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Distribution of Drugs and Toxic Chemicals in Blood
3. Forens. Sci. Sac.
(1980), 20, 187
Received 11th March 1980
Distribution of Drugs and Toxic Chemicals in Blood M. D. OSSELTON,* M. D. HAMMOND and A. C. MOFFAT Home O f i e Central Research Establishment, Aldermaston, Reading, Berkshire, England, RG7 4PN
The distribution of 61 drugs and toxic chemicals in dzfferent blood components has been examined. Data are presented which demonstrate the wide dayerence in distribution of drugs between dzfferent blood components. A recommendation is made that unfractionated blood should be used for forensic analyses. Introduction A number of recently published articles have drawn attention to the possible association between road traffic accidents and drug taking by road users (Bestic, 1979; Moffat, 1977; Raffle, 1977; Silverstone, 1977). One estimation of the number of drivers taking psychoactive drugs in Britain (excluding alcohol) has placed the number between 3% and 5% i.e., some 500,000 drivers (Silverstone, 1977). Since it is an offence to drive or attempt to drive or to be in charge of a vehicle whilst unfit to drive through drugs, the forensic toxicologist may be requested to perform drug analyses on blood specimens submitted by police officers where drug taking has been implicated or suspected. Because the quantity of blood submitted for analysis is likely to be limited and because drugs are not uniformly distributed between different blood components, we have investigated the available data on the distribution of drugs in blood in order to decide whether analyses should be performed on serum or plasma, as in most clinical and pharmacokinetic laboratories, or whether unfractionated blood should be examined. The published data have been supplemented by values determined experimentally for this study. During a recent literature search we noted that out of 92 papers concerned with methods for the analysis of drugs in blood only 12 presented results from the analysis of whole blood. I t was also noted that 5 of the authors of these 12 papers were employed in forensic science laboratories and out of the remaining 80 papers describing the analysis of blood plasma for drugs, only one forensic science laboratory was cited. Materials and Methods Blood Sambles Blood samples were provided by informed volunteers receiving drug therapy at Park Prewitt Hospital, Basingstoke during venepuncture extraction of blood for routine biochemical examination. Blood was collected into heparinised containers. Procedures involving human volunteers were approved by the hospital medical ethics committee. Aliquots of whole blood were removed from the heparinised containers and stored frozen prior to analysis. Plasma from each sample was collected and stored a t - 20°C. Reagents Trizma base (Sigma Chemical Co.) was dissolved in water to yield a 1.OM solution (pH 10.5). *Present address: Home Office Forensic Science Laboratory, Shakespeare Street, Nottingham, England, NGl 4FR. 187
n-Butyl acetate (Analytical Reagent Grade, B.D.H. Ltd.) was redistilled before use. Drug standards (lmg/ml) were prepared in absolute alcohol and diluted to working strength with redistilled butyl acetate. Extraction of Drugs from Blood 50p1 of Trizma base was added to a glass tube (50 x 6mm) containing 50 to 2 0 0 ~ 1blood or plasma. Butyl acetate (50 to 100~1)containing prazepam internal standard (0.2mg/l) was next added and the tube contents were mixed for 30 seconds using a 'Whirlimixer'. Specimens were centrifuged (12,000 rpm) for 2 minutes and aliquots (2 to 5p1) of the upper solvent phase were used for gas chromatographic analysis. Gas-Liquid Chromatography (GLC) Drugs were analysed using a column of 3% w/w OV-17 on Chromosorb W H P (80-100 mesh) in a Pye Series 104 gas chromatograph. Nitrogen carrier gas maintained at a flow rate of 50ml/minute and a column temperature of 264°C was used for chromatography of benzodiazepines. The column effluent was split 50 : 50 to a flame ionisation detector (FID) and a 63Ni electron capture detector (ECD). Make-up gas (nitrogen, 50ml/min) was used to supplement gas flow through the ECD. Blood extracts were also analysed using a Perkin Elmer F33 gas chromatograph equipped with a phosphorus/nitrogen detector (PND) and FID. Nitrogen carrier gas was passed through a l m x 4mm column of 3% w/w OV-17 on Chromosorb W H P (100-120 mesh) at a flow of 40ml/minute. Gas leaving the column was split 50 : 50 to the PND and F I D detectors.
