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Simple and sensitive spectrofluorimetric determination of pyrimethamine in biological fluids
Analytica Chimica Acta, 169 (1985) 361-365 Elsevier Science Publishers B.V., Amsterdam - Printed in Tbe Netherlands
Short Communication
SIMPLE AND SENSITIVE SPECTROFLUORIMETRIC OF PYRIMETHAMINE IN BIOLOGICAL FLUIDS
DETERMINATION
0. R. IDOWU* and 0. A. DADA Department
of Chemistry,
University of Zbadan, Zbadan (Nigeria)
(Received 7 th August 1984)
Summary. The method is based on the reaction of pyrimethamine with chloroacetaldehyde and measurement of the fluorescence of the resulting pyrimidoimidazole derivative. The limits of detection for pyrimethamine in ethanol, plasma and saliva were 2.7, 8.4 and 6.6 ng ml-‘, respectively. Chloroquine and quinine do not interfere.
Pyrimethamine (I; 2,4-diamino-5-(3-chlorophenyl)-6-ethylpyrimidine) has long been in use in antimalarial therapy as a suppressive, often in combination with a sulphonamide such as sulphadoxme. It is also used as a coccidiostat in rabbit and poultry feeds [l] and occasionally, in the treatment of meningeal leukemia [2] . Renewed interest in the antimalarial activity of pyrimethamine has arisen recently because of the emergence of plasmodial resistance to a number of chloroquine-like drugs at present in use, and the observation that the development of plasmodial resistance is markedly de layed when a pyrimethamine/sulphonamide combination is used along with another antimalarial such as quinine or mefloquine, a recent quinoline derivative [3] . Interest in the pharmacokinetics of mefloquine suggested a study of the pharmacokinetics of pyrimethamine when used in combination with mefloquine and a sulphonamide. A rapid, sensitive and selective method was therefore needed for the determination of pyrimethamine in plasma and, especially, in saliva, which is a more convenient sampling fluid. Various methods have been reported for the determination of pyrimethamine in feeding stuffs, chicken tissue and body fluids. These include gas chromatography [4-71, thin-layer chromatography in combination with spectrophotodensitometry [2] and spectrofluorodensitometry [8, 91, and high-performance liquid chromatography with ultra-violet absorbance detection [l, 10,111 or fluorescence detection [12]. There is, however, no report on the determination of pyrimethamine by solution spectrofluorimetry, probably because pyrimethamine possesses only a very weak native fluorescence. However, a spectrofluorimetric method offers the advantages of simplicity and cheapness, which may be important in areas of endemic malaria where economic and technological difficulties may render published methods
inaccessible. This communication describes a simple, sensitive and rapid spectrofluorimetric method for quantifying pyrimethamine in plasma and saliva based on its reaction with chloroacetaldehyde. The method is applicable to the determination of pyrimethamine in the presence of sulphadoxine, mefloquine and quinine. Experimental Reagents and apparatus. Chloroacetal, 1,2-dichloroethane and anhydrous quinine sulphate were all reagent grade whereas ethanol was of analytical grade (BDH Chemicals Ltd.). Quinine sulphate was recrystallized twice from hot water before use. Pyrimethamine was obtained by crushing 20 tablets of Daraprim (Wellcome) in 2 ml of 0.1 M sodium hydroxide, extracting the slurry twice with 30 ml of chloroform, and drying the extract over anhydrous sodium sulphate before evaporation, The pyrimethamme obtained had a melting point of 237-238°C (lit. 239-242°C [3]). (Found, C = 57.9% H = 5.35%,N=21.9%,Cl= 14.2%;expected,C=57.95%,H= 5.3%,N=22.5%, Cl = 14.25%) An acetate buffer (pH 4.5) containing 0.2 M sodium chloride was prepared. All fluorescence measurements were made on a Perkin-Elmer 204 spectrofluorimeter. Preparation of aqueous solution of chloroacetaldehyde. A solution of chloroacetal (6 g) in 30 ml of 5% (w/v) sulphuric acid was reflexed gently for 45 min and the mixture was distilled using an ice-water-cooled doublesurface reflux condenser, with the receiver cooled in an ice-water bath. The pH of the distillate was adjusted to ca. 4.5 by dropwise addition of 1 M sodium hydroxide and the mixture was distilled as before. The distillate (30 ml) was used in the following investigations. When not in use, the solution was kept in a freezer at -5”C, where it