Ann. clin. Biochem. 13 (1976) 435-437
Rapid Determination of Plasma Paracetamol GEORGE STANLEY WILKINSON
Pathology Department, Clatterbridge Hospital, Bebington, Wirral £63 4JY A simple colorimetric method for the determination of plasma paracetamol in emergency cases of overdose is described. A comparison with an ultraviolet method was performed.
Paracetamol is normally a safe drug, but cases of overdose are becoming more frequent and may lead to hepatic necrosis. An early reliable guide to the severity of the poisoning can be obtained from repeat assays 8 to 12 hours after the assay obtained on admission. If the paracetamol half life is less than 4 hours, liver damage is most unlikely, while a value exceeding 12 hours gives warning of severe and possibly fatal hepatic failure. Plasma paracetamol concentrations greater than 20 mg per 100 ml at 4 hours or 7.5 mg per 100 ml at 12 hours are associated with serious liver damage, while patients with plasma concentrations less than 12 mg per 100 ml at 4 hours have no clinical evidence of liver damage (Simpson and Stewart, 1973). Requests for estimations of plasma levels often reach the laboratory during on-call periods and are assayed by staff who are not always whole-time members of the biochemistry department. A simple, accurate, and rapid test for estimating plasma paracetamol is needed. Methods for estimating paracetamol include: ultraviolet scans of extracts (Routh et al., 1968; Gibson, 1972), gas liquid chromatography (Prescott, 1971; Groves, 1971; Stewart and Willis, 1975), and a colorimetric assay using the indophenol reaction (Tompsett, 1969). In the procedure described perchloric acid replaces hydrochloric acid used in the Tompsett (1969) method. Perchloric acid deproteinizes the plasma and yields a supernatant ideally suited to bring about acid hydrolysis of N-acetyl p-aminophenol. Solvent extraction is essential in the Tompsett (1969) procedure but now is no longer necessary when perchloric acid is used. The effectiveness of perchloric acid over hydrochloric acid in the rapid development of the indophenol reaction is demonstrated. The ultraviolet method of Routh et al. (1968) was performed on the same serum samples so as to compare recovery, precision, and accuracy. MATERIALS AND METHODS
Reagents (1) Perchloric acid 0.33 mol/l,
(2) Ammonium hydroxide 4 mol/I. (3) O-Cresol 1 % w/v in distilled water. (4) Standard solution of paracetamol (N-acetyl p-aminophenol) 20 rng in 100 ml distilled water.
Method (1) To 0.5 ml plasma is added 4.5 ml 0.33 mol/l
(2) (3) (4)
perchloric acid. 0.5 ml water and 0.5 ml standard solution (100 p.g) are treated similarly. The tubes are mixed and centrifuged. 1.0 ml supernatant fluid is pipetted into a Bl9 boiling tube and the neck of the tube covered with aluminium foil. N-acetyl p-aminophenol and its conjugates are hydrolysed by autoclaving for 15 min at a temperature of 135 and a pressure of 30 Ib/in 2 ("Little Sister" automatic autoclave, Surgical Equipment Supplies Ltd., Westfields Road, London W3). Alternatively hydrolysis can be achieved by boiling in a water bath for 40 min. Allow to cool and add 1.0 ml o-cresol solution and 2.0 ml ammonium hydroxide solution. Mix and read the blue colour formed at 615nm. 0
(5) (6)
Evaluation of the method Perchloric acid is a protein precipitant and has similar acidic properties as hydrochloric acid. To assess its suitability as a hydrolytic agent paracetamol was hydrolysed with perchloric acid and hydrochloric acid under identical conditions. The effectiveness of hydrolysis was apparent during the final stages of the analysis. Both acids achieve maximum hydrolysis of N-acetyl p-aminophenol in IS min at elevated temperature and pressure and in 40 min in boiling water. The production of the blue indophenol reaction depends on the conversion of N-acetyl p-aminophenol to p-aminophenol and its reaction with o-cresol and ammonium hydroxide. Perchloric acid is superior to hydrochloric acid in this respect, since the colour production is almost instantaneous and
435
Downloaded from acb.sagepub.com by guest on June 5, 2016
436
Wilkinson
containing 20 mg and 5 mg paracetamol per 100 m respectively. The accuracy of assay was examined by measuring the recovery of paracetamol added to control horse serum.