Results and Discussion The major functions of blood in relation to drug therapy are transport and storage, and any drugs present in blood are distributed in a non-uniform manner between different blood components (Curry, 1977). They may be transported as complexes bound to plasma proteins such as albumin or aglycoproteins (Meyer and Guttman, 1968; Schneider et al., 1979) or associated with other blood components, for example erythrocytes, platelets and leucocytes (Maren et al., 1960; Beerman et al., 1975; Ahtee and Paasonan, 1966; Rollo, 1965). Data relating to the distribution of 61 substances in different blood components are presented in Table 1 which may aid the correlation of whole blood and plasma drug levels. Results obtained in our laboratory have been supplemented by information retrieved from published data. Since the percentage of blood volume occupied by cellular constituents varies between 43% v/v and 48% v/v according to age and sex (Diem and Lentner, 1970) a mean value of 46% v/v was assumed for our calculations. Using this figure, the distribution of the drugs was calculated as 1-85 for plasma: whole blood in the situation where drugs were confined to the plasma. Where drugs were equally distributed between blood fractions a value of 0.93 was obtained. Ratios lower than 0.93 indicated that more drug was present in the cellular fraction than in the plasma. Many published figures on drug distribution in blood are presented as percentage of drug in plasma without providing ratios. I n an attempt to provide a uniform presentation of the data in Table 1, we have also calculated the percentage of drug in plasma from available data on plasma: whole blood ratios using 1-85 as the figure representing 100% of drug in the plasma. Figures concerning percentage of drug in any fraction should be regarded as approximate since the effects of age, sex, disease and the presence of other drugs may cause significant changes in the plasma levels of many drugs (e.g., Crooks et al., 1976; Johnsson and Regardh, 1976). For example Chan et al. (1975) reported that pethidine binds with high affinity to the red cells of young patients to a significantly higher extent than in older patients and suggested that such differences could be used to explain the high
TABLE 1 DISTRIBUT'ION OF DRUGS AND TOXIC CHEMICALS IN BI.OOD Ruth Compound
Plasma : Whole Blood
Acetazolamide
-
Aldrin Alprenolol
-
Amitriptyline Amphetamine
1.21
-
Ampicillin
-
Arsenic Atenolol
--
-
Plasma : Erythrocyte Concentrated in red cells -
80 85
-
-
65
96
Approximately 0.8 but varies with time after dose -
Even distribution 0.83
-
Even distribution
Carbamazepine
-
Red cell level 1 5 2 0 % of plasma level
Chlordecone Chlorpromazine
0.57
Chlorthalidone
-
Clomipramine Cocaine Cyanide
1.17
Fluoride Gold Imipramine Indomethacin Lanatoside C Medazepam Mercury Metaprolol 3-Methyl Digexin Oxalate Pentachlorophenol Pentazocine Pentobarbitone Pethidine
-
-
Observed in red cells Red cell level x 50-100 that in plasma
Phenobarbitone
-
-
Almost all in plasma 50 41.5
25
Ehrnebo (1978)
43 50 -
Binds to red cells in young subiects
-
Baselt (1978) Johnsson and Regardh (1976) 46-58 Bender et al. (1975) - Stewart and Stolman (1960) 72 Pynnonen and Yrjana (1977) 91-99
-
Baselt (1978) Zingales (1969)
-
-
FIeuren and van Rossum (1978)
-
This study Javaid et al. (1978) Baselt (1978)
30
-
-
-
-
62 Distribution variable Most whole blood CN- in erythrocytes 3.4 1.66 89 98 1.83 75 98 - Equal distribution 50 68 1.26 40 0.60.9 72 -Restricted to serum in most patients 53 0.98 100 -16 1-85 99 5 -0.83 41.5
1.3 Whole blood level higher than plasma level
Reference Maren and Robinson (1960) Baselt (1978) ,Johnsson and Regardh (1976) This study Beckett et al., (1969)
-
-
Phenacetin
Protein Bound
-
-
Deslanoside Desmethyldiazepam Diazepam Dieldrin Digitoxin Digoxin Diphenhydramine Ethambutol
%
zn Plasma
-
Benzylpenicillin Caffeine
-
%Drug
-
-
-
95 23 79 8
Fletcher et al. (1979) This study This study Baselt (1978) Baselt (1978) Baselt (1978) This study Lee et al. (1977)
-
Baselt (1978) Baselt (1978)
96
-
75-96 This study 90 McArthur et al. (1971) - Fletcher et al. (1979) - This study - Baselt (1978) 12 Johnsson and Regardh f 1976) - ~ l d t c h e et r al. (1979) - Baselt (1978) -
99 50 50
-
-
-
70 50
40 30
50
33
-
Baselt (1978) Ehrnebo, et al. (1974) Stewart & Stolman (1960) Chan, et al. (1975) Morganetal.(1978) Stewart & Stolman (1960) Stewart & Stolman (1960)
TABLE 1-Continued
Compound Phenylbutazone Phenytoin
Plasma : Whole Blood 1.64
Ratios Plasma : Eythrocyte A
% Drug zn Plasma 96 88
