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Correction for salicylate interference in the colorimetric paracetamol assay
301
Clinica Chimica Acta, 122 (1982) 301-304 Elsevier Biomedical Press
CCA 2157
Brief technical
note
Correction for salicylate interference in the calorimetric paracetamol assay J.E. Buttery Depurtmeut
*, E.A. Braiotta
of Cliuicul Chemisiy,
(Received
and P.R.
The Queen Ehzuheth Hosp,tul. (Austruhu)
November
3 1st. 198 I : revision
Pannall
Woodoille. South Au.wolru.
February
501 I
17th. 1982)
Introduction The calorimetric nitration method for plasma paracetamol (acetaminophen) estimation [ 1,2] is simple and rapid and is well suited to emergency and after-hours requests. The method is unaffected by most drugs except salicylate and salicylamide [3,4]. Salicylate interference is the more likely as several proprietary preparations contain both paracetamol and acetylsalicylic acid. Mace and Walker [5] proposed two ways of minimising this salicylate interference. One, measuring the absorbance at 450 nm rather than the usual 430 nm was said to reduce interference by 60%. Alternatively, both paracetamol and salicylate may be assayed in the sample. The apparent paracetamoi concentration (P) due to salicylate interference (S) can be derived from the equation P (pmol/l) = 64 S (mmol/l) f 46, and is then subtracted from the measured paracetamol concentration to give the correct result. We observed that whereas, in most samples, the paracetamol concentrations (P) measured at 450 nm were similar, in others they were lower when measured at 450 nm. The latter specimens were subsequently shown to contain salicylate. It seemed that when the concentrations measured at the two wave-lengths were similar, the sample contained only paracetamol and that any difference was probably due to salicylate, and that the difference increased with increasing salicylate levels. Based on these observations, we devised a method to correct for salicylate interference in the paracetamol assay. Using a pre-constructed graph this difference can be equated to a ‘paracetamol’ concentration, which, when subtracted from the measured paracetamol concentration gives the correct result. Method The procedure of Glynn and Kendal II] as modified by Wiener [2] was followed, with an aqueous paracetamol solution (2000 pmol/l) as standard. In initial tests, the * To whom correspondence 0009498
l/82/0000-0000/$02.75
should
be addressed.
8 I982 Elsevier Biomedical
Press
302
absorbance values of serum base standard and aqueous standard were similar, consequently the latter was chosen for routine use. The interference of salicylate measured as paracetamol by the above method was investigated using a range of salicylate standards (l-5 mmol/l) prepared in serum free of paracetamol and salicylate. The absorbances were measured at 430 nm and 450 nm. Sera containing known concentrations of paracetamol (100-2000 pmol/l) and salicylate (2-4 mmol/l) were prepared and assayed for paracetamol using the two wave-lengths. Both the within-day and the day-to-day precision of our method was evaluated by assaying 20 times each a specimen containing 1000 pmol/l paracetamol and 4 mmol/l salicylate. Salicylate was determined by the method of Trinder [6]. Results The absorption spectra of nitrated samples containing paracetamol, salicylate and a mixture of both are shown in Fig. 1. The sample containing paracetamol showed absorbance characteristics similar to the paracetamol standard, but the sample containing the mixture had a higher absorbance at 430 nm than at 450 nm. The ‘paracetamol’ concentrations, in sera containing only salicylate, measured at the two wave-lengths, are shown in Table I. The difference between these values (AP), plotted against the apparent concentration of paracetamol measured at 430 nm shows a linear relationship. Fig. 2 is a typical correction graph. We have found it to be reproducible provided measurements are taken on the same spectrophotometer. Recalibration with serum base salicylate standards is recommended when reagents are changed. The application of this approach to serum samples containing both paracetamol and salicylate is shown in Table II. The difference (A P) is converted from the graph in Fig. 2 to give a measure of salicylate interference in terms of paracetamol. This is then subtracted from the paracetamol calculated at 430 nm to give a corrected concentration which agrees well with the expected result. Paracetamol values cor-
0.4
L
P (Std)
0.3
0.2
0.1
450
400
WAVELENGTH
Fig. I. Absorption
spectra
500 nm
of nitrated
compounds
of paracetamol
(P),
salicylate
(S) and mixture
303 TABLE SERUM
I SALICYLATE
Salicylate (mmol/l)
MEASURED
Apparent
AS PARACETAMOL
paracetamol
(P)
AP
(rmol/l)
1 2 3 4 5
AT 2 WAVE-LENGTHS
(amol/l)
430 nm
450 nm
66 165 253 347 435
‘35 93 151 215 267
31 72 102 132 168
rected for salicylate interference by the method of Mace and Walker [5] are also compared. The within-day precision (mean 1014 pmol/l, SD 25, CV 2.5%) and the day-to-day precision (mean 1017 pmol/l, SD 29, CV 2.8%) of our method was excellent. The salicylate concentrations measured on the samples containing salicylate and paracetamol by Trinder’s method gives results which agree very closely with the expected values.