remained stable for at least a month. Examination of the reaction between chlomacetaldehyde and pyrimethamine. A 0.4-ml aliquot of a 50 pg ml-’ solution of pyrimethamine in ethanol was evaporated to dryness in a test tube and the residue was mixed with 0.2 ml of the chloroacetaldehyde solution and 0.4 ml of the acetate buffer. The mixture was heated in a boiling water bath for 30 min and, after cooling to room temperature for 5 min, the mixture was examined at 366 nm for fluorescence, by excitation at 254 nm. A further 3 ml of acetate buffer was added to the mixture and the excitation and emission wavelengths which gave maximum fluorescence intensity were established as 320 and 400 nm, respectively. Estimation of detection limits for pyrimethamine in ethanol, saliva and plasma. A 2.0-ml aliquot of ethanol was evaporated to dryness in a test tube and the residue was treated with 0.2 ml of the chloroacetaldehyde solution and 0.5 ml of the acetate buffer. The mixture was heated in a boiling waterbath for 30 min and cooled for 5 min at room temperature, and a further 2.0 ml of acetate buffer was added. The fluorescence was measured at excita-
363
tion and emission wavelengths of 320 and 450 nm, respectively. The procedure was repeated ten times. This gave a measure of the blank fluorescence. For the determination of detection limits in saliva or plasma, 1.0 ml of drug-free mixed saliva (or plasma) was placed in a test tube and made alkaline (pH 12) by addition of 0.5 ml of 2.0 M sodium hydroxide. After addition of 6.0 ml of 1,2dichloroethane, the mixture was shaken for 1 min on a vortex mixer and centrifuged at 2500 rpm for 10 min. The organic phase was removed with a Pasteur pipette and evaporated to dryness, and the residue was treated with chloroacetaldehyde and acetate buffer. The fluorescence was then measured as described above for the ethanol blank. Detection limits were calculated as the concentrations of pyrimethamine that gave intensities two standard deviations of the blank above the mean blank signal. Preparation of calibmtion‘graphs forpyrimethamine in ethanol, saliva and plasma. A stock solution containing 100 r_cgml-’ pyrimethamine was diluted serially to give a working solution of 100 ng ml-’ pyrimethamine in ethanol. Aliquots (0.1-2.0 ml) of the working solution, corresponding to 10-200 ng of pyrimethamine were placed in test tubes and the ethanol was evaporated by keeping the tubes in a beaker of warm water (60°C) for a few minutes. The residues were treated with chloroacetaldehyde and acetate buffer as described above for ethanol blanks. The fluorescence of the reaction mixtures was measured as above after diluting with 2.0 ml of acetate buffer. The procedure was repeated 5 times, and 5 blank determinations were also done. For the preparation of calibration graphs for pyrimethamine in mixed saliva (or plasma), 0.1-2.0 ml aliquots of the above working solution (lo200 ng of pyrimethamine) were evaporated to dryness as described above. To each residue was added 1.0 ml of drug-free mixed saliva (or plasma) and the mixture was shaken on a vortex mixer for 30 s. The resulting solutions were made alkaline with 0.5 ml of 2.0 M sodium hydroxide and treated as described above for the determination of detection limits in these fluids. The procedure was repeated 4 times for saliva and 5 times for plasma. To correct for the blank fluorescence, five blank determinations were also done. Results and discussion Determination of pyrimethamine by reaction with chloroacetaldehyde. NHeteroaromatic compounds, including pyridines and pyrimidines, possessing a primary amino group which is ortho to the heterocyclic nitrogen atom react readily with a-halocarbonyl compounds to give the corresponding imidazol[1,2-a] -pyridine and imidazol[ 1,2-a] pyrimidine derivatives, respectively. Thus, the reaction of 2aminopyrimidine with chloro- or bromo-acetaldehyde in aqueous alcoholic solution in the presence of sodium hydrogencarbonate gives a quantitative yield of imidazole[ 1,2-a] pyrimidine [ 131. Several of these bridge-head imidazole derivatives have been reported to exhibit a blue fluorescence under U.V. irradiation. The nucleosides, adenosine and cytidine, also react readily with chloroacetaldehyde [ 141 and the intense blue fluorescence of the resulting imidazole derivatives has been used for the sensitive determination of these nucleosides [U-17] .