Prilocaine Promethazine Propranolol 1.23 Quinidine
-
Quinine Salicylic Acid
-
Spironolactone Sulphadiazine Temazepam Theophylline
-
2 .o
-
1.87 1.22
a 9 Tetrahydro cannabinol Thiopentone
-
Thioridazine
-
Tiflorax Tolomol
-
Trimiprarnine Tubocurarine Warfarin
-
1.23
-
-
Plasma level > red cell level Equally distributed Appreciable amounts in red cells -
-
Little drug bound to red cells Observed in red cells 0.16-0.25 2 .O -
Confined to plasma
-
66 -
50
-
100 50 100 66
%
Protein Reference Bound W 9 8 Wallace, et al. (1976) 87 Kurata and Wilkinson (1974) 50 Baselt (1978) 75 This study 90 Isaacs and Schoenwald ( 1974) Baselt (1978) 60-80 Isaacs and Schoenwald (1974) 75-90 Raselt (1978) McArthur, et al. (1971) 75 74-94 Rorga, et al. (1977) - Karim, et al. (1976) 20-68 Wallace, et al. (1976) - This study 15 Mitenko and Ogilvie (1973) 87-90 Widman, et al. (1974)
-
75
-
-
13 90
A
66 100
-
94
98.6
97
Stewart & Stolman (1960) Zingales (1969) Biachetti et al. (1978) Johnsson and Regardh (1976) This study Stewart & Stolman ( 1960) Crooks, et a1 (1976)
serum levels of this drug observed in older patients. Age related differences in drug absorption, metabolism and excretion could also have significant effects on drug levels in the blood of old and young subjects. Results published by Piafsky and Borga (1977) reported that women taking oral contraceptives had lower concentrations of albumin and a,-acid glycoprotein and lower levels of alprenolol and imipramine bound to plasma proteins than women not using oral contraceptives. The data in Table 1 clearly reveal that drugs are widely distributed between various blood components and that considerable amounts of some drugs are present in red cells, e.g., acetazolamide, chlorthalidone (> go%), ethambutol (60%), pentazocine (50%) and tiflorax (87%). Thus, drugs are not always primarily associated with plasma proteins. Fletcher et al. (1979) reported that digoxin binds to red cell membranes and haemoglobin in addition to being present in plasma. They found it necessary to haemolyse whole blood and to remove red cell ghosts before assaying for digoxin by radioimmunoassay since the membrane-bound drug was only released slowly over a period of several hours. Consideration should also be given to changes which may occur during the period between collection of the blood specimen and subsequent analysis. Very slight haemolysis of blood samples (undetectable to the eye) containing compounds such as chlorthalidone (red cell levels 50 to 100 times that in plasma) could produce significant changes in the measured plasma level of these drugs and subsequently result in false interpretation of the plasma levels. Cotham and Shand (1975) demonstrated that plasticisers in Vacutainer blood collection tubes displaced propranolol bound to plasma proteins into the red cells resulting
in the measurement of 'spuriously low plasma propranolol levels'. The major impurities shown to enter blood and to interfere with analyses have been identified as di-(2-ethylhexy1)-phthalate and trisbutoxyethyl phosphate. These substances may be introduced from tube stoppers, stopper lubricants, polyvinyl chloride blood transfusion storage bags and filter papers (Dickson et al., 1974; Piafsky and Borga, 1976; Ramsey et al., 1980). I t was later shown that the lowering of the plasma concentrations of alprenolol and imipramine by displacement from plasma proteins by these impurities is from 76% to 16% and 69% to 13% respectively (Borga et al., 1977). The data in Table 1 are presented as a histogram in Figure 1 which illustrates that more drugs are concentrated in the plasma than in the erythrocytes. Plasma appears to be the most favoured phase to be used in drug analysis by clinical chemists and pharmacokineticists, and hence more information is available to aid the interpretation of results. However, the analysis of plasma alone may result in failure to identify some drugs which are present. I n view of the lack of published information on drug levels in whole blood, plasma : whole blood or plasma : erythrocyte ratios may be used to correlate whole blood levels with published plasma drug levels and hence may provide a useful guideline for the interpretation of results. I n many instances the forensic analyst will have no choice between analysing whole blood and plasma since specimens are frequently haemolysed on arrival at the laboratory. However, if the choice is available, with the exception of some immunoassay analyses, unfractionated blood should be selected in order to (a) detect as many com-
% Drug in Plasma Figure 1.