TABLE
II
MEASUREMENT OF PARACETAMOL ON SPECIMENS TIONS OF PARACETAMOL AND SALICYLATES Specimen
containing
Paracetamol
Corrected paracetamol
(pmol/l) paracetamol (pmol/l)
salicylate (mmol/l)
CONTAINING
(pmol/l) 430 nm
450 nm
KNOWN
CONCENTRA-
Paracetamol (pmol/l) Mace and Walker
100 100
2 4
280 371
208 271
105 118
114 82
200 200
2 4
399 527
313 403
186 212
229 235
500 500
2 4
662 813
598 692
507 506
491 519
1000 1000
2 4
1 176 1359
1 103 1220
996 1001
1004 1064
1500 I 500
2 4
1686 1870
1616 1726
1516 1500
1513
2000 2000
2 4
2221 2356
2138 2223
2014 2016
2047 2058
1574
[S]
AP 100 (pmolll)
100
APPARENT
200 PARACETAMOL
300 CONCENTRATION
500
400 (YmOl/I
Fig. 2. The salicylate induced difference in apparent paracetamol against the concentration of apparent paracetamol at 430 nm.
)
concentration
( AP) at two wave-lengths
Discussion The measurement of paracetamol in serum containing a mixture of paracetamol and salicylate shows that salicylate falsely increases values (Table 11). This interference is less when the measurement is done at 450 nm rather than 430 nm, because salicylate absorbs less at 450 nm (Fig. 1). We have not, however, been able to reduce interference by 60% by reading at 450 nm as claimed by Mace and Walker [5]. Like Wiener [3], we found a much smaller reduction in interference. When the salicylate interference is corrected using the formula of Mace and Walker [5], the paracetamol results are closer to the expected values but are significantly higher (t = 2.29, p -C0.05) than those derived by our method (Table 11). Our method, besides providing a more accurate way of correcting for salicylate interference, does not require a salicylate estimation to be done. A markedly raised serum paracetamol (e.g. 1000 pmol/l) indicates a high value regardless of salicylate interference. It is in the case of moderate elevations (300-800 pmol/l) that it is important to ascertain the true value. We believe that the method described in this paper offers a rapid way to do so. References I Glynn JP, Kendal SE. Paracetamol measurement. Lancet 1975; 1: 1147-I 148. 2 Wiener K. Paracetamol estimation: comparison of a quick calorimetric method with a standard spectrophotometric method. Ann Clin B&hem 1977: 14: 55-5X. 3 Wiener K. A review of methods for plasma paracetamol estimation. Ann Clin Biochem 1978: 15: 1X7-196. 4 Chafetz L, Daly RE, Schriftman H, Lomner JJ. Selective calorimetric determination of acetaminophen. J Pharm Sci 1911; 60: 463-466. 5 Mace PFK, Walker G. Salicylate interference with plasma-paracetamol method. Lancet 1976; 2: 1362. 6 Trinder P. Rapid determination of salicylate in biological fluids. Biochem J 1954: 57: 301-303.