0.6
0.5
00
REsULTS
o.q 0.3
0.2
0.1
12
16
20
Zq
MINUTES
Fig. t.-Development of the indophenol colour in the presence of bydrocbloric acid and perchloric acid.
- - - Perchloric acid. - - - Hydrochloric acid. stable. With hydrochloric acid the colour is also stable but takes 25 min to reach its maximum intensity. To show that the method is specific for paracetamol plasma samples from patients on therapeutic doses of drugs other than paracetamol were put through the procedure. No positive tests were obtained from plasma samples containing therapeutic or overdose amounts of drugs. Linearity experiments were carried out on pooled plasma to which was added paracetamol over the range 2 mg to 30 mg per 100 m!. Precision of the method was estimated by repeatedly assaying aliquots from two serum pools Table 1. Drugs shown to have given negative o-cresol tests Amylobarbitone Butobarbitone Pentobarbitone Quinalbarbitone Cyclobarbitone Phenobarbitone Salicylate Chlorpromazine Diazepam
Frusemide Imipramine Lithium Phenytoin Primidone Sulthiame Thyroxine Glutethimide
Linearity.-The addition of aqueous paracetamol to pooled plasma over the range 2-30 mg per 100 ml gave calibration curves which were linear. Rate of colour formation. Fig. 1 shows the effectiveness of perchloric acid in the development of the blue indophenol reaction. With the former maximum colour formation is almost instantaneous, while with the latter it takes 25 min to reach maximum colour. Interference from drugs.--Of the drugs tested only those able to liberate p-aminophenol have given a positive alkaline o-cresol reaction. Table 1 shows those substances which have given negative results with this procedure. Recovery.-{i) A mean recovery of 96 % (range 92-103) was obtained for serum containing 20 rng paracetamol per 100m!. (ii) A mean recovery of 100% (range 93-106) was obtained for serum containing 5 mg paracetamol per 100m!. Precision.-{i) The within-batch coefficient of variation was 2.6% and 4.2% for the 20 mg and the 5 mg paracetamol per 100 ml respectively. (ii) The between-batch coefficient of variation was 3.4 % and 4.6 % for the 20 mg and S mg paracetamol per 100 ml respectively. DISCUSSION
The method of choice for the determination of paracetamol in emergency cases must be easy to perform, sensitive, and accurate. These qualities are especially necessary when non-wholetime biochemical staff are concerned. The method described entails minimal manipulations, avoids the use of highly volatile solvents, and can be completed in a relatively short time with simple equipment. Procedural steps involve protein precipitation, heating the supernatant, adding reagents, and reading the absorbance. Perchloric acid used in the procedure has three functions. Firstly, it effects deproteinization; secondly, it yields an acid solution which on heating converts paracetamol to p-aminophenol; and, thirdly, its presence appears to be important in bringing about the almost instantaneous formation of the blue indophenol colour. No positive tests were given by plasmas containing drugs listed in Table 1 in therapeutic or excessive dosage. This result confirms the observations of Simpson and Stewart (1973).
Downloaded from acb.sagepub.com by guest on June 5, 2016
Rapid Determination ofPlasma Paraeetamol 437 As a comparison the ultraviolet method of Routh et al. (1968) was performed at the same time as the described procedure. Experiments including recoveries and within-batch and between-batch coefficients of variation show that both procedures compare favourably as regards recovery, sensitivity, precision, and accuracy.
REFERENCES
Gibson, P. F. Haemodialysis in paracetamol self poisoning. Lancet 2 (1972) 607. Groves, J. Gas liquid chromatography of N-acetyl-paminophenol (paracetamol) in plasma and urine.
J. Chromat, 59 (1971) 289. Prescott, L. F. The gas-liquid chromatographic estimation of paracetamol. J. Pharm. Pharmacol. 23 (1971) 807. Routh, J. I., Shane, N. A., Arredondo, E. G., Paul, W. D. Determination of N-acetyl-p-aminophenol in plasma. Clin. Chem. 14 (1968) 882. Simpson, E., Stewart, M. J. Screening for paracetamol poisoning. Ann. din. Biochem, 10 (1973) 171. Stewart, J. J., Willis, R. G. Simplified gas chromatographic assay for paracetamol, Ann. din. Biochem. 12 (1975)4. Tompsett, S. L. The detection and determination of phenacetin and N-acetyl-p-aminophenol (paracetamol) in blood, serum and urine. Ann. din. Biachem. 6 (1969) 81.