Histogram of the distribution of fifty-six drugs and toxic chemicals in blood. 191
pounds as possible in a given blood sample and (b) provide a more realistic picture of blood drug levels for the interpretation of analytical findings. Conclusions The distribution of drug substances between blood components varies considerably and may be affected by such factors as age, sex, disease, presence of other drugs, storage conditions and age of the specimen. In order to detect as many compounds as possible in a given blood sample unfractionated blood should be selected for analysis. Since relatively little data are available to guide analysts with the interpretation of the levels for drugs found in unfractionated blood, whole blood : plasma ratios or erythrocyte : plasma ratios may be used to aid interpretation of results. References AHTEE,L. and PAASONAN, M. K., 1966, J. Pharm. Pharmacol., 18, 126. BASELT, R. C., 1978, Disposition of Toxic Drugs and Chemicals in Man, Biomedical Publications. Canton.. Conn.. U.S.A. M., 1969, J. Pharm. Pharmacol., BECKETT, A. H'., SALMON, J. A. and MITCHARD, 21. 25 1. BEERMAN, B., HELLSTROM, K., LINDSTROM, B. and ROSEN,A., 1975, Clin. Pharmacol., Ther., 17, 424. BENDER, A. D., POST,A., MEIER,J. P., HIGSON, J. E. and REICHARD, G., 1975, 3. Pharm. Sci., 64, 1711. BESTIC, A., 1979, Drive, No. 55, p. 32, A.A. Publications Ltd. BIACHETTI, G., MITCHARD, M. and MORSELLI, P. L., 1978, J. Chromatogr., 152, 87. BORGA,O., PIAFSKY, K. M. and NILSEN,0. G., 1977, Clin. Pharmacol. Ther., 22, 539. M., WELLS,W. D. E. and VICKERS, CHAN,K., KENDALL, M. J., MITCHARD, M. D., 1975, Br. J. Clin. Pharmacol., 2, 297. COTHAM, R. H. and SHAND,D., 1975, Clin. Pharmacol. Ther., 18, 535. I. H., 1976, Clin. Pharmacokinet., 1, CROOKS, J., O'MALLEY,K. and STEVENSON, 280. CURRY,S. H., 1977, Drug Disposition and Pharmacokinetics, 2nd Ed., p. 26, Blackwell, Oxford. DICKSON, S. J., MISSEN, A. W. and DOWN,G. J., 1974, Forens. Sci., 4, 155. DIEM,K. and LENTNER, C., 1970, Eds., Docurnenta Geigy, Scientijic Tables, 7th Edn., p. 617, Geigy, Basle, Switzerland. E. and LONROTH, U., EHRNEBO, M., AGURELL, S., BIREUS,L. O., GORDON, 1974, Clin. Pharmacol. Ther., 16, 424. EHRNEBO, M., 1978,J. Pharm. Pharmacol., 30, 730. G. J. and MOFFAT, A. C., 1979,J. Forens. Sci. Soc., FLETCHER, S. M., LAWSON, 19, 183. J. M., 1978, J.Chromatogr., 152,41. FLEUREN, H. L. J. M. and VAN ROSSUM, ISAACS, V. E. and SCHOENWALD, R. D., 1974, J. Pharm. Sci., 63, 1267. JAVAID, J. I., DEKIRMENJIAN, H., DAVIS,J. M. and SCHUSTER, C. R., 1978, J. Chromatogr., 152, 105. JOHNSSON, G. and REGARDH, C . G., 1976, Clin. Pharmacokinet., 1, 233. J. and DANBY, M., 1976, Clin. Pharmacol. KARIM, A., ZAGARELLA, B. A., HRIBAR, Ther., 19, 158. KURATA, D. and WILKINSON, G. R., 1974, Clin. Pharmacol. Ther., 16, 355. J. G., BRATER, D. C. and BENET,L. Z., 1977, Clin. LEE, C. S., GAMBERTOGLIO. Pharmacol. Ther., 22, 615. MAREN,T. H. and ROBINSON, B., 1960, Bull. J. Hopkins Hosp., 106, 1. MCARTHUR, J. N., DAWKINS, P. D. and SMITH,M. J. H., 1971, J. Pharm. Pharmacol., 23, 32. 192
MEYER,M. C. and GUTTMAN, D. E., 1968, J. Pharm. Sci., 57, 895. P. A. and OGILVIE, R. I., 1973, Clin. Pharmacol. Ther., 14,569. MITENKO, MOFFAT, A. C., 1977, Pharm. J., 219, 310. E., 1978, Clin. Pharmacol. MORGAN, D., MOORE,G., THOMAS, J. and TRIGGS, Ther., 23, 288. PIAFSKY, K. M. and BORGA,O., 1976, Lancet, ii, 963. PIAFSKY, K. M. and BORGA, O., 1977, Clin. Pharmacol. Ther., 22, 545. PYNNONEN, S. and YRJANA, T., 1977, Int. 3. Clin. Pharmacol., 15, 222. RAFFLE,P. A. B., 1977, Pharm. J., 219, 3 11. RAMSEY, J. D., LEE, D. T., OSSELTON, M. D. and MOFFAT,A. C., 1980, J. Chromatogr., 184, 185. L. S. and GILMAN, A., Pharmacological Basis ROLLO,I. M., 1965, in GOODMAN, of Therapeutics, 3rd Ed., p. 1058, Macmillan, New York. R. E., BISHOP, H., HAWKINS, C. F. and KITIS,G., 1979, Lancet, ii, SCHNEIDER, 554. J. T.. 1977, Pharm. J., 219, 309. SILVERSTONE, C. P. and STOLMAN, A., 1960, Eds., Toxicology-Mechanisms and STEWART, Analytical Methods, Vol. 1, Part 1, Academic Press, New York, London. WALLACE, S., WHITING, B. and RUNCIE, J., 1976,J . Clin. Pharmacol., 3, 327. WIDMAN, M., AGURELL, S., EHRNEBO, M. and JONES,G., 1974, J . Pharm. Pharmacol., 26, 9 14. I. A., 1969, J. Chromato,qr., 44, 547. ZINGALES,
(1980), 20, 187
Received 11th March 1980
