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Determination of barbiturates in biological fluids
CLINICA
CHIMICA
547
.4CTA
ccA 4817
Determination
of barbiturates
in biological fluids
Some of the most widely employed methods for the determination of barbiburates are based on the well kown reaction of dithizone with Hgr1-barbiturate complex. In some procedures a combined one-step extraction of barbiturates in the presence of mercuric chloride is used’, which according to our experience gives false positive results caused mainly by interference of chlorides from biological material. L%modified procedure avoiding such interference and using a simple method for the qualitative and semi-quantitative determination of the Hg1i-barbiturate complex is suggested in this paper. Cheniicals nnd rccommendecl jwoccdure 0.5 Sl phosphate buffer solution pH 6.8-7.2 Chloroform G.R., redistilled 5 x IO-” RI water solution of mercuric chloride 1.75 x IO-~ M dithizone solution in chloroform,
prepared freshly every day.
Add 0.2 ml of buffer solution and 5 ml of chloroform to the 2 ml of sample in a 5oml separatory funnel. Shake vigorously I nun and after phase separation transfer the chloroform layer into another separatory funnel. Add to it I ml of the same buffer, 0.5 ml of H&l, solution and 3.5 ml of water. Shake I min, separate and centrifuge the chloroform layer, remove traces of aqueous phase by a strip of filter paper. Put 5 ml of dithizone reagent into a test tube. Take 3.5 ml of CHCl, extract with a 5-n11 graduated pipette and titrate slowly reagent with extract. Stop the titration as soon as the green colour of the reagent is changed to bright orange. From the consumption of CHCl, extract read the amount of barbiturate in the sample from a calibration curve obtained by analysis of standard aqueous solution of barbiturates (I-IO mg per IOO ml). RESULTS
Chloroform was found to be the most suitable organic solvent for extraction. For phase ratio chloroform-water 2 : I and pH 669, barbiturates with longer aliphatic chain or aryl-substituted derivatives are extracted with recovery better than 9896, the efficiency of extraction of 5,5-diethylbarbituric acid being about 75%. To change barbiturate into Hg*r-barbiturate complex, the chloroform layer is treated with an excess of mercuric chloride in water solution. However, mercuric chloride (depending on pH, concentration and phase ratio) is extracted into chloroform too and extraction increases greatly in the presence of chlorides. Thus, for example in 0.01 bl NaCl the extraction of mercuric chloride causes false positive results comparable with results obtained with a sample containing about 2 mg of barbiturate per IOO ml. Similar effect can be attained increasing the phase ratio of chloroform-water. Recause of the high concentration of chlorides in biological material this gives false positive results in modifications1 using one-step extraction in the presence of mercuric chloride. To avoid interference of chlorides, separate extraction of barbiturates from
548
RRIEF
SOTES
a sample is recommended. Xs optimal conditions enabling utilization of a single buffer solution for both extraction steps and preventing the interference of mercuric chloride were found phase ratio I :I, pH 6.8-7.2 and about tenfold excess of mercuric chloride to barbiturate. For the detection of Hg 11 in complex, back titration of standard solution with HgI’ -barbiturate extract was used enabling simultaneously
dithizone the semi-
quantitative interpretation of the results. By the procedure described, serum or whole blood can be directly analysed, urine and other specimens have to be brought to pH 6.5-7.5
The scatter of results is about & 0.3 mg for lower and r I mg for higher (:~-Io mg per IOO ml) levels of barbiturate in a sample. Full agreement with thin-layer chromatograpy and no risk of false negative results was experimentally confirmed. HOW ever, the interference of substances common to all methods inlrolving Hg”-barbturate
complexes
Research
Institute
PostgYaduatc
Received Clin.
