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Rapid spectrofluorimetric determination of plasma salicylate with edta and terbium
Analytica Chimica Acta, 201(198’7) 335-338 Elsevier Science Publishers, B.V., Amsterdam - Printed in The Netherlands
Short Communication RAPID SPECTROFLUORIMETRIC DETERMINATION SALICYLATE WITH EDTA AND TERBIUM
M. P. BAILEY
OF PLASMA
and B. F. ROCKS*
Biochemistry Department, Royal Sussex County Brighton BN2 5BE (Great Britain)
Hospital,
Eastern Road,
C. RILEY Centre for Medical Research, University Sussex BNl 9RF (Great Britain) (Received 19th February
of Sussex, Falmer,
1987)
Summary. A rapid single-reagent spectrofluorimetric assay for salicylate in human blood plasma is reported. The method is based on ternary complex formation with terbium and EDTA in alkaline solution; it requires only 10 ~1 of plasma, and protein precipitation is not necessary. Within-assay relative standard deviations were better than 2.5%, and correlation with the Trinder method was excellent. The procedure is particularly suited for emergency use in cases of suspected aspirin poisoning.
A simple rapid method for the determination of salicylate in blood plasma is required in cases of accidental or suicidal overdose with aspirin. Determination of salicylate is also useful in monitoring blood concentrations in patients on long-term aspirin therapy, Therapeutic concentrations are usually below 200 mg 1-l while, above 300 mg l-l, symptoms of toxicity appear. Concentrations of salicylate in plasma exceeding 600 mg 1-l are usually lethal. Techniques described for the assay of salicylate include fluorimetry of plasma extracts [l] , liquid chromatography [2], enzymatic methods [ 31, and immunoassay [4]. By far the most commonly used method, however, is the calorimetric procedure of Trinder [5], which is based on the use of a reagent containing iron(II1) nitrate, mercury(I1) chloride and hydrochloric acid, which precipitates the proteins and simultaneously reacts with salicylate to produce a purple solution. After centrifugation, the absorbance of the supematant liquid, which contains the soluble iron(III)/salicylate complex, is measured at 540 nm. Unfortunately, this simple method suffers from interference by a number of drugs and metabolites which give colour reactions similar to that for salicylate [6, 71. A fluorescent ternary complex of terbium with ethylenediaminetetraacetic acid (EDTA) and sulphosalicylic acid was reported by Charles and Riedel [8] in 1966. A similar fluorescent complex is formed from terbium, EDTA and salicylic acid in alkaline solution. This complex absorbs radiation at 320 nm and transfers the energy to the terbium ion, which emits a narrowline spectrum with maxima at 488, 547 and 596 nm (see Fig. 1). In this 0003-2670/87/$03.50
0 1987 Elsevier Science Publishers B.V.
336
go
80
70
20
10
/
;
0 250
I!. .-.. .h A *......._
.__.“...
300
350
LOO
Wavelength
Fig. 1. Excitation
(. *. ) and emission (-
L50
500
550
600
(nm)
) spectra of the terbium/EDTA/salicylate
complex.
communication, a reagent containing terbium and EDTA at pH 12 is used to determine salicylate spectrofluorimetrically, with greater selectivity than the Trinder method.
Experimental Reagents. Demineralized water was used throughout. Sodium salicylate and EDTA, tetrasodium salt, were laboratory reagents and sodium hydroxide was AnalaR grade (BDH). Terbium chloride was obtained from Aldrich. A stock 10 g 1-l salicylate standard was prepared by dissolving 292 mg of sodium salicylate in 25 ml of water. Working standards were prepared by dilution of this solution with 0.05 mol 1-l sodium hydroxide to give concentrations of 200, 400, 600,800 and 1000 mg 1-l salicylic acid. Alkaline terbium/EDTA reagent was prepared by dissolving 1.86 g of terbium chloride hexahydrate and 4.1 g of EDTA (tetrasodium salt) in water, adding 20 ml of 1 mol 1-l sodium hydroxide, and diluting to 500 ml with water. The terbium chloride and EDTA should be completely dissolved
331
