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A non-ionic resin extraction of drugs in blood
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Clinica Chimica Acta, 53 (1974) 389-390 @Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BRIEF TECHNICAL NOTE CCA 6316
A NON-IONIC RESIN EXTRACTION OF DRUGS IN BLOOD
A.W. MISSEN and J.F. LEWIN Chemistry Division, Department Zealand)
of Scientific and Industrial Research, Petone, (New
(Received November 12th, 1973)
The non-ionic resin Amberlite XAD-2 has been shown to be suitable for the extraction of drugs from urine [l] . Its application to the extraction of drugs from whole blood is presented in comparison with the effectiveness of solvent extraction and protein precipitation procedures. Methods
Amberlite XAD-2 extraction A 5 ml blood sample was diluted to 20 ml with water and stirred with 3 ml of an XAD-2 resin slurry (thoroughly pre-washed with acetone and water) on a rolling mill for 1 hour. The diluted blood was passed through a sintered glass filter and the trapped resin was washed with water. After removing residual water, drugs adsorbed onto the resin were eluted with 3 X 10 ml redistilled acetone. The acetone solution was evaporated down and drug recoveries were determined using gas-liquid chromatography (GLC).
Solvent extraction A 5 ml blood sample was diluted to 20 ml with 1% sodium chloride solution, acidified with 1.5 ml of 1N sulphuric acid, and extracted with 100 ml ether on a rolling mill for 1 hour. The ether phase was separated, washed with 2 X 25 ml of 2% sodium hydrogen carbonate solution to remove pigments, and was concentrated under vacuum to give weakly acidic and neutral drugs. The diluted blood was made alkaline with 3 ml of 1N sodium hydroxide, and again extracted with 100 ml ether on the rolling mill. Concentration of the ether phase under vacuum gave basic drugs.
Fro tein precipitation The method outlined by Curry [2] was followed in extracting weakly acidic and neutral drugs. A 5 ml blood sample was stirred with 30ml of 0.35% sodium hydroxide solution. After standing for 10 minutes, 10 ml of 10% sodium
390
tungstate solution followed by 3.5 ml of 10% sulphuric acid were added dropwise to the stirred solution. The mixture was heated at 100” for 10 minutes, and the precipitated protein was filtered off. The cooled filtrate was extracted with 2 X 50 ml ether, and the ether phases were concentrated to give weakly acidic and neutral drugs. To obtain information on the recoveries of drugs using the various extraction methods, drug standards were prepared in human transfusion blood. One standard contained 30 pg/ml amylobarbitone with 10 pg/ml methaqualone, and the other contained 10 pg/ml amitriptyline. These drugs are representative of a wide range of weakly acidic, neutral, and basic drugs which are commonly encountered in post-mortem analyses. Each extraction procedure was applied four times to both of the standard solutions. Recoveries were calculated in each case using a 3% OV-17 column in a Perkin-Elmer Fll gas chromato~aph. Results and discussion Drug recoveries achieved by the different extraction methods are tabulated below. Method
70Recovery + Amylobarbitone
Methaqualone
Amitriptyline
XAD-2 Solvent
81 (70-96) 75 (65-90)
Protein precipitation
72 (63-80)
74 (68-85) 73 (66-78) 36 (32-40)
61 (57-65) 17 (15-21) Not determined
* Mean, and range in brackets.
