after extracting) and water (twice). The preextracted ether was used only on the day of preparation. 51
Chloroform Analytical grade chloroform (50 ml to 100 ml) was evaporated, and the residue dissolved in 2 ml of 0.45N NaOH. The ultraviolet spectrum showed a slight interference effect between 240 and 290 mp. This interference was completely eliminated by preliminary distillation, rejecting the first and last portions. Sodium Sulfihate The common use of sodium sulphate as a drying agent for solvents saturated with water is, in this application, questionable. Not only did the use of sodium sulphate cause barbiturate recovery to be lowered, but it constituted a further source of interference. Various grades of anhydrous sodium sulphate, including analytical grade, and general purpose reagent, both granular and powdered were compared with analytical grade sodium sulphate purified by soxhlet extraction with chloroform, followed by heating a t 600°C overnight. Comparable quantities of each grade of sodium sulphate and the purified sample were extracted with approximately 50 ml of ether (purified as above). The ether was poured off, evaporated and the residues dissolved in 2 ml of 0.45N NaOH. The ultraviolet spectra of these solutions showed that interference with barbiturate spectra would result from all grades ranging from serious interference from the granular grade to only slight interference from the pre-treated sodium sulphate. The use of sodium sulphate is therefore not recommended. Recovery of Phenobarbitone from Aqueous Solutions (a) Direct Extraction: Aqueous solutions containing 25 micrograms (pg) of phenobarbitone in 5 ml of acidulated water were extracted by the solvents diethyl ether, chloroform, and carbon tetrachloride. The solvent extracts were either evaporated direct, or the barbiturate was isolated in the weak acid fraction (fraction B, Curry, 1963). In each case, the residue was dissolved in 2 ml of 0.45N sodium hydroxide, and the ultraviolet spectra recorded successively a t pH levels of 13, 10 and 2. Spectra from phenobarbitone solutions of known concentration were compared for quantitative estimation of barbiturate recovery. The recoveries obtained are shown in Table 1. TABLE 1 RECOVERY (PER CENT.) O F T'HENOBARBITONE FROM AQUEOUS SOLUTION Solvent Total Extract Weak Acid Fraction 90-100 70-7 5 Diethyl ether 20-35* 75-80 Chloroform Carbon tetrachloride 0 *This figure of 35 per cent. represents the maximum recovery that could be obtained, as a result of very thorough chloroform extraction a t each stage, and chloroform washing of the sodium bicarbonate extract.
The solvents were not dried, and evaporations were carried out on a rotary evaporator under reduced pressure, at a temperature a little below the boiling point of each solvent. (b) Tungstate Precipitation : Recoveries of phenobarbitone from aqueous solution, treated as for a protein removal by a tungstate precipitation procedure (Stewart and Stolman, 1961) were all in cases comparable to those by direct extraction.
Eva$oration of Solvents: Solutions of known amounts of phenobarbitone in diethyl ether and chloroform were divided and evaporated by the two following alternative procedures: (a) in a stoppered flask in a rotary evaporator, under reduced pressure and a little below the boiling point, and (b) in an open evaporating basin on a water bath. 52
The solvent solutions were still saturated with water, and evaporations were taken just to complete dryness. The recoveries were good from the ether solutions evaporated by either method and the cl~loroformsolutions evaporated in the rotary evaporator. The barbiturate recovery was, however, lower from the chloroform evaporated in the evaporating basin with losses of up to 40 per cent. being obtained. Drying of solvents: In all cases, the addition of anhydrous sodium sulphate to solutions of phenobarbitone in solvent resulted in incomplete recovery of the barbiturate. Even repeated extractions of the sodium sulphate failed to remove all the barbiturate. Due to the accompanying interference from the sodium sulphate, exact losses could not be measured, but can be estimated to be in the range of 20-40 per cent.
Interference from Blood Extraction of blood, unbuffered, buffered a t pH7, or acidified, both by chloroform and ether, gave rise to highly coloured direct solvent extracts. Even after isolation of the weak acid fraction, interfering effects on the ultraviolet spectra were marked. The chromogen removal method of McCallum (1954) for plasma or serum, and the Plaa and Hine (1956) method for blood were tried on whole blood without success a t the level of barbiturate being investigated. The tungstate precipitation method (Stewart, 1961) was found completely satisfactory when taken to the weak acid fraction stage.