Distribution of Drugs and Toxic Chemicals in Blood M. D. OSSELTON,* M. D. HAMMOND and A. C. MOFFAT Home O f i e Central Research Establishment, Aldermaston, Reading, Berkshire, England, RG7 4PN
The distribution of 61 drugs and toxic chemicals in dzfferent blood components has been examined. Data are presented which demonstrate the wide dayerence in distribution of drugs between dzfferent blood components. A recommendation is made that unfractionated blood should be used for forensic analyses. Introduction A number of recently published articles have drawn attention to the possible association between road traffic accidents and drug taking by road users (Bestic, 1979; Moffat, 1977; Raffle, 1977; Silverstone, 1977). One estimation of the number of drivers taking psychoactive drugs in Britain (excluding alcohol) has placed the number between 3% and 5% i.e., some 500,000 drivers (Silverstone, 1977). Since it is an offence to drive or attempt to drive or to be in charge of a vehicle whilst unfit to drive through drugs, the forensic toxicologist may be requested to perform drug analyses on blood specimens submitted by police officers where drug taking has been implicated or suspected. Because the quantity of blood submitted for analysis is likely to be limited and because drugs are not uniformly distributed between different blood components, we have investigated the available data on the distribution of drugs in blood in order to decide whether analyses should be performed on serum or plasma, as in most clinical and pharmacokinetic laboratories, or whether unfractionated blood should be examined. The published data have been supplemented by values determined experimentally for this study. During a recent literature search we noted that out of 92 papers concerned with methods for the analysis of drugs in blood only 12 presented results from the analysis of whole blood. I t was also noted that 5 of the authors of these 12 papers were employed in forensic science laboratories and out of the remaining 80 papers describing the analysis of blood plasma for drugs, only one forensic science laboratory was cited. Materials and Methods Blood Sambles Blood samples were provided by informed volunteers receiving drug therapy at Park Prewitt Hospital, Basingstoke during venepuncture extraction of blood for routine biochemical examination. Blood was collected into heparinised containers. Procedures involving human volunteers were approved by the hospital medical ethics committee. Aliquots of whole blood were removed from the heparinised containers and stored frozen prior to analysis. Plasma from each sample was collected and stored a t - 20°C. Reagents Trizma base (Sigma Chemical Co.) was dissolved in water to yield a 1.OM solution (pH 10.5). *Present address: Home Office Forensic Science Laboratory, Shakespeare Street, Nottingham, England, NGl 4FR. 187
n-Butyl acetate (Analytical Reagent Grade, B.D.H. Ltd.) was redistilled before use. Drug standards (lmg/ml) were prepared in absolute alcohol and diluted to working strength with redistilled butyl acetate. Extraction of Drugs from Blood 50p1 of Trizma base was added to a glass tube (50 x 6mm) containing 50 to 2 0 0 ~ 1blood or plasma. Butyl acetate (50 to 100~1)containing prazepam internal standard (0.2mg/l) was next added and the tube contents were mixed for 30 seconds using a 'Whirlimixer'. Specimens were centrifuged (12,000 rpm) for 2 minutes and aliquots (2 to 5p1) of the upper solvent phase were used for gas chromatographic analysis. Gas-Liquid Chromatography (GLC) Drugs were analysed using a column of 3% w/w OV-17 on Chromosorb W H P (80-100 mesh) in a Pye Series 104 gas chromatograph. Nitrogen carrier gas maintained at a flow rate of 50ml/minute and a column temperature of 264°C was used for chromatography of benzodiazepines. The column effluent was split 50 : 50 to a flame ionisation detector (FID) and a 63Ni electron capture detector (ECD). Make-up gas (nitrogen, 50ml/min) was used to supplement gas flow through the ECD. Blood extracts were also analysed using a Perkin Elmer F33 gas chromatograph equipped with a phosphorus/nitrogen detector (PND) and FID. Nitrogen carrier gas was passed through a l m x 4mm column of 3% w/w OV-17 on Chromosorb W H P (100-120 mesh) at a flow of 40ml/minute. Gas leaving the column was split 50 : 50 to the PND and F I D detectors.