Downloaded from acb.sagepub.com by guest on June 5, 2016
Rapid Determination of Plasma Paracetamol GEORGE STANLEY WILKINSON
Pathology Department, Clatterbridge Hospital, Bebington, Wirral £63 4JY A simple colorimetric method for the determination of plasma paracetamol in emergency cases of overdose is described. A comparison with an ultraviolet method was performed.
Paracetamol is normally a safe drug, but cases of overdose are becoming more frequent and may lead to hepatic necrosis. An early reliable guide to the severity of the poisoning can be obtained from repeat assays 8 to 12 hours after the assay obtained on admission. If the paracetamol half life is less than 4 hours, liver damage is most unlikely, while a value exceeding 12 hours gives warning of severe and possibly fatal hepatic failure. Plasma paracetamol concentrations greater than 20 mg per 100 ml at 4 hours or 7.5 mg per 100 ml at 12 hours are associated with serious liver damage, while patients with plasma concentrations less than 12 mg per 100 ml at 4 hours have no clinical evidence of liver damage (Simpson and Stewart, 1973). Requests for estimations of plasma levels often reach the laboratory during on-call periods and are assayed by staff who are not always whole-time members of the biochemistry department. A simple, accurate, and rapid test for estimating plasma paracetamol is needed. Methods for estimating paracetamol include: ultraviolet scans of extracts (Routh et al., 1968; Gibson, 1972), gas liquid chromatography (Prescott, 1971; Groves, 1971; Stewart and Willis, 1975), and a colorimetric assay using the indophenol reaction (Tompsett, 1969). In the procedure described perchloric acid replaces hydrochloric acid used in the Tompsett (1969) method. Perchloric acid deproteinizes the plasma and yields a supernatant ideally suited to bring about acid hydrolysis of N-acetyl p-aminophenol. Solvent extraction is essential in the Tompsett (1969) procedure but now is no longer necessary when perchloric acid is used. The effectiveness of perchloric acid over hydrochloric acid in the rapid development of the indophenol reaction is demonstrated. The ultraviolet method of Routh et al. (1968) was performed on the same serum samples so as to compare recovery, precision, and accuracy. MATERIALS AND METHODS
Reagents (1) Perchloric acid 0.33 mol/l,
(2) Ammonium hydroxide 4 mol/I. (3) O-Cresol 1 % w/v in distilled water. (4) Standard solution of paracetamol (N-acetyl p-aminophenol) 20 rng in 100 ml distilled water.
Method (1) To 0.5 ml plasma is added 4.5 ml 0.33 mol/l
(2) (3) (4)
perchloric acid. 0.5 ml water and 0.5 ml standard solution (100 p.g) are treated similarly. The tubes are mixed and centrifuged. 1.0 ml supernatant fluid is pipetted into a Bl9 boiling tube and the neck of the tube covered with aluminium foil. N-acetyl p-aminophenol and its conjugates are hydrolysed by autoclaving for 15 min at a temperature of 135 and a pressure of 30 Ib/in 2 ("Little Sister" automatic autoclave, Surgical Equipment Supplies Ltd., Westfields Road, London W3). Alternatively hydrolysis can be achieved by boiling in a water bath for 40 min. Allow to cool and add 1.0 ml o-cresol solution and 2.0 ml ammonium hydroxide solution. Mix and read the blue colour formed at 615nm. 0
(5) (6)
Evaluation of the method Perchloric acid is a protein precipitant and has similar acidic properties as hydrochloric acid. To assess its suitability as a hydrolytic agent paracetamol was hydrolysed with perchloric acid and hydrochloric acid under identical conditions. The effectiveness of hydrolysis was apparent during the final stages of the analysis. Both acids achieve maximum hydrolysis of N-acetyl p-aminophenol in IS min at elevated temperature and pressure and in 40 min in boiling water. The production of the blue indophenol reaction depends on the conversion of N-acetyl p-aminophenol to p-aminophenol and its reaction with o-cresol and ammonium hydroxide. Perchloric acid is superior to hydrochloric acid in this respect, since the colour production is almost instantaneous and
435
Downloaded from acb.sagepub.com by guest on June 5, 2016
436
Wilkinson
containing 20 mg and 5 mg paracetamol per 100 m respectively. The accuracy of assay was examined by measuring the recovery of paracetamol added to control horse serum.
0.6
0.5
00
REsULTS
o.q 0.3
0.2
0.1
12
16
20
Zq
MINUTES
Fig. t.-Development of the indophenol colour in the presence of bydrocbloric acid and perchloric acid.