364
As expected, pyrimethamine (I) reacted with chloroacetaldehyde to give a pyrimidoimidazole derivative (II) which had a blue fluorescence. The reaction
III)
(I)
took place readily in the acetate buffer or in a hydrogencarbonate buffer (pH 10). Acetate buffer containing chloride was selected because this allowed greater reagent stability, and because it eliminated interference from chloroquine and quinine. Heating the mixture for >30 min did not appreciably increase the intensity. The reaction of 10 ng of pyrimethamine with chloroacetaldehyde as described above yielded a solution giving a fluorescence intensity that corresponded to 13.9% of the fluorescence of a 1 pg ml-’ solution of quinine sulphate in 0.05 M sulphuric acid, when measured at the appropriate maximum wavelengths. Spectrofluorimetric measurement of the pyrimido-imidazole derivative of pyrimethamine (II) provided a suitable indirect determination of the drug after its extraction from plasma or mixed saliva. Linear calibration graphs were obtained when standard solutions of the drug in these fluids were treated as described. The results are shown in Table 1. A comparison of the regression calibration equations shows the recovery of the drug from plasma to be 93.9% and from saliva to be 95.0%. TABLE 1 Calibration results for pyrimethamine Pyrimethamine taken (ng ml-‘) 0
10 50 100 150 200
in ethanol, saliva and plasma
Fluorescence
intensitya
Ethanol
Plasma
Saliva
5.3 (34) 6.3 (4.3) 17.0 (7.0) 21.7 (9.0) 31.1(8.1) 43.0 (5.2)
3.8 5.7 13.6 17.5 30.6 37.9
2.0 (116) 6.1 (6.7) 9.6 (4.3) 14.5 (7.6) 30.1(4.1) 37.0 (1.7)
(40) (4.8) (7.5) (5.9) (2.1) (6.0)
aEach value is the mean of 5 determinations for ethanol and plasma and 4 determinations for saliva. The relative standard deviations (%) are in parentheses. Regression equations are: for ethanol, y =: 0.182x(*0.015) + 5.26(*1.83), where y is the fluorescence intensity and x (ng ml-‘) is the concentration of pyrimethsmine, standard error = 6.08, correlation coefficient r = 0.9822; for plasma, y = 0.170x(+0.012) t 3.75(*1.57). standard error = 4.46, r = 0.9849; and for saliva y = 0.172x(~O.O19) t 1.95(*2.37), standard error = 2.79, r = 0.9752.