Results and Discussion The major functions of blood in relation to drug therapy are transport and storage, and any drugs present in blood are distributed in a non-uniform manner between different blood components (Curry, 1977). They may be transported as complexes bound to plasma proteins such as albumin or aglycoproteins (Meyer and Guttman, 1968; Schneider et al., 1979) or associated with other blood components, for example erythrocytes, platelets and leucocytes (Maren et al., 1960; Beerman et al., 1975; Ahtee and Paasonan, 1966; Rollo, 1965). Data relating to the distribution of 61 substances in different blood components are presented in Table 1 which may aid the correlation of whole blood and plasma drug levels. Results obtained in our laboratory have been supplemented by information retrieved from published data. Since the percentage of blood volume occupied by cellular constituents varies between 43% v/v and 48% v/v according to age and sex (Diem and Lentner, 1970) a mean value of 46% v/v was assumed for our calculations. Using this figure, the distribution of the drugs was calculated as 1-85 for plasma: whole blood in the situation where drugs were confined to the plasma. Where drugs were equally distributed between blood fractions a value of 0.93 was obtained. Ratios lower than 0.93 indicated that more drug was present in the cellular fraction than in the plasma. Many published figures on drug distribution in blood are presented as percentage of drug in plasma without providing ratios. I n an attempt to provide a uniform presentation of the data in Table 1, we have also calculated the percentage of drug in plasma from available data on plasma: whole blood ratios using 1-85 as the figure representing 100% of drug in the plasma. Figures concerning percentage of drug in any fraction should be regarded as approximate since the effects of age, sex, disease and the presence of other drugs may cause significant changes in the plasma levels of many drugs (e.g., Crooks et al., 1976; Johnsson and Regardh, 1976). For example Chan et al. (1975) reported that pethidine binds with high affinity to the red cells of young patients to a significantly higher extent than in older patients and suggested that such differences could be used to explain the high
TABLE 1 DISTRIBUT'ION OF DRUGS AND TOXIC CHEMICALS IN BI.OOD Ruth Compound
Plasma : Whole Blood
Acetazolamide
-
Aldrin Alprenolol
-
Amitriptyline Amphetamine
1.21
-
Ampicillin
-
Arsenic Atenolol
--
-
Plasma : Erythrocyte Concentrated in red cells -
80 85
-
-
65
96
Approximately 0.8 but varies with time after dose -
Even distribution 0.83
-
Even distribution
Carbamazepine
-
Red cell level 1 5 2 0 % of plasma level
Chlordecone Chlorpromazine
0.57
Chlorthalidone
-
Clomipramine Cocaine Cyanide
1.17
Fluoride Gold Imipramine Indomethacin Lanatoside C Medazepam Mercury Metaprolol 3-Methyl Digexin Oxalate Pentachlorophenol Pentazocine Pentobarbitone Pethidine
-
-
Observed in red cells Red cell level x 50-100 that in plasma
Phenobarbitone
-
-
Almost all in plasma 50 41.5
25
Ehrnebo (1978)
43 50 -
Binds to red cells in young subiects
-
Baselt (1978) Johnsson and Regardh (1976) 46-58 Bender et al. (1975) - Stewart and Stolman (1960) 72 Pynnonen and Yrjana (1977) 91-99
-
Baselt (1978) Zingales (1969)
-
-
FIeuren and van Rossum (1978)
-
This study Javaid et al. (1978) Baselt (1978)
30
-
-
-
-
62 Distribution variable Most whole blood CN- in erythrocytes 3.4 1.66 89 98 1.83 75 98 - Equal distribution 50 68 1.26 40 0.60.9 72 -Restricted to serum in most patients 53 0.98 100 -16 1-85 99 5 -0.83 41.5
1.3 Whole blood level higher than plasma level
Reference Maren and Robinson (1960) Baselt (1978) ,Johnsson and Regardh (1976) This study Beckett et al., (1969)
-
-
Phenacetin
Protein Bound
-
-
Deslanoside Desmethyldiazepam Diazepam Dieldrin Digitoxin Digoxin Diphenhydramine Ethambutol
%
zn Plasma
-
Benzylpenicillin Caffeine
-
%Drug
-
-
-
95 23 79 8
Fletcher et al. (1979) This study This study Baselt (1978) Baselt (1978) Baselt (1978) This study Lee et al. (1977)
-
Baselt (1978) Baselt (1978)
96
-
75-96 This study 90 McArthur et al. (1971) - Fletcher et al. (1979) - This study - Baselt (1978) 12 Johnsson and Regardh f 1976) - ~ l d t c h e et r al. (1979) - Baselt (1978) -
99 50 50
-
-
-
70 50
40 30
50
33
-
Baselt (1978) Ehrnebo, et al. (1974) Stewart & Stolman (1960) Chan, et al. (1975) Morganetal.(1978) Stewart & Stolman (1960) Stewart & Stolman (1960)
TABLE 1-Continued
Compound Phenylbutazone Phenytoin
Plasma : Whole Blood 1.64
Ratios Plasma : Eythrocyte A
% Drug zn Plasma 96 88
