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Determination of β-adrenoreceptor antagonists in urine by high-performance liquid chromatography with diode-array spectrophotometric detection
Analytica Chimica Acta, 196 (1987) 255-258 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Short Communication
DETERMINATION OF P-ADRENORECEPTOR ANTAGONISTS IN URINE BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE-ARRAY SPECTROPHOTOMETRIC DETECTION
LI YE and ZHANG XIANGXI*
Labomtory ofAnalytical Chemistry, Institute of Forensic Sciences, lUinistry of Public Security, P.O. Box 2808-13, Beijing (China) (Received 31st July 1986)
The determination of pindolol, oxprenolol and propranolol in human urine is described. The drugs are isolated with a GDX-502 resin-packed column, separated on a C,, (5-Nrn) reversed-phase column with methanol/aqueous acetic acid as mobile phase and quantified with diode-array spectrophotometric detector. The recovery was >93%, and detection limits were 2 ng for pindolol, 12 ng for oxprenolol and 2 ng for propranolol. Results are given for urine from healthy volunteers who had received the drugs orally.
Summary.
fi-adrenoreceptor antagonists such as pinodolol, oxprenolol and propran0101 are widely used in the treatment of cardiac arrhythmias, hypertension, angina and thyrotoxicosis, but are also sometimes used as doping agents in sport. Various methods are available for determining these drugs in urine or other biological liquids involving either spectrofluorimetry [ 1,2], thin-layer chromatography [ 3, 41, gas chromatography [ 5-121, gas chromatography/ mass spectrometry [13-l 51 or high-performance liquid chromatography (h.p.1.c.) [16-261. These methods require a time-consuming extraction precedure and are for determination of a single drug and its metabolites. ln this communication, a simple h.p.1.c. method is described for determining pindolol, oxprenolol and propranolol in human urine, Experimental Reagents. All reagents were of analytical grade. Methanol, ethanol and acetic acid were purchased from Beijing Chemical Works (China). The water was twice-distilled from glass. Oxprenolol and propranolol were obtained from Beijing Second Pharmaceutic Works (China). Pindolol was a gift from Shijiazhuang Pharmaceutic Works (Hebei, China). Chromatogmphic system. The Hewlett-Packard HP1090 liquid chromatograph used was equipped with a DR5 solvent delivery system, variablevolume auto-injector, autosampler, HP3392A integrator, HP7470A plotter and HP1040A diode-array detector. The column (150 X 2.1 mm i.d.) was packed with 5 pm GYQGC18 (Beijing Factory of Chemical Reagents, China). Other conditions were as follows: mobile phase, methanol/2% acetic 0003-2670/87/$03.60
0 1987 Elsevier Science Publishers B.V.
256
acid (9:1, v/v); flow rate, 0.5 ml min”; column temperature, 30°C; detector wavelength, 260 nm (band-width 80 nm). Standard solution and urine samples. Standard stock solutions of pindolol, oxprenolol