365
The determination of pyrimethamine in saliva has been applied in a study of the plasma pharmacokinetics of the drug [ 181. When simultaneous plasma and saliva samples have to be examined, standard solutions of the drug in mixed saliva, which is more readily available, can be used for calibration because statistical comparison of the regression equations (Table 1) shows there is no significant difference between the equations for plasma and saliva (F = 5.32; p Q 0.01). The limits of detection for pyrimethamine in ethanol, plasma and saliva were estimated as 2.7, 8.4 and 6.6 ng ml-‘, respectively. REFERENCES 1 G. B. Cox and K. Sugden, Analyst (London), 102 (1977) 29. 2 R. L. De Angelis, W. S. Simmons and C. A. Nichol, Chromatographia, 106 (1975) 41. 3 A. G. Goodman, L. S. Gordman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, 6th edn., MacMillan, New York, 1980, p. 1050. 4 P. C. Caia, N. R. Trenner, R. P. Buhs, G. V. Downing, J. L. Smith and W. J. A. Van den Henvel, J. Agric. Food Chem., 20 (1972) 377. 5 J. R. Harris, P. G. Baker and P. G. Munday, Analyst (London), 102 (1977) 873. 6 G. Royere, J. Dufoir and J. Faure, Analusis, 1 (1972) 362. 7 C. R. Jones, P. P. Ryle and B. C. Weatherley, J. Chromatogr., 224 (1981) 492. 8 C. R. Jones and L. A. King, Biochem. Med., 2 (1968) 251. 9 W. S. Simmons and R. L. DeAngelis, Anal. Chem., 45 (1973) 1538. 10 C. R. Jones and S. M. Ovenell, J. Chromatogr., 163 (1979) 179. 11 E. M. Levin, R. B. Meyer and V. A. Levin, J. Chromatogr., 156 (1981) 181. 12 U. Tim and E. Weidekam, J. Chromatogr., 230 (1982) 107. 13 W. L. Moshy, The Chemistry of Heterocyclic Compounds: Heterocyclic Compounds with Bridgehead Nitrogen, Interscience, New York, 1961, Part I, p. 461, Part II, p. 802. 14 N. K. Kochetkov, V. N. Shibaev and A. A. Kost, Tetrahedron Lett., 22 (1971) 1993. 15 J. R. Barrio, J. A. Secrist and M. J. Leornard, Biochem. Biophys. Res. Commun., 46 (1972) 597. 16 G. Avigad and S. Damle, Anal. Biochem., 50 (1972) 321. 17 M. Yoshioka and Z. Tamura, J. Chromatogr., 123 (1976) 220. 18 R. A. Ahmad and H. J. Rogers, Brit. J. Clin. Pharmacol., 11 (1981) 101.
Short Communication
SIMPLE AND SENSITIVE SPECTROFLUORIMETRIC OF PYRIMETHAMINE IN BIOLOGICAL FLUIDS
DETERMINATION
0. R. IDOWU* and 0. A. DADA Department
of Chemistry,
University of Zbadan, Zbadan (Nigeria)
(Received 7 th August 1984)
Summary. The method is based on the reaction of pyrimethamine with chloroacetaldehyde and measurement of the fluorescence of the resulting pyrimidoimidazole derivative. The limits of detection for pyrimethamine in ethanol, plasma and saliva were 2.7, 8.4 and 6.6 ng ml-‘, respectively. Chloroquine and quinine do not interfere.