Prilocaine Promethazine Propranolol 1.23 Quinidine
-
Quinine Salicylic Acid
-
Spironolactone Sulphadiazine Temazepam Theophylline
-
2 .o
-
1.87 1.22
a 9 Tetrahydro cannabinol Thiopentone
-
Thioridazine
-
Tiflorax Tolomol
-
Trimiprarnine Tubocurarine Warfarin
-
1.23
-
-
Plasma level > red cell level Equally distributed Appreciable amounts in red cells -
-
Little drug bound to red cells Observed in red cells 0.16-0.25 2 .O -
Confined to plasma
-
66 -
50
-
100 50 100 66
%
Protein Reference Bound W 9 8 Wallace, et al. (1976) 87 Kurata and Wilkinson (1974) 50 Baselt (1978) 75 This study 90 Isaacs and Schoenwald ( 1974) Baselt (1978) 60-80 Isaacs and Schoenwald (1974) 75-90 Raselt (1978) McArthur, et al. (1971) 75 74-94 Rorga, et al. (1977) - Karim, et al. (1976) 20-68 Wallace, et al. (1976) - This study 15 Mitenko and Ogilvie (1973) 87-90 Widman, et al. (1974)
-
75
-
-
13 90
A
66 100
-
94
98.6
97
Stewart & Stolman (1960) Zingales (1969) Biachetti et al. (1978) Johnsson and Regardh (1976) This study Stewart & Stolman ( 1960) Crooks, et a1 (1976)
serum levels of this drug observed in older patients. Age related differences in drug absorption, metabolism and excretion could also have significant effects on drug levels in the blood of old and young subjects. Results published by Piafsky and Borga (1977) reported that women taking oral contraceptives had lower concentrations of albumin and a,-acid glycoprotein and lower levels of alprenolol and imipramine bound to plasma proteins than women not using oral contraceptives. The data in Table 1 clearly reveal that drugs are widely distributed between various blood components and that considerable amounts of some drugs are present in red cells, e.g., acetazolamide, chlorthalidone (> go%), ethambutol (60%), pentazocine (50%) and tiflorax (87%). Thus, drugs are not always primarily associated with plasma proteins. Fletcher et al. (1979) reported that digoxin binds to red cell membranes and haemoglobin in addition to being present in plasma. They found it necessary to haemolyse whole blood and to remove red cell ghosts before assaying for digoxin by radioimmunoassay since the membrane-bound drug was only released slowly over a period of several hours. Consideration should also be given to changes which may occur during the period between collection of the blood specimen and subsequent analysis. Very slight haemolysis of blood samples (undetectable to the eye) containing compounds such as chlorthalidone (red cell levels 50 to 100 times that in plasma) could produce significant changes in the measured plasma level of these drugs and subsequently result in false interpretation of the plasma levels. Cotham and Shand (1975) demonstrated that plasticisers in Vacutainer blood collection tubes displaced propranolol bound to plasma proteins into the red cells resulting
in the measurement of 'spuriously low plasma propranolol levels'. The major impurities shown to enter blood and to interfere with analyses have been identified as di-(2-ethylhexy1)-phthalate and trisbutoxyethyl phosphate. These substances may be introduced from tube stoppers, stopper lubricants, polyvinyl chloride blood transfusion storage bags and filter papers (Dickson et al., 1974; Piafsky and Borga, 1976; Ramsey et al., 1980). I t was later shown that the lowering of the plasma concentrations of alprenolol and imipramine by displacement from plasma proteins by these impurities is from 76% to 16% and 69% to 13% respectively (Borga et al., 1977). The data in Table 1 are presented as a histogram in Figure 1 which illustrates that more drugs are concentrated in the plasma than in the erythrocytes. Plasma appears to be the most favoured phase to be used in drug analysis by clinical chemists and pharmacokineticists, and hence more information is available to aid the interpretation of results. However, the analysis of plasma alone may result in failure to identify some drugs which are present. I n view of the lack of published information on drug levels in whole blood, plasma : whole blood or plasma : erythrocyte ratios may be used to correlate whole blood levels with published plasma drug levels and hence may provide a useful guideline for the interpretation of results. I n many instances the forensic analyst will have no choice between analysing whole blood and plasma since specimens are frequently haemolysed on arrival at the laboratory. However, if the choice is available, with the exception of some immunoassay analyses, unfractionated blood should be selected in order to (a) detect as many com-
% Drug in Plasma Figure 1.