before the sodium hydroxide is added, otherwise terbium hydroxide is precipitated and is extremely slow to redissolve. The pH of the reagent should be ca. 12. Procedure. A lo-p1 portion of standard or sample was mixed with 3.0 ml of reagent and the fluorescent intensity was measured in a 1 X 1 cm quartz cuvette with a Perkin-Elmer MPF-3L spectrofluorimeter. The instrument settings were: excitation wavelength 320 nm; excitation slitwidth 10 nm; emission wavelength 547 nm; emission slitwidth 16 nm; instrument sensitivity 1.0. The emission peak is very narrow, and is best located initially by manual scanning in the region around 545 nm. Results and discussion The fluorescence intensity increased linearly with salicylate concentration up to at least 1000 mg 1-i. The reagent blank reading was less than 0.5% of the reading given by the 1000 mg 1-l standard. Table 1 shows the recovery of added salicylate from 10 plasma specimens. The mean recovery, in specimens with salicylate concentrations corresponding to the upper limit of the therapeutic range, was 100.3% with a range from 98 to 103%. The within-assay relative standard deviation for 20 replicate samples from each of two pools of spiked plasma was 2.4% at 50 mg 1-l and 1.4% at 500 mg 1-l. The between-assay r.s.d. (n = 8) was 7.2% at 50 mg 1-l and 2.3% at 500 mg 1-l. The limit of detection (30) was 0.4 mg 1-l. Comparison between the present method (X) and the Trinder calorimetric method [7] (our routine assay, Y) for 16 plasma samples received in the emergency laboratory from patients with possible salicylate poisoning, gave a linear correlation equation of X = 1.06Y - 21 (in mg 1-l) with a correlation coefficient of 0.997 (n = 16). In addition, 6 plasma samples which were reported as “not detected” by the calorimetric method showed salicylate concentrations of less than 2 mg 1-l by the fluorimetric method. Several compounds tested which showed substantial interference in the Trinder method [7] showed no fluorescence with terbium and EDTA. The following concentrations (mg 1-l) gave less fluorescence intensity than 2 mg 1-l TABLE
1
Recovery of salicylate added to plasma samples Salicylate concentration (me 1-l1 Expected
Measured
189 213 232 222 247
188 209 230 223 245
Recovery (%I
99.5 98.1 99.1 100.5 99.2
Salicylate concentration (me 1-l1 Expected
Measured
202 185 217 253 211
205 188 216 260 213
Recovery WI
101.5 101.6 99.5 102.8 100.9
338
salicylate : acetoacetate (10 300), p-acetaminophenol (200), 3-hydroxytyramine (500), 3,4dihydroxybenzylamine (1300), 3,4dihydroxycinnamic acid (1400), 5-hydroxyindoleacetic acid (660), p-hydroxybenzoic acid (7 50), salicylurid acid (200) and gentisic acid (240). Enhancement of terbium fluorescence by catechol derivatives has been observed under conditions when the catechol is in excess, but the complete absence of interference by such compounds in the present method probably reflects their extreme instability in dilute oxygenated alkaline solution. Moderate amounts of lipaemia, icterus or haemolysis did not interfere with the estimation of salicylate in plasma. The use of a whole blood specimen yields a solution which rapidly becomes clear, but is strongly coloured and gives a measured concentration about 30% of the true concentration. The reaction mixture in this instance had an absorbance of 0.59 at 320 nm, indicating that the underestimate was a result of absorption of the exciting radiation. By using a shorter excitation pathlength, or by measuring frontsurface emission, it should be possible to use the present method to measure salicylate in whole blood. It is worth noting that Trinder’s reagent is a highly corrosive and toxic solution. Salicylate estimation is frequently requested as an emergency out-of-hours procedure: the assay should therefore be rapid and simple. The method presented here uses a single stable reagent and requires no sample extraction, protein precipitation or blank correction. The test can be read immediately after reagent addition, and the calibration is sufficiently stable that a single graph could be used for determinations over several days. The small sample volume ensures that even paediatric specimens should present no problem. We thank the South-East Thames Regional Health Authority for financial support under a Locally Organized Research Scheme. We also thank the staff of the emergency laboratory, and the on-call staff, for their assistance in collecting blood samples from patients with suspected aspirin overdose. REFERENCES 1 D. O’Brien, F. A. Ibbott and D. 0. Rodgerson, Laboratory Manual of Paediatric Microbiochemical Techniques, Harper and Row, New York, 1968. 2 P. J. Twitchett, A. E. P. Gorvin, A. C. Moffat, P. L. Williams and A. T. Sullivan, in P. F. Dixon, C. H. Gray, C. K. Lim and M. S. Stoll (Eds.), High Pressure Liquid Chromatography in Clinical Chemistry, Academic, London, 1976. 3 K. You and J. A. Bittikofer, Clin. Chem., 30 (1984) 1549. 4 C. L. Keegan, F. Ungemach, J. Simpson and M. Aden, Clin. Chem., 31(1985) 942. 5 P. Trinder, Biochem. J., 57 (1954) 301. 6 K. D. Mutchie, G. H. Saunders, A. S. Hanissan and T. E. Poe, J. Rheumatol., 7 (1980) 737. 7 E. S. Kang, T. A. Todd, M. T. Capaci, K. Schwenzer and J. T. Jabbour, Clin. Chem., 29 (1983) 1012. 8 R. G. Charles and E. P. Riedel, J. Inorg. Nucl. Chem., 28 (1966) 527.