Pyrimethamine (I; 2,4-diamino-5-(3-chlorophenyl)-6-ethylpyrimidine) has long been in use in antimalarial therapy as a suppressive, often in combination with a sulphonamide such as sulphadoxme. It is also used as a coccidiostat in rabbit and poultry feeds [l] and occasionally, in the treatment of meningeal leukemia [2] . Renewed interest in the antimalarial activity of pyrimethamine has arisen recently because of the emergence of plasmodial resistance to a number of chloroquine-like drugs at present in use, and the observation that the development of plasmodial resistance is markedly de layed when a pyrimethamine/sulphonamide combination is used along with another antimalarial such as quinine or mefloquine, a recent quinoline derivative [3] . Interest in the pharmacokinetics of mefloquine suggested a study of the pharmacokinetics of pyrimethamine when used in combination with mefloquine and a sulphonamide. A rapid, sensitive and selective method was therefore needed for the determination of pyrimethamine in plasma and, especially, in saliva, which is a more convenient sampling fluid. Various methods have been reported for the determination of pyrimethamine in feeding stuffs, chicken tissue and body fluids. These include gas chromatography [4-71, thin-layer chromatography in combination with spectrophotodensitometry [2] and spectrofluorodensitometry [8, 91, and high-performance liquid chromatography with ultra-violet absorbance detection [l, 10,111 or fluorescence detection [12]. There is, however, no report on the determination of pyrimethamine by solution spectrofluorimetry, probably because pyrimethamine possesses only a very weak native fluorescence. However, a spectrofluorimetric method offers the advantages of simplicity and cheapness, which may be important in areas of endemic malaria where economic and technological difficulties may render published methods
inaccessible. This communication describes a simple, sensitive and rapid spectrofluorimetric method for quantifying pyrimethamine in plasma and saliva based on its reaction with chloroacetaldehyde. The method is applicable to the determination of pyrimethamine in the presence of sulphadoxine, mefloquine and quinine. Experimental Reagents and apparatus. Chloroacetal, 1,2-dichloroethane and anhydrous quinine sulphate were all reagent grade whereas ethanol was of analytical grade (BDH Chemicals Ltd.). Quinine sulphate was recrystallized twice from hot water before use. Pyrimethamine was obtained by crushing 20 tablets of Daraprim (Wellcome) in 2 ml of 0.1 M sodium hydroxide, extracting the slurry twice with 30 ml of chloroform, and drying the extract over anhydrous sodium sulphate before evaporation, The pyrimethamme obtained had a melting point of 237-238°C (lit. 239-242°C [3]). (Found, C = 57.9% H = 5.35%,N=21.9%,Cl= 14.2%;expected,C=57.95%,H= 5.3%,N=22.5%, Cl = 14.25%) An acetate buffer (pH 4.5) containing 0.2 M sodium chloride was prepared. All fluorescence measurements were made on a Perkin-Elmer 204 spectrofluorimeter. Preparation of aqueous solution of chloroacetaldehyde. A solution of chloroacetal (6 g) in 30 ml of 5% (w/v) sulphuric acid was reflexed gently for 45 min and the mixture was distilled using an ice-water-cooled doublesurface reflux condenser, with the receiver cooled in an ice-water bath. The pH of the distillate was adjusted to ca. 4.5 by dropwise addition of 1 M sodium hydroxide and the mixture was distilled as before. The distillate (30 ml) was used in the following investigations. When not in use, the solution was kept in a freezer at -5”C, where it remained stable for at least a month. Examination of the reaction between chlomacetaldehyde and pyrimethamine. A 0.4-ml aliquot of a 50 pg ml-’ solution of pyrimethamine in ethanol was evaporated to dryness in a test tube and the residue was mixed with 0.2 ml of the chloroacetaldehyde solution and 0.4 ml of the acetate buffer. The mixture was heated in a boiling water bath for 30 min and, after cooling to room temperature for 5 min, the mixture was examined at 366 nm for fluorescence, by excitation at 254 