Clinica Chimica Acta, 122 (1982) 301-304 Elsevier Biomedical Press
CCA 2157
Brief technical
note
Correction for salicylate interference in the calorimetric paracetamol assay J.E. Buttery Depurtmeut
*, E.A. Braiotta
of Cliuicul Chemisiy,
(Received
and P.R.
The Queen Ehzuheth Hosp,tul. (Austruhu)
November
3 1st. 198 I : revision
Pannall
Woodoille. South Au.wolru.
February
501 I
17th. 1982)
Introduction The calorimetric nitration method for plasma paracetamol (acetaminophen) estimation [ 1,2] is simple and rapid and is well suited to emergency and after-hours requests. The method is unaffected by most drugs except salicylate and salicylamide [3,4]. Salicylate interference is the more likely as several proprietary preparations contain both paracetamol and acetylsalicylic acid. Mace and Walker [5] proposed two ways of minimising this salicylate interference. One, measuring the absorbance at 450 nm rather than the usual 430 nm was said to reduce interference by 60%. Alternatively, both paracetamol and salicylate may be assayed in the sample. The apparent paracetamoi concentration (P) due to salicylate interference (S) can be derived from the equation P (pmol/l) = 64 S (mmol/l) f 46, and is then subtracted from the measured paracetamol concentration to give the correct result. We observed that whereas, in most samples, the paracetamol concentrations (P) measured at 450 nm were similar, in others they were lower when measured at 450 nm. The latter specimens were subsequently shown to contain salicylate. It seemed that when the concentrations measured at the two wave-lengths were similar, the sample contained only paracetamol and that any difference was probably due to salicylate, and that the difference increased with increasing salicylate levels. Based on these observations, we devised a method to correct for salicylate interference in the paracetamol assay. Using a pre-constructed graph this difference can be equated to a ‘paracetamol’ concentration, which, when subtracted from the measured paracetamol concentration gives the correct result. Method The procedure of Glynn and Kendal II] as modified by Wiener [2] was followed, with an aqueous paracetamol solution (2000 pmol/l) as standard. In initial tests, the * To whom correspondence 0009498
l/82/0000-0000/$02.75
should
be addressed.
8 I982 Elsevier Biomedical
Press
302
absorbance values of serum base standard and aqueous standard were similar, consequently the latter was chosen for routine use. The interference of salicylate measured as paracetamol by the above method was investigated using a range of salicylate standards (l-5 mmol/l) prepared in serum free of paracetamol and salicylate. The absorbances were measured at 430 nm and 450 nm. Sera containing known concentrations of paracetamol (100-2000 pmol/l) and salicylate (2-4 mmol/l) were prepared and assayed for paracetamol using the two wave-lengths. Both the within-day and the day-to-day precision of our method was evaluated by assaying 20 times each a specimen containing 1000 pmol/l paracetamol and 4 mmol/l salicylate. Salicylate was determined by the method of Trinder [6]. Results The absorption spectra of nitrated samples containing paracetamol, salicylate and a mixture of both are shown in Fig. 1. The sample containing paracetamol showed absorbance characteristics similar to the paracetamol standard, but the sample containing the mixture had a higher absorbance at 430 nm than at 450 nm. The ‘paracetamol’ concentrations, in sera containing only salicylate, measured at the two wave-lengths, are shown in Table I. The difference between these values (AP), plotted against the apparent concentration of paracetamol measured at 430 nm shows a linear relationship. Fig. 2 is a typical correction graph. We have found it to be reproducible provided measurements are taken on the same spectrophotometer. Recalibration with serum base salicylate standards is recommended when reagents are changed. The application of this approach to serum samples containing both paracetamol and salicylate is shown in Table II. The difference (A P) is converted from the graph in Fig. 2 to give a measure of salicylate interference in terms of paracetamol. This is then subtracted from the paracetamol calculated at 430 nm to give a corrected concentration which agrees well with the expected result. Paracetamol values cor-
0.4
L
P (Std)
0.3
0.2
0.1
450
400
WAVELENGTH
Fig. I. Absorption
spectra
500 nm
of nitrated
compounds
of paracetamol
(P),
salicylate
(S) and mixture
303 TABLE SERUM
I SALICYLATE
Salicylate (mmol/l)
MEASURED
Apparent
AS PARACETAMOL
paracetamol
(P)
AP
(rmol/l)
1 2 3 4 5
AT 2 WAVE-LENGTHS
(amol/l)
430 nm
450 nm
66 165 253 347 435
‘35 93 151 215 267
31 72 102 132 168
rected for salicylate interference by the method of Mace and Walker [5] are also compared. The within-day precision (mean 1014 pmol/l, SD 25, CV 2.5%) and the day-to-day precision (mean 1017 pmol/l, SD 29, CV 2.8%) of our method was excellent. The salicylate concentrations measured on the samples containing salicylate and paracetamol by Trinder’s method gives results which agree very closely with the expected values.