Anticoagulants and Preservatives The effect of the anticoagulants and preservatives commonly used in blood samples, on recovery of phenobarbitone was first ascertained from aqueous solutions. Citrate-dextrose, sodium fluoride and potassium oxalate, mercuric chloride, heparin and disodium ethylene diamine tetraacetate were added to aqueous solutions of phenobarbitone in concentrations normally used in blood. These solutions were extracted with ether and with chloroform both directly and after tungstate treatment, the solvents evaporated, and spectra run in 0.45 N sodium hydroxide. No interference was experienced. Portions of blood containing these anticoagulants and preservatives were treated by the sodium tungstate protein precipitation procedure and extracted with ether and the weak acid fraction (fraction B, Curry, 1963) isolated. The only interference experienced was from the heparinised blood, where high background spectra were obtained. S9ectra at $H 10: The procedure of conversion of the 0.45N sodium hydroxide to pH 10 by addition of 16 per cent. ammonium chloride (Curry, 1963) was found to be very useful. At low barbiturate concentrations in the 0.45N NaOH, the peak a t 254 mp may be almost imperceptible. A peak shift to 239 mp provides very useful confirmation of the presence of a barbiturate. Acidification of Alkaline Solutions in Cells: The addition of acid to the solution in the spectrophotometer cell is known to be effective in nullifying the absorption due to the barbiturate. However, it was frequently found that acidification produced a turbidity, sometimes almost immediately and sometimes after a minute or two. Despite trials on purity of reagents, completeness of evaporation of solvent, cleanliness of apparatus and pre-extraction of reagents, this tendency could not reliably be prevented. The turbidity sometimes occurred in reagent checks with no blood present. This is an effect that would not be noticed a t higher dilutions. The only effective way to overcome it was by quick addition of acid in a standardised amount, followed immediately by readings a t 239 and 254 my, before turbidity could develop. 53
Recommendations A synopsis of the recommended procedures which enables a quantitative estimation of low levels of barbiturate in small samples of blood, by measurement of their ultraviolet spectra, is given below: (a) Heparinised blood is unsuitable, but the presence of other common anticoagulants and preservatives does not interfere. (b) Best results are obtained with the sodium tiingstate precipitation method carried to the weak acid fraction stage, using diethyl ether as solvent. (c) The diethyl ether requires pre-extraction with alkali to remove interfering substances. (d) Ether extracts should not be dried with sodium sulphate, but may be filtered through filter paper to remove water droplets. (e) The ether should be evaporated just to dryness in a rotary evaporator under reduced pressure, and a t a temperature below the boiling point. (f) Conversion of the alkaline solution in a spectrophotometer cell from pH 13 to pH 10 with ammonium chloride confirms the presence of barbiturate. (g) Acidification of a cell solution to obtain a non-barbiturate blank may produce cloudiness; this may be overcomc by taking rcadings a t 239 mp and 254 mp, immediately after addition of acid. References CURRY,A. S., 1963, Poison Detectiot8 i n H u m a n Organs, p. 42, Springfield, Illinois. Charles C. Thomas. MCCALLUM, N. E. W., 1954, J. Pharm, andI'harmacol., 6, 733. PLAA,G. L., and HINE,C. H., 1956, J. Lab. and Clin. Med., 47 (4), 649-57. STEWART, C. P., and STOLMAN, A., 1961, Toxicology, Mechanisms and Analytical Methods, Vol. 2, p. 158, New York and London. Academic Press. STOLMAN, A., 1963, Progress in Chemical Toxicology, Vol 1, p. 136, New York and London. Academic Press.
