Simplified Gas Chromatographic Assay for Paracetamol

Transcription

1 Ann. clin. Biochem. 12 (1975) 4 Simplified Gas Chromatographic Assay for Paracetamol M. J. STEWART AND R. G. WILLIS Department of' Clinical Chemistry, Ninewells Hospital and Medical School, Dundee, DD2 IUD A rapid gas liquid chromatography method has been developed which is suitable for emergency estimations of paracetamol in overdose. The method is compared with a standard ultraviolet method. Plasma paracetamol levels have been used for some years in the study of cases of self-poisoning with this drug, following reports that the levels could be used to predict the extent of liver damage (Proudfoot and Wright, 1970; Prescott et al., 1971). Two types of estimation have been used to date, those involving ultraviolet (UV) scanning of plasma extracts, as described by Routh et "I. (1968) and later as modified by Gibson (1970), and gas chromatographic methods described by Prescott (1971) and Grove (1971). The gas liquid chromatography (GLC) methods have considerable advantages in terms of precision and specificity, but have not been found to be sufficiently rapid for emergency use. Consequently the UV methods have been used preferably in these situations. The development of treatment for paracetamol poisoning (Prescott, 1974) has led to an increased requirement for the accurate and rapid determination of plasma paracetamol levels on-call, as at present the decision as to whether or not to treat the patient is based on knowledge ofthe paracetamol concentration and its relationship to the time of overdose. Our requirement was for a method which could be carried out by non-specialist staff in a crowded emergency laboratory. The method had to be accurate especially in the range jlg/ml, as it is in this area that decisions as to treatment have to be made. In addition, the use of ether was precluded due to the close proximity of flame photometers. Chloroform was utilised, the less efficient extraction from plasma being compensated for by the use of an internal standard of similar extractability to paracetamol. MATERIALS AND METHODS Internal Standard N-Butyryl-p-aminophenol (prepared by reacting butyric anhydride with p-hydroxy aniline.) The recrystallised product was dissolved in ethanol at a concentration of 1 jlg/ml for overdose levels or 0.1 jlg/ml for therapeutic levels. Standard Paracetamol (a) Stock solution (1 mg{ml N-acetyl-p-aminophenol in water) (b) Working Standards prepared from stock by dilution with distilled water to final concentrations of 20, 50, and 200 jlg{m1. To a Bl9 Q and Q tube the following were added: 2.5 ml salt-saturated 0.2 M HCl; 10 ILl internal standard and 0.5 ml of horse serum, standard, quality control, or test plasma; 10 ml chloroform. After extraction for 2 min, the aqueous layer was removed by suction and the chloroform filtered through Whatman No. 1 paper into a second B19 tube and evaporated to dryness at 60 C under a stream of air. The residue was redissolved in 20 jll ethanol and 5 jll applied to the column. GLC operating conditions Equipment: Pye 104 with FtD head Column: Glass coil 91 em x 5 mm I D silanised Packing: 3% cyclohexane dimethanol succinate on acid-washed, silanised Chromosorb W Temperatures: Column oven 240 C Detectors 260 C Injectors 250 C Gas flow rates: Argon (carrier) 60 ml/rnin hydrogen 60 air 200 Reagents Chloroform A.R. BDH O.2M Hydrochloric acid saturated with sodium chloride (BDH A.R.) 4 Preparation of Quality Control sera N-acetyl p-arninophenol (20 mg) was dissolved ina minimalamount ofwaterand made up to too ml with Wellcome horse serum NO.5. The solution was

2 5 stirred gently for 2 hours, divided into 1 ml aliquots and stored at -100e. Specimens were allowed to thaw at room temperature before use. 15 Calculation A plot of plasma paracetamol concentration versus the ratio of peak heights of paracetamol to internal standard was constructed, and the values for QC and unknown specimens read from this. In an emergency a single standard is set up, with a value of 200 J1-g/ul, and the value of QC and test specimens calculated by direct proportion. RESULTS Calibration. Calibration curves prepared using both concentrations of internal standard gave straight lines. The ranges covered were f-lg/ml and 2-30 f-lg/ml. Specificity. A trace obtained from a standard solution is shown in fig. 1. There are few contaminaing peaks, and none which interfere with either the paracetamol or internal standard peaks. A trace from a patient with an overdose of paracetamol is shown in fig. 2. Endogenous peaks are found but again no interference is caused. Fig. 3 shows a trace from quality control serum to which had been added a mixture of seven common barbiturates, each at a final concentration of 20 jj.g/mj. None of the barbiturate peaks interfere. Accuracy. Quality control specimens were assayed by both the above method and that of Routh (1968). The results are shown in table 1. It can be seen that the relative recovery using the internal standard was 98% compared with the order of90% for the UV method. Table 1. Recoveries obtained from weighed-in quality control sera using the GLC and UV methods B 10 5 Method Paracetamol No. of Mean % concen- specimens value Recovery tration assayed obtained GLe 200!J.g/ml % so!j.g/m % UV 200!J.g/ml % SO!J.g/ml % I Precision. The method was checked by analysing 20 each of the high and low quality control sera o Fig. t.-trace obtained using an aqueous standard.