It was found that the solvent method extracted a significant qu~tity of fats, which could cause interference with the GLC analysis of many drugs. In contrast, the XAD-2 resin technique gave very clean extracts, as long as redistilled solvents were used. Cholesterol was extracted to some extent with the resin but it did not interfere with the GLC analyses. The solvent extraction method gave a very low recovery for the basic drug amitriptyline, and the protein precipitation method gave an unsatisfactory recovery for methaqu~one. The XAD-2 resin gave the most efficient recovery for all the drugs and was the least complicated of the extraction techniques. The Amberlite XAD-2 resin extraction has proved, in our experience, to be most suitable for the routine screening and analysis of drugs in blood. References 1 LB. Hetland, D.A. Knowlton and D. Couri, Clin. Chim. Acta. 36 (1972) 473. 2 A.S. Curry. in C.P. Stewart and A. Stolman (Eds). Toxicology - Mechanisms and Analytical Methods, Vol. 2, Academic Press, New York and London, 1961, p. 158
Clinica Chimica Acta, 53 (1974) 389-390 @Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BRIEF TECHNICAL NOTE CCA 6316
A NON-IONIC RESIN EXTRACTION OF DRUGS IN BLOOD
A.W. MISSEN and J.F. LEWIN Chemistry Division, Department Zealand)
of Scientific and Industrial Research, Petone, (New
(Received November 12th, 1973)
The non-ionic resin Amberlite XAD-2 has been shown to be suitable for the extraction of drugs from urine [l] . Its application to the extraction of drugs from whole blood is presented in comparison with the effectiveness of solvent extraction and protein precipitation procedures. Methods
Amberlite XAD-2 extraction A 5 ml blood sample was diluted to 20 ml with water and stirred with 3 ml of an XAD-2 resin slurry (thoroughly pre-washed with acetone and water) on a rolling mill for 1 hour. The diluted blood was passed through a sintered glass filter and the trapped resin was washed with water. After removing residual water, drugs adsorbed onto the resin were eluted with 3 X 10 ml redistilled acetone. The acetone solution was evaporated down and drug recoveries were determined using gas-liquid chromatography (GLC).
Solvent extraction A 5 ml blood sample was diluted to 20 ml with 1% sodium chloride solution, acidified with 1.5 ml of 1N sulphuric acid, and extracted with 100 ml ether on a rolling mill for 1 hour. The ether phase was separated, washed with 2 X 25 ml of 2% sodium hydrogen carbonate solution to remove pigments, and was concentrated under vacuum to give weakly acidic and neutral drugs. The diluted blood was made alkaline with 3 ml of 1N sodium hydroxide, and again extracted with 100 ml ether on the rolling mill. Concentration of the ether phase under vacuum gave basic drugs.
Fro tein precipitation The method outlined by Curry [2] was followed in extracting weakly acidic and neutral drugs. A 5 ml blood sample was stirred with 30ml of 0.35% sodium hydroxide solution. After standing for 10 minutes, 10 ml of 10% sodium
390
tungstate solution followed by 3.5 ml of 10% sulphuric acid were added dropwise to the stirred solution. The mixture was heated at 100” for 10 minutes, and the precipitated protein was filtered off. The cooled filtrate was extracted with 2 X 50 ml ether, and the ether phases were concentrated to give weakly acidic and neutral drugs. To obtain information on the recoveries of drugs using the various extraction methods, drug standards were prepared in human transfusion blood. One standard contained 30 pg/ml amylobarbitone with 10 pg/ml methaqualone, and the other contained 10 pg/ml amitriptyline. These drugs are representative of a wide range of weakly acidic, neutral, and basic drugs which are commonly encountered in post-mortem analyses. Each extraction procedure was applied four times to both of the standard solutions. Recoveries were calculated in each case using a 3% OV-17 column in a Perkin-Elmer Fll gas chromato~aph. Results and discussion Drug recoveries achieved by the different extraction methods are tabulated below. Method
70Recovery + Amylobarbitone
Methaqualone
Amitriptyline
XAD-2 Solvent
81 (70-96) 75 (65-90)
Protein precipitation
72 (63-80)
74 (68-85) 73 (66-78) 36 (32-40)
61 (57-65) 17 (15-21) Not determined
* Mean, and range in brackets.
It was found that the solvent method extracted a significant qu~tity of fats, which could cause interference with the GLC analysis of many drugs. In contrast, the XAD-2 resin technique gave very clean extracts, as long as redistilled solvents were used. Cholesterol was extracted to some extent with the resin but it did not interfere with the GLC analyses. The solvent extraction method gave a very low recovery for the basic drug amitriptyline, and the protein precipitation method gave an unsatisfactory recovery for methaqu~one. The XAD-2 resin gave the most efficient recovery for all the drugs and was the least complicated of the extraction techniques. The Amberlite XAD-2 resin extraction has proved, in our experience, to be most suitable for the routine screening and analysis of drugs in blood. References 1 LB. Hetland, D.A. Knowlton and D. Couri, Clin. Chim. Acta. 36 (1972) 473. 2 A.S. Curry. in C.P. Stewart and A. Stolman (Eds). Toxicology - Mechanisms and Analytical Methods, Vol. 2, Academic Press, New York and London, 1961, p. 158