TABLE
II
MEASUREMENT OF PARACETAMOL ON SPECIMENS TIONS OF PARACETAMOL AND SALICYLATES Specimen
containing
Paracetamol
Corrected paracetamol
(pmol/l) paracetamol (pmol/l)
salicylate (mmol/l)
CONTAINING
(pmol/l) 430 nm
450 nm
KNOWN
CONCENTRA-
Paracetamol (pmol/l) Mace and Walker
100 100
2 4
280 371
208 271
105 118
114 82
200 200
2 4
399 527
313 403
186 212
229 235
500 500
2 4
662 813
598 692
507 506
491 519
1000 1000
2 4
1 176 1359
1 103 1220
996 1001
1004 1064
1500 I 500
2 4
1686 1870
1616 1726
1516 1500
1513
2000 2000
2 4
2221 2356
2138 2223
2014 2016
2047 2058
1574
[S]
AP 100 (pmolll)
100
APPARENT
200 PARACETAMOL
300 CONCENTRATION
500
400 (YmOl/I
Fig. 2. The salicylate induced difference in apparent paracetamol against the concentration of apparent paracetamol at 430 nm.
)
concentration
( AP) at two wave-lengths
Discussion The measurement of paracetamol in serum containing a mixture of paracetamol and salicylate shows that salicylate falsely increases values (Table 11). This interference is less when the measurement is done at 450 nm rather than 430 nm, because salicylate absorbs less at 450 nm (Fig. 1). We have not, however, been able to reduce interference by 60% by reading at 450 nm as claimed by Mace and Walker [5]. Like Wiener [3], we found a much smaller reduction in interference. When the salicylate interference is corrected using the formula of Mace and Walker [5], the paracetamol results are closer to the expected values but are significantly higher (t = 2.29, p -C0.05) than those derived by our method (Table 11). Our method, besides providing a more accurate way of correcting for salicylate interference, does not require a salicylate estimation to be done. A markedly raised serum paracetamol (e.g. 1000 pmol/l) indicates a high value regardless of salicylate interference. It is in the case of moderate elevations (300-800 pmol/l) that it is important to ascertain the true value. We believe that the method described in this paper offers a rapid way to do so. References I Glynn JP, Kendal SE. Paracetamol measurement. Lancet 1975; 1: 1147-I 148. 2 Wiener K. Paracetamol estimation: comparison of a quick calorimetric method with a standard spectrophotometric method. Ann Clin B&hem 1977: 14: 55-5X. 3 Wiener K. A review of methods for plasma paracetamol estimation. Ann Clin Biochem 1978: 15: 1X7-196. 4 Chafetz L, Daly RE, Schriftman H, Lomner JJ. Selective calorimetric determination of acetaminophen. J Pharm Sci 1911; 60: 463-466. 5 Mace PFK, Walker G. Salicylate interference with plasma-paracetamol method. Lancet 1976; 2: 1362. 6 Trinder P. Rapid determination of salicylate in biological fluids. Biochem J 1954: 57: 301-303.