3 p... El --- e t) t) '-' '-'..c:: ClI) ~..c:: ClI) 0..>II..>II <U <U Q) Q) p., p.. := 5 5 B o I o Fig. 2.-Trace obtained from a patient with a pure paracetamol overdose. Fig. 3.-Trace obtained from a quality control specimen containing paracetamol at a concentrate of 50 1J.8:/m1 and also seven common barbiturates, each at a concentration of 20!Jog/mi.

4 7 (200 p.g/ml and 50 p.g/ml) in five separate batches. The same samples were assayed by the Gibson method. The between-batch precision is shown for each method in table 2. Time required. In the emergency situation this is a serious consideration. The average time required for a batch of UV estimations, including centrifugation to remove emulsions (q.v.), was 20 min to 30 min for a set of standards, a quality control, and a specimen. The limiting factor on gas chromatographic analysis is the time taken for chromatography. Using the method as above it was possible to obtain the results on specimens with a poor history in 45 minutes including I standard and I quality control. Where the history indicated no other drugs the oven temperature was raised to 260 C and the results were obtained in 30 minutes. DISCUSSION The UV method for the estimation of paracetamol will probably continue for some time to be the most convenient method for the emergency situation, as it is rapid, sufficiently accurate for the decision to be made as to whether to discharge or keep and treat the patient. and can be carried out using a recording UV spectrophotometer, which is now commonly available and simple to operate. The disadvantages of the method are: (a) there is a difference in the percentage recovery of paracetamol from aqueous and plasma or serum samples, which is partly due to the tendency for emulsions to form when using ether and proteinaceous solutions; (b) the amount of manual work is considerable and involves the removal of an accurate volume of ether after extraction; (c) the formation of emulsions generally means that there is a requirement to centrifuge for at least 5 minutes, which adds to the time taken; (d) the use of ether in an emergency laboratory, which normally contains flame photometers, is a dangerous procedure which was regarded as not acceptable in this hospital group. The procedure suggested here, while taking a longer time to complete, involves the staff in a minimum amount of actual manipulation. The use of the internal standard means that volumes are not important after the initial additions and therefore less glassware and time is taken. Much of the time taken is due to the requirement for evaporation of solvent and elution from the gas chromatograph. This can be minimised by efficient planning, and in a busy laboratory by leaving GLC ovens on overnight. The major advantage of this system over the UV method is the use of non-inflammable solvent, coupled with increased recovery and greater precision. We have so far suffered no interference from other drugs. Comparison with previously published GLC methods show that this method is more rapid than that described by Prescott (1971) for which preparation of a derivative is required. The method is no more rapid than that of Grove (1971), but the use of an internal standard which has similar extraction characteristics to paracetamol makes the use of chloroform as extractant a possibility, with the advantage that this leads to less contaminating peaks. It is appreciated that many laboratories do not have GLC equipment available for emergency estimations. For those who do, this method is presented as one which has given rise to few problems in our hands over the past year. Table 2. Between batch precision figures for weighed-in quality control sera using the GLe and UV methods Method Mean Value No. of specimens Standard deviation Coefficient of variation GLC 197!Lg/ml 20 ± % 49.3!Lg/ml 20 ± % UV 182!Lg/ml 20 ± % 43.4!Lg/m1 20 ± %

5 8 REFERENCES Gibson, P. F. Haemodialysis in paracetamol self-poisoning. Lancet, 2 (1972) 607. Grove, J. Gas liquid chromatography of N-acetyl-paminophenol (paracetamol) in plasma and urine. J. Chromatog, 59 (1971) 289. Prescott, L. F. The gas chromatographic estimation of phenacetin and paracetamol in plasma and urine. J. Pharm. Pharmac. 23 (1971) 111. Prescott, L. F. The gas-liquid chromatographic estimation of paracetamol. J. Pharm, Pharmacol. 23 (1971) 807. Prescott, L. F., Wright, N., Roscoe, P., Brown, S. S. Plasma paracetamol halflife and hepatic necrosis in.patients with paracetamol overdosage. Lancet, 1 (1971) 519. Prescott, L. F., et al., Successful treatment of severe paracetamol overdosage with cysteamine. Lancet, 1 (1974) 588. Proudfoot, A. T., Wright, N. Acute paracetamol poisoning. Brit. med. J. 4 (1970) 557. Routh, J. I., et al., Determination of N-acetyl-p -arninophenol in plasma. Clin. Chem. 14 (1968) 882.