An investigation has been carried out on the determination of low levels of barbiturate in small samples of blood by measurement of ultraviolet spectra. Methods are compared and conditions and precautions described which allow a marked improvement in recovery with very little interference from blood chromogens,reagents, and common anticoagulants and preservatives. Introduction Many methods (Stolman, 1963) have been published describing the determination of barbiturates in blood, by means of their ultraviolet absorption spectra in alkaline solutions. Where blood barbiturate levels are more than 1milligram per 100 millilitres (mg/100 ml), and 10 ml or more of blood is available, very little trouble is experienced with these methods, although high recovery of barbiturate may not always be achieved. In the present investigation it was found that with blood samples of 5 ml or less, and phenobarbitone levels of less than 1 mg/100 ml of blood, considerable interference from reagents and blood chromogens was experienced. Interference from these sources was investigated and it was found that it could completely mask the barbiturate ultraviolet spectral peaks. In addition, depending on choice of solvent and extraction method, the recovery of barbiturate was found to be very variable. The effect of various anticoagulants and ~i.eservativesin the blood on possible interference to ultraviolet spectra of barbiturates, has also been investigated. As a result of this investigation, a method has been adopted which will allow the determination of 10 micrograms of barbiturate in 5 ml of blood (a level of 0.2 mg/100 ml), whereas previously, barbiturate present at this concentration could not be demonstrated adequately. At higher levels recovery was considerably improved. In a particular case, analysis by the modified procedure, of 1 ml of whole blood gave a barbiturate level of 5.5 mg/100 ml, whereas analysis 10 weeks previously by a conventional sodium tungstate procedure (Stewart, 1961) on a 5 ml sample of whole blood showed approximately 2.0 mg/100 ml. Experimental All spectra were run on a Beckman DK2A recording spectrophotometer. Diethyl ether Three common British sources of analytical reagent grade diethyl ether were examined. Ether from one source interfered with the ultraviolet spectra. When twenty-five ml were evaporated, the residue dissolved in 2 ml of 0.45N NaOH, and the spectrum recorded, a strong peak was present at 240 millimicrons (mp) and a weaker peak a t 315 mp. Pre-extraction with N sodium hydroxide solution only partly removed this interference. Each of the other sources of diethyl ether when extracted with sodium hydroxide imparted an intense brown colour to the alkali. This interference could be removed by extracting successively with 0.1N NaOH (twice) very dilute hydrochloric acid (once; this must still be acid after extracting) and water (twice). The preextracted ether was used only on the day of preparation. 51
Chloroform Analytical grade chloroform (50 ml to 100 ml) was evaporated, and the residue dissolved in 2 ml of 0.45N NaOH. The ultraviolet spectrum showed a slight interference effect between 240 and 290 mp. This interference was completely eliminated by preliminary distillation, rejecting the first and last portions. Sodium Sulfihate The common use of sodium sulphate as a drying agent for solvents saturated with water is, in this application, questionable. Not only did the use of sodium sulphate cause barbiturate recovery to be lowered, but it constituted a further source of interference. Various grades of anhydrous sodium sulphate, including analytical grade, and general purpose reagent, both granular and powdered were compared with analytical grade sodium sulphate purified by soxhlet extraction with chloroform, followed by heating a t 600°C overnight. Comparable quantities of each grade of sodium sulphate and the purified sample were extracted with approximately 50 ml of ether (purified as above). The ether was poured off, evaporated and the residues dissolved in 2 ml of 0.45N NaOH. The ultraviolet spectra of these solutions showed that interference with barbiturate spectra would result from all grades ranging from serious interference from the granular grade to only slight interference from the pre-treated sodium sulphate. The use of sodium sulphate is therefore not recommended. Recovery of Phenobarbitone from Aqueous Solutions (a) Direct Extraction: Aqueous solutions containing 25 micrograms (pg) of phenobarbitone in 5 ml of acidulated water were extracted by the solvents diethyl ether, chloroform, and carbon tetrachloride. The solvent extracts were either evaporated direct, or the barbiturate was isolated in the weak acid fraction (fraction B, Curry, 1963). In each case, the residue was dissolved in 2 ml of 0.45N sodium hydroxide, and the ultraviolet spectra recorded successively a t pH levels of 13, 10 and 2. Spectra from phenobarbitone solutions of known concentration were compared for quantitative estimation of barbiturate recovery. The recoveries obtained are shown in Table 1. TABLE 1 RECOVERY (PER CENT.) O F T'HENOBARBITONE FROM AQUEOUS SOLUTION Solvent Total Extract Weak Acid Fraction 90-100 70-7 5 Diethyl ether 20-35* 75-80 Chloroform Carbon tetrachloride 0 *This figure of 35 per cent. represents the maximum recovery that could be obtained, as a result of very thorough chloroform extraction a t each stage, and chloroform washing of the sodium bicarbonate extract.
The solvents were not dried, and evaporations were carried out on a rotary evaporator under reduced pressure, at a temperature a little below the boiling point of each solvent. (b) Tungstate Precipitation : Recoveries of phenobarbitone from aqueous solution, treated as for a protein removal by a tungstate precipitation procedure (Stewart and Stolman, 1961) were all in cases comparable to those by direct extraction.
Eva$oration of Solvents: Solutions of known amounts of phenobarbitone in diethyl ether and chloroform were divided and evaporated by the two following alternative procedures: (a) in a stoppered flask in a rotary evaporator, under reduced pressure and a little below the boiling point, and (b) in an open evaporating basin on a water bath. 52
The solvent solutions were still saturated with water, and evaporations were taken just to complete dryness. The recoveries were good from the ether solutions evaporated by either method and the cl~loroformsolutions evaporated in the rotary evaporator. The barbiturate recovery was, however, lower from the chloroform evaporated in the evaporating basin with losses of up to 40 per cent. being obtained. Drying of solvents: In all cases, the addition of anhydrous sodium sulphate to solutions of phenobarbitone in solvent resulted in incomplete recovery of the barbiturate. Even repeated extractions of the sodium sulphate failed to remove all the barbiturate. Due to the accompanying interference from the sodium sulphate, exact losses could not be measured, but can be estimated to be in the range of 20-40 per cent.