Chirn.
must be taken into accounPT. of Puvc Chnicals,
Medical
September .I&,
Institute,
24, 1071
37 (197~)
517~ .jl’
Pmgw
Lachenza,
&T.c‘., Bmo
(Cscchosloi~af~ia)
\.. CHKO.\I+ J. S. B.413~1~
CHIMICA
547
.4CTA
ccA 4817
Determination
of barbiturates
in biological fluids
Some of the most widely employed methods for the determination of barbiburates are based on the well kown reaction of dithizone with Hgr1-barbiturate complex. In some procedures a combined one-step extraction of barbiturates in the presence of mercuric chloride is used’, which according to our experience gives false positive results caused mainly by interference of chlorides from biological material. L%modified procedure avoiding such interference and using a simple method for the qualitative and semi-quantitative determination of the Hg1i-barbiturate complex is suggested in this paper. Cheniicals nnd rccommendecl jwoccdure 0.5 Sl phosphate buffer solution pH 6.8-7.2 Chloroform G.R., redistilled 5 x IO-” RI water solution of mercuric chloride 1.75 x IO-~ M dithizone solution in chloroform,
prepared freshly every day.
Add 0.2 ml of buffer solution and 5 ml of chloroform to the 2 ml of sample in a 5oml separatory funnel. Shake vigorously I nun and after phase separation transfer the chloroform layer into another separatory funnel. Add to it I ml of the same buffer, 0.5 ml of H&l, solution and 3.5 ml of water. Shake I min, separate and centrifuge the chloroform layer, remove traces of aqueous phase by a strip of filter paper. Put 5 ml of dithizone reagent into a test tube. Take 3.5 ml of CHCl, extract with a 5-n11 graduated pipette and titrate slowly reagent with extract. Stop the titration as soon as the green colour of the reagent is changed to bright orange. From the consumption of CHCl, extract read the amount of barbiturate in the sample from a calibration curve obtained by analysis of standard aqueous solution of barbiturates (I-IO mg per IOO ml). RESULTS
Chloroform was found to be the most suitable organic solvent for extraction. For phase ratio chloroform-water 2 : I and pH 669, barbiturates with longer aliphatic chain or aryl-substituted derivatives are extracted with recovery better than 9896, the efficiency of extraction of 5,5-diethylbarbituric acid being about 75%. To change barbiturate into Hg*r-barbiturate complex, the chloroform layer is treated with an excess of mercuric chloride in water solution. However, mercuric chloride (depending on pH, concentration and phase ratio) is extracted into chloroform too and extraction increases greatly in the presence of chlorides. Thus, for example in 0.01 bl NaCl the extraction of mercuric chloride causes false positive results comparable with results obtained with a sample containing about 2 mg of barbiturate per IOO ml. Similar effect can be attained increasing the phase ratio of chloroform-water. Recause of the high concentration of chlorides in biological material this gives false positive results in modifications1 using one-step extraction in the presence of mercuric chloride. To avoid interference of chlorides, separate extraction of barbiturates from
548
RRIEF
SOTES
a sample is recommended. Xs optimal conditions enabling utilization of a single buffer solution for both extraction steps and preventing the interference of mercuric chloride were found phase ratio I :I, pH 6.8-7.2 and about tenfold excess of mercuric chloride to barbiturate. For the detection of Hg 11 in complex, back titration of standard solution with HgI’ -barbiturate extract was used enabling simultaneously
dithizone the semi-
quantitative interpretation of the results. By the procedure described, serum or whole blood can be directly analysed, urine and other specimens have to be brought to pH 6.5-7.5
The scatter of results is about & 0.3 mg for lower and r I mg for higher (:~-Io mg per IOO ml) levels of barbiturate in a sample. Full agreement with thin-layer chromatograpy and no risk of false negative results was experimentally confirmed. HOW ever, the interference of substances common to all methods inlrolving Hg”-barbturate
complexes
Research
Institute
PostgYaduatc
Received Clin.
Chirn.
must be taken into accounPT. of Puvc Chnicals,
Medical
September .I&,
Institute,
24, 1071
37 (197~)
517~ .jl’
Pmgw
Lachenza,
&T.c‘., Bmo
(Cscchosloi~af~ia)
\.. CHKO.\I+ J. S. B.413~1~