Histogram of the distribution of fifty-six drugs and toxic chemicals in blood. 191
pounds as possible in a given blood sample and (b) provide a more realistic picture of blood drug levels for the interpretation of analytical findings. Conclusions The distribution of drug substances between blood components varies considerably and may be affected by such factors as age, sex, disease, presence of other drugs, storage conditions and age of the specimen. In order to detect as many compounds as possible in a given blood sample unfractionated blood should be selected for analysis. Since relatively little data are available to guide analysts with the interpretation of the levels for drugs found in unfractionated blood, whole blood : plasma ratios or erythrocyte : plasma ratios may be used to aid interpretation of results. References AHTEE,L. and PAASONAN, M. K., 1966, J. Pharm. Pharmacol., 18, 126. BASELT, R. C., 1978, Disposition of Toxic Drugs and Chemicals in Man, Biomedical Publications. Canton.. Conn.. U.S.A. M., 1969, J. Pharm. Pharmacol., BECKETT, A. H'., SALMON, J. A. and MITCHARD, 21. 25 1. BEERMAN, B., HELLSTROM, K., LINDSTROM, B. and ROSEN,A., 1975, Clin. Pharmacol., Ther., 17, 424. BENDER, A. D., POST,A., MEIER,J. P., HIGSON, J. E. and REICHARD, G., 1975, 3. Pharm. Sci., 64, 1711. BESTIC, A., 1979, Drive, No. 55, p. 32, A.A. Publications Ltd. BIACHETTI, G., MITCHARD, M. and MORSELLI, P. L., 1978, J. Chromatogr., 152, 87. BORGA,O., PIAFSKY, K. M. and NILSEN,0. G., 1977, Clin. Pharmacol. Ther., 22, 539. M., WELLS,W. D. E. and VICKERS, CHAN,K., KENDALL, M. J., MITCHARD, M. D., 1975, Br. J. Clin. Pharmacol., 2, 297. COTHAM, R. H. and SHAND,D., 1975, Clin. Pharmacol. Ther., 18, 535. I. H., 1976, Clin. Pharmacokinet., 1, CROOKS, J., O'MALLEY,K. and STEVENSON, 280. CURRY,S. H., 1977, Drug Disposition and Pharmacokinetics, 2nd Ed., p. 26, Blackwell, Oxford. DICKSON, S. J., MISSEN, A. W. and DOWN,G. J., 1974, Forens. Sci., 4, 155. DIEM,K. and LENTNER, C., 1970, Eds., Docurnenta Geigy, Scientijic Tables, 7th Edn., p. 617, Geigy, Basle, Switzerland. E. and LONROTH, U., EHRNEBO, M., AGURELL, S., BIREUS,L. O., GORDON, 1974, Clin. Pharmacol. Ther., 16, 424. EHRNEBO, M., 1978,J. Pharm. Pharmacol., 30, 730. G. J. and MOFFAT, A. C., 1979,J. Forens. Sci. Soc., FLETCHER, S. M., LAWSON, 19, 183. J. M., 1978, J.Chromatogr., 152,41. FLEUREN, H. L. J. M. and VAN ROSSUM, ISAACS, V. E. and SCHOENWALD, R. D., 1974, J. Pharm. Sci., 63, 1267. JAVAID, J. I., DEKIRMENJIAN, H., DAVIS,J. M. and SCHUSTER, C. R., 1978, J. Chromatogr., 152, 105. JOHNSSON, G. and REGARDH, C . G., 1976, Clin. Pharmacokinet., 1, 233. J. and DANBY, M., 1976, Clin. Pharmacol. KARIM, A., ZAGARELLA, B. A., HRIBAR, Ther., 19, 158. KURATA, D. and WILKINSON, G. R., 1974, Clin. Pharmacol. Ther., 16, 355. J. G., BRATER, D. C. and BENET,L. Z., 1977, Clin. LEE, C. S., GAMBERTOGLIO. Pharmacol. Ther., 22, 615. MAREN,T. H. and ROBINSON, B., 1960, Bull. J. Hopkins Hosp., 106, 1. MCARTHUR, J. N., DAWKINS, P. D. and SMITH,M. J. H., 1971, J. Pharm. Pharmacol., 23, 32. 192
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