nm. A further 3 ml of acetate buffer was added to the mixture and the excitation and emission wavelengths which gave maximum fluorescence intensity were established as 320 and 400 nm, respectively. Estimation of detection limits for pyrimethamine in ethanol, saliva and plasma. A 2.0-ml aliquot of ethanol was evaporated to dryness in a test tube and the residue was treated with 0.2 ml of the chloroacetaldehyde solution and 0.5 ml of the acetate buffer. The mixture was heated in a boiling waterbath for 30 min and cooled for 5 min at room temperature, and a further 2.0 ml of acetate buffer was added. The fluorescence was measured at excita-
363
tion and emission wavelengths of 320 and 450 nm, respectively. The procedure was repeated ten times. This gave a measure of the blank fluorescence. For the determination of detection limits in saliva or plasma, 1.0 ml of drug-free mixed saliva (or plasma) was placed in a test tube and made alkaline (pH 12) by addition of 0.5 ml of 2.0 M sodium hydroxide. After addition of 6.0 ml of 1,2dichloroethane, the mixture was shaken for 1 min on a vortex mixer and centrifuged at 2500 rpm for 10 min. The organic phase was removed with a Pasteur pipette and evaporated to dryness, and the residue was treated with chloroacetaldehyde and acetate buffer. The fluorescence was then measured as described above for the ethanol blank. Detection limits were calculated as the concentrations of pyrimethamine that gave intensities two standard deviations of the blank above the mean blank signal. Preparation of calibmtion‘graphs forpyrimethamine in ethanol, saliva and plasma. A stock solution containing 100 r_cgml-’ pyrimethamine was diluted serially to give a working solution of 100 ng ml-’ pyrimethamine in ethanol. Aliquots (0.1-2.0 ml) of the working solution, corresponding to 10-200 ng of pyrimethamine were placed in test tubes and the ethanol was evaporated by keeping the tubes in a beaker of warm water (60°C) for a few minutes. The residues were treated with chloroacetaldehyde and acetate buffer as described above for ethanol blanks. The fluorescence of the reaction mixtures was measured as above after diluting with 2.0 ml of acetate buffer. The procedure was repeated 5 times, and 5 blank determinations were also done. For the preparation of calibration graphs for pyrimethamine in mixed saliva (or plasma), 0.1-2.0 ml aliquots of the above working solution (lo200 ng of pyrimethamine) were evaporated to dryness as described above. To each residue was added 1.0 ml of drug-free mixed saliva (or plasma) and the mixture was shaken on a vortex mixer for 30 s. The resulting solutions were made alkaline with 0.5 ml of 2.0 M sodium hydroxide and treated as described above for the determination of detection limits in these fluids. The procedure was repeated 4 times for saliva and 5 times for plasma. To correct for the blank fluorescence, five blank determinations were also done. Results and discussion Determination of pyrimethamine by reaction with chloroacetaldehyde. NHeteroaromatic compounds, including pyridines and pyrimidines, possessing a primary amino group which is ortho to the heterocyclic nitrogen atom react readily with a-halocarbonyl compounds to give the corresponding imidazol[1,2-a] -pyridine and imidazol[ 1,2-a] pyrimidine derivatives, respectively. Thus, the reaction of 2aminopyrimidine with chloro- or bromo-acetaldehyde in aqueous alcoholic solution in the presence of sodium hydrogencarbonate gives a quantitative yield of imidazole[ 1,2-a] pyrimidine [ 131. Several of these bridge-head imidazole derivatives have been reported to exhibit a blue fluorescence under U.V. irradiation. The nucleosides, adenosine and cytidine, also react readily with chloroacetaldehyde [ 141 and the intense blue fluorescence of the resulting imidazole derivatives has been used for the sensitive determination of these nucleosides [U-17] .
364
As expected, pyrimethamine (I) reacted with chloroacetaldehyde to give a pyrimidoimidazole derivative (II) which had a blue fluorescence. The reaction
III)
(I)