- - - Perchloric acid. - - - Hydrochloric acid. stable. With hydrochloric acid the colour is also stable but takes 25 min to reach its maximum intensity. To show that the method is specific for paracetamol plasma samples from patients on therapeutic doses of drugs other than paracetamol were put through the procedure. No positive tests were obtained from plasma samples containing therapeutic or overdose amounts of drugs. Linearity experiments were carried out on pooled plasma to which was added paracetamol over the range 2 mg to 30 mg per 100 m!. Precision of the method was estimated by repeatedly assaying aliquots from two serum pools Table 1. Drugs shown to have given negative o-cresol tests Amylobarbitone Butobarbitone Pentobarbitone Quinalbarbitone Cyclobarbitone Phenobarbitone Salicylate Chlorpromazine Diazepam
Frusemide Imipramine Lithium Phenytoin Primidone Sulthiame Thyroxine Glutethimide
Linearity.-The addition of aqueous paracetamol to pooled plasma over the range 2-30 mg per 100 ml gave calibration curves which were linear. Rate of colour formation. Fig. 1 shows the effectiveness of perchloric acid in the development of the blue indophenol reaction. With the former maximum colour formation is almost instantaneous, while with the latter it takes 25 min to reach maximum colour. Interference from drugs.--Of the drugs tested only those able to liberate p-aminophenol have given a positive alkaline o-cresol reaction. Table 1 shows those substances which have given negative results with this procedure. Recovery.-{i) A mean recovery of 96 % (range 92-103) was obtained for serum containing 20 rng paracetamol per 100m!. (ii) A mean recovery of 100% (range 93-106) was obtained for serum containing 5 mg paracetamol per 100m!. Precision.-{i) The within-batch coefficient of variation was 2.6% and 4.2% for the 20 mg and the 5 mg paracetamol per 100 ml respectively. (ii) The between-batch coefficient of variation was 3.4 % and 4.6 % for the 20 mg and S mg paracetamol per 100 ml respectively. DISCUSSION
The method of choice for the determination of paracetamol in emergency cases must be easy to perform, sensitive, and accurate. These qualities are especially necessary when non-wholetime biochemical staff are concerned. The method described entails minimal manipulations, avoids the use of highly volatile solvents, and can be completed in a relatively short time with simple equipment. Procedural steps involve protein precipitation, heating the supernatant, adding reagents, and reading the absorbance. Perchloric acid used in the procedure has three functions. Firstly, it effects deproteinization; secondly, it yields an acid solution which on heating converts paracetamol to p-aminophenol; and, thirdly, its presence appears to be important in bringing about the almost instantaneous formation of the blue indophenol colour. No positive tests were given by plasmas containing drugs listed in Table 1 in therapeutic or excessive dosage. This result confirms the observations of Simpson and Stewart (1973).
Downloaded from acb.sagepub.com by guest on June 5, 2016
Rapid Determination ofPlasma Paraeetamol 437 As a comparison the ultraviolet method of Routh et al. (1968) was performed at the same time as the described procedure. Experiments including recoveries and within-batch and between-batch coefficients of variation show that both procedures compare favourably as regards recovery, sensitivity, precision, and accuracy.
REFERENCES
Gibson, P. F. Haemodialysis in paracetamol self poisoning. Lancet 2 (1972) 607. Groves, J. Gas liquid chromatography of N-acetyl-paminophenol (paracetamol) in plasma and urine.
J. Chromat, 59 (1971) 289. Prescott, L. F. The gas-liquid chromatographic estimation of paracetamol. J. Pharm. Pharmacol. 23 (1971) 807. Routh, J. I., Shane, N. A., Arredondo, E. G., Paul, W. D. Determination of N-acetyl-p-aminophenol in plasma. Clin. Chem. 14 (1968) 882. Simpson, E., Stewart, M. J. Screening for paracetamol poisoning. Ann. din. Biochem, 10 (1973) 171. Stewart, J. J., Willis, R. G. Simplified gas chromatographic assay for paracetamol, Ann. din. Biochem. 12 (1975)4. Tompsett, S. L. The detection and determination of phenacetin and N-acetyl-p-aminophenol (paracetamol) in blood, serum and urine. Ann. din. Biachem. 6 (1969) 81.
Downloaded from acb.sagepub.com by guest on June 5, 2016