Interference from Blood Extraction of blood, unbuffered, buffered a t pH7, or acidified, both by chloroform and ether, gave rise to highly coloured direct solvent extracts. Even after isolation of the weak acid fraction, interfering effects on the ultraviolet spectra were marked. The chromogen removal method of McCallum (1954) for plasma or serum, and the Plaa and Hine (1956) method for blood were tried on whole blood without success a t the level of barbiturate being investigated. The tungstate precipitation method (Stewart, 1961) was found completely satisfactory when taken to the weak acid fraction stage.
Anticoagulants and Preservatives The effect of the anticoagulants and preservatives commonly used in blood samples, on recovery of phenobarbitone was first ascertained from aqueous solutions. Citrate-dextrose, sodium fluoride and potassium oxalate, mercuric chloride, heparin and disodium ethylene diamine tetraacetate were added to aqueous solutions of phenobarbitone in concentrations normally used in blood. These solutions were extracted with ether and with chloroform both directly and after tungstate treatment, the solvents evaporated, and spectra run in 0.45 N sodium hydroxide. No interference was experienced. Portions of blood containing these anticoagulants and preservatives were treated by the sodium tungstate protein precipitation procedure and extracted with ether and the weak acid fraction (fraction B, Curry, 1963) isolated. The only interference experienced was from the heparinised blood, where high background spectra were obtained. S9ectra at $H 10: The procedure of conversion of the 0.45N sodium hydroxide to pH 10 by addition of 16 per cent. ammonium chloride (Curry, 1963) was found to be very useful. At low barbiturate concentrations in the 0.45N NaOH, the peak a t 254 mp may be almost imperceptible. A peak shift to 239 mp provides very useful confirmation of the presence of a barbiturate. Acidification of Alkaline Solutions in Cells: The addition of acid to the solution in the spectrophotometer cell is known to be effective in nullifying the absorption due to the barbiturate. However, it was frequently found that acidification produced a turbidity, sometimes almost immediately and sometimes after a minute or two. Despite trials on purity of reagents, completeness of evaporation of solvent, cleanliness of apparatus and pre-extraction of reagents, this tendency could not reliably be prevented. The turbidity sometimes occurred in reagent checks with no blood present. This is an effect that would not be noticed a t higher dilutions. The only effective way to overcome it was by quick addition of acid in a standardised amount, followed immediately by readings a t 239 and 254 my, before turbidity could develop. 53
Recommendations A synopsis of the recommended procedures which enables a quantitative estimation of low levels of barbiturate in small samples of blood, by measurement of their ultraviolet spectra, is given below: (a) Heparinised blood is unsuitable, but the presence of other common anticoagulants and preservatives does not interfere. (b) Best results are obtained with the sodium tiingstate precipitation method carried to the weak acid fraction stage, using diethyl ether as solvent. (c) The diethyl ether requires pre-extraction with alkali to remove interfering substances. (d) Ether extracts should not be dried with sodium sulphate, but may be filtered through filter paper to remove water droplets. (e) The ether should be evaporated just to dryness in a rotary evaporator under reduced pressure, and a t a temperature below the boiling point. (f) Conversion of the alkaline solution in a spectrophotometer cell from pH 13 to pH 10 with ammonium chloride confirms the presence of barbiturate. (g) Acidification of a cell solution to obtain a non-barbiturate blank may produce cloudiness; this may be overcomc by taking rcadings a t 239 mp and 254 mp, immediately after addition of acid. References CURRY,A. S., 1963, Poison Detectiot8 i n H u m a n Organs, p. 42, Springfield, Illinois. Charles C. Thomas. MCCALLUM, N. E. W., 1954, J. Pharm, andI'harmacol., 6, 733. PLAA,G. L., and HINE,C. H., 1956, J. Lab. and Clin. Med., 47 (4), 649-57. STEWART, C. P., and STOLMAN, A., 1961, Toxicology, Mechanisms and Analytical Methods, Vol. 2, p. 158, New York and London. Academic Press. STOLMAN, A., 1963, Progress in Chemical Toxicology, Vol 1, p. 136, New York and London. Academic Press.