took place readily in the acetate buffer or in a hydrogencarbonate buffer (pH 10). Acetate buffer containing chloride was selected because this allowed greater reagent stability, and because it eliminated interference from chloroquine and quinine. Heating the mixture for >30 min did not appreciably increase the intensity. The reaction of 10 ng of pyrimethamine with chloroacetaldehyde as described above yielded a solution giving a fluorescence intensity that corresponded to 13.9% of the fluorescence of a 1 pg ml-’ solution of quinine sulphate in 0.05 M sulphuric acid, when measured at the appropriate maximum wavelengths. Spectrofluorimetric measurement of the pyrimido-imidazole derivative of pyrimethamine (II) provided a suitable indirect determination of the drug after its extraction from plasma or mixed saliva. Linear calibration graphs were obtained when standard solutions of the drug in these fluids were treated as described. The results are shown in Table 1. A comparison of the regression calibration equations shows the recovery of the drug from plasma to be 93.9% and from saliva to be 95.0%. TABLE 1 Calibration results for pyrimethamine Pyrimethamine taken (ng ml-‘) 0
10 50 100 150 200
in ethanol, saliva and plasma
Fluorescence
intensitya
Ethanol
Plasma
Saliva
5.3 (34) 6.3 (4.3) 17.0 (7.0) 21.7 (9.0) 31.1(8.1) 43.0 (5.2)
3.8 5.7 13.6 17.5 30.6 37.9
2.0 (116) 6.1 (6.7) 9.6 (4.3) 14.5 (7.6) 30.1(4.1) 37.0 (1.7)
(40) (4.8) (7.5) (5.9) (2.1) (6.0)
aEach value is the mean of 5 determinations for ethanol and plasma and 4 determinations for saliva. The relative standard deviations (%) are in parentheses. Regression equations are: for ethanol, y =: 0.182x(*0.015) + 5.26(*1.83), where y is the fluorescence intensity and x (ng ml-‘) is the concentration of pyrimethsmine, standard error = 6.08, correlation coefficient r = 0.9822; for plasma, y = 0.170x(+0.012) t 3.75(*1.57). standard error = 4.46, r = 0.9849; and for saliva y = 0.172x(~O.O19) t 1.95(*2.37), standard error = 2.79, r = 0.9752.
365
The determination of pyrimethamine in saliva has been applied in a study of the plasma pharmacokinetics of the drug [ 181. When simultaneous plasma and saliva samples have to be examined, standard solutions of the drug in mixed saliva, which is more readily available, can be used for calibration because statistical comparison of the regression equations (Table 1) shows there is no significant difference between the equations for plasma and saliva (F = 5.32; p Q 0.01). The limits of detection for pyrimethamine in ethanol, plasma and saliva were estimated as 2.7, 8.4 and 6.6 ng ml-‘, respectively. REFERENCES 1 G. B. Cox and K. Sugden, Analyst (London), 102 (1977) 29. 2 R. L. De Angelis, W. S. Simmons and C. A. Nichol, Chromatographia, 106 (1975) 41. 3 A. G. Goodman, L. S. Gordman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, 6th edn., MacMillan, New York, 1980, p. 1050. 4 P. C. Caia, N. R. Trenner, R. P. Buhs, G. V. Downing, J. L. Smith and W. J. A. Van den Henvel, J. Agric. Food Chem., 20 (1972) 377. 5 J. R. Harris, P. G. Baker and P. G. Munday, Analyst (London), 102 (1977) 873. 6 G. Royere, J. Dufoir and J. Faure, Analusis, 1 (1972) 362. 7 C. R. Jones, P. P. Ryle and B. C. Weatherley, J. Chromatogr., 224 (1981) 492. 8 C. R. Jones and L. A. King, Biochem. Med., 2 (1968) 251. 9 W. S. Simmons and R. L. DeAngelis, Anal. Chem., 45 (1973) 1538. 10 C. R. Jones and S. M. Ovenell, J. Chromatogr., 163 (1979) 179. 11 E. M. Levin, R. B. Meyer and V. A. Levin, J. Chromatogr., 156 (1981) 181. 12 U. Tim and E. Weidekam, J. Chromatogr., 230 (1982) 107. 13 W. L. Moshy, The Chemistry of Heterocyclic Compounds: Heterocyclic Compounds with Bridgehead Nitrogen, Interscience, New York, 1961, Part I, p. 461, Part II, p. 802. 14 N. K. Kochetkov, V. N. Shibaev and A. A. Kost, Tetrahedron Lett., 22 (1971) 1993. 15 J. R. Barrio, J. A. Secrist and M. J. Leornard, Biochem. Biophys. Res. Commun., 46 (1972) 597. 16 G. Avigad and S. Damle, Anal. Biochem., 50 (1972) 321. 17 M. Yoshioka and Z. Tamura, J. Chromatogr., 123 (1976) 220. 18 R. A. Ahmad and H. J. Rogers, Brit. J. Clin. Pharmacol., 11 (1981) 101.