Short Communication RAPID SPECTROFLUORIMETRIC DETERMINATION SALICYLATE WITH EDTA AND TERBIUM
M. P. BAILEY
OF PLASMA
and B. F. ROCKS*
Biochemistry Department, Royal Sussex County Brighton BN2 5BE (Great Britain)
Hospital,
Eastern Road,
C. RILEY Centre for Medical Research, University Sussex BNl 9RF (Great Britain) (Received 19th February
of Sussex, Falmer,
1987)
Summary. A rapid single-reagent spectrofluorimetric assay for salicylate in human blood plasma is reported. The method is based on ternary complex formation with terbium and EDTA in alkaline solution; it requires only 10 ~1 of plasma, and protein precipitation is not necessary. Within-assay relative standard deviations were better than 2.5%, and correlation with the Trinder method was excellent. The procedure is particularly suited for emergency use in cases of suspected aspirin poisoning.
A simple rapid method for the determination of salicylate in blood plasma is required in cases of accidental or suicidal overdose with aspirin. Determination of salicylate is also useful in monitoring blood concentrations in patients on long-term aspirin therapy, Therapeutic concentrations are usually below 200 mg 1-l while, above 300 mg l-l, symptoms of toxicity appear. Concentrations of salicylate in plasma exceeding 600 mg 1-l are usually lethal. Techniques described for the assay of salicylate include fluorimetry of plasma extracts [l] , liquid chromatography [2], enzymatic methods [ 31, and immunoassay [4]. By far the most commonly used method, however, is the calorimetric procedure of Trinder [5], which is based on the use of a reagent containing iron(II1) nitrate, mercury(I1) chloride and hydrochloric acid, which precipitates the proteins and simultaneously reacts with salicylate to produce a purple solution. After centrifugation, the absorbance of the supematant liquid, which contains the soluble iron(III)/salicylate complex, is measured at 540 nm. Unfortunately, this simple method suffers from interference by a number of drugs and metabolites which give colour reactions similar to that for salicylate [6, 71. A fluorescent ternary complex of terbium with ethylenediaminetetraacetic acid (EDTA) and sulphosalicylic acid was reported by Charles and Riedel [8] in 1966. A similar fluorescent complex is formed from terbium, EDTA and salicylic acid in alkaline solution. This complex absorbs radiation at 320 nm and transfers the energy to the terbium ion, which emits a narrowline spectrum with maxima at 488, 547 and 596 nm (see Fig. 1). In this 0003-2670/87/$03.50
0 1987 Elsevier Science Publishers B.V.
336
go
80
70
20
10
/
;
0 250
I!. .-.. .h A *......._
.__.“...
300
350
LOO
Wavelength
Fig. 1. Excitation
(. *. ) and emission (-
L50
500
550
600
(nm)
) spectra of the terbium/EDTA/salicylate
complex.
communication, a reagent containing terbium and EDTA at pH 12 is used to determine salicylate spectrofluorimetrically, with greater selectivity than the Trinder method.
Experimental Reagents. Demineralized water was used throughout. Sodium salicylate and EDTA, tetrasodium salt, were laboratory reagents and sodium hydroxide was AnalaR grade (BDH). Terbium chloride was obtained from Aldrich. A stock 10 g 1-l salicylate standard was prepared by dissolving 292 mg of sodium salicylate in 25 ml of water. Working standards were prepared by dilution of this solution with 0.05 mol 1-l sodium hydroxide to give concentrations of 200, 400, 600,800 and 1000 mg 1-l salicylic acid. Alkaline terbium/EDTA reagent was prepared by dissolving 1.86 g of terbium chloride hexahydrate and 4.1 g of EDTA (tetrasodium salt) in water, adding 20 ml of 1 mol 1-l sodium hydroxide, and diluting to 500 ml with water. The terbium chloride and EDTA should be completely dissolved
331
before the sodium hydroxide is added, otherwise terbium hydroxide is precipitated and is extremely slow to redissolve. The pH of the reagent should be ca. 12. Procedure. A lo-p1 portion of standard or sample was mixed with 3.0 ml of reagent and the fluorescent intensity was measured in a 1 X 1 cm quartz cuvette with a Perkin-Elmer MPF-3L spectrofluorimeter. The instrument settings were: excitation wavelength 320 nm; excitation slitwidth 10 nm; emission wavelength 547 nm; emission slitwidth 16 nm; instrument sensitivity 1.0. The emission peak is very narrow, and is best located initially by manual scanning in the region around 545 nm. Results and discussion The fluorescence intensity increased linearly with salicylate concentration up to at least 1000 mg 1-i. The reagent blank reading was less than 0.5% of the reading given by the 1000 mg 1-l standard. Table 1 shows the recovery of added salicylate from 10 plasma specimens. The mean recovery, in specimens with salicylate concentrations corresponding to the upper limit of the therapeutic range, was 100.3% with a range from 98 to 103%. The within-assay relative standard deviation for 20 replicate samples from each of two pools of spiked plasma was 2.4% at 50 mg 1-l and 1.4% at 500 mg 1-l. The between-assay r.s.d. (n = 8) was 7.2% at 50 mg 1-l and 2.3% at 500 mg 1-l. The limit of detection (30) was 0.4 mg 1-l. Comparison between the present method (X) and the Trinder calorimetric method [7] (our routine assay, Y) for 16 plasma samples received in the emergency laboratory from patients with possible salicylate poisoning, gave a linear correlation equation of X = 1.06Y - 21 (in mg 1-l) with a correlation coefficient of 0.997 (n = 16). In addition, 6 plasma samples which were reported as “not detected” by the calorimetric method showed salicylate concentrations of less than 2 mg 1-l by the fluorimetric method. Several compounds tested which showed substantial interference in the Trinder method [7] showed no fluorescence with terbium and EDTA. The following concentrations (mg 1-l) gave less fluorescence intensity than 2 mg 1-l TABLE
1
Recovery of salicylate added to plasma samples Salicylate concentration (me 1-l1 Expected
Measured
189 213 232 222 247
188 209 230 223 245
Recovery (%I
99.5 98.1 99.1 100.5 99.2
Salicylate concentration (me 1-l1 Expected
Measured
202 185 217 253 211
205 188 216 260 213
Recovery WI
101.5 101.6 99.5 102.8 100.9
338
salicylate : acetoacetate (10 300), p-acetaminophenol (200), 3-hydroxytyramine (500), 3,4dihydroxybenzylamine (1300), 3,4dihydroxycinnamic acid (1400), 5-hydroxyindoleacetic acid (660), p-hydroxybenzoic acid (7 50), salicylurid acid (200) and gentisic acid (240). Enhancement of terbium fluorescence by catechol derivatives has been observed under conditions when the catechol is in excess, but the complete absence of interference by such compounds in the present method probably reflects their extreme instability in dilute oxygenated alkaline solution. Moderate amounts of lipaemia, icterus or haemolysis did not interfere with the estimation of salicylate in plasma. The use of a whole blood specimen yields a solution which rapidly becomes clear, but is strongly coloured and gives a measured concentration about 30% of the true concentration. The reaction mixture in this instance had an absorbance of 0.59 at 320 nm, indicating that the underestimate was a result of absorption of the exciting radiation. By using a shorter excitation pathlength, or by measuring frontsurface emission, it should be possible to use the present method to measure salicylate in whole blood. It is worth noting that Trinder’s reagent is a highly corrosive and toxic solution. Salicylate estimation is frequently requested as an emergency out-of-hours procedure: the assay should therefore be rapid and simple. The method presented here uses a single stable reagent and requires no sample extraction, protein precipitation or blank correction. The test can be read immediately after reagent addition, and the calibration is sufficiently stable that a single graph could be used for determinations over several days. The small sample volume ensures that even paediatric specimens should present no problem. We thank the South-East Thames Regional Health Authority for financial support under a Locally Organized Research Scheme. We also thank the staff of the emergency laboratory, and the on-call staff, for their assistance in collecting blood samples from patients with suspected aspirin overdose. REFERENCES 1 D. O’Brien, F. A. Ibbott and D. 0. Rodgerson, Laboratory Manual of Paediatric Microbiochemical Techniques, Harper and Row, New York, 1968. 2 P. J. Twitchett, A. E. P. Gorvin, A. C. Moffat, P. L. Williams and A. T. Sullivan, in P. F. Dixon, C. H. Gray, C. K. Lim and M. S. Stoll (Eds.), High Pressure Liquid Chromatography in Clinical Chemistry, Academic, London, 1976. 3 K. You and J. A. Bittikofer, Clin. Chem., 30 (1984) 1549. 4 C. L. Keegan, F. Ungemach, J. Simpson and M. Aden, Clin. Chem., 31(1985) 942. 5 P. Trinder, Biochem. J., 57 (1954) 301. 6 K. D. Mutchie, G. H. Saunders, A. S. Hanissan and T. E. Poe, J. Rheumatol., 7 (1980) 737. 7 E. S. Kang, T. A. Todd, M. T. Capaci, K. Schwenzer and J. T. Jabbour, Clin. Chem., 29 (1983) 1012. 8 R. G. Charles and E. P. Riedel, J. Inorg. Nucl. Chem., 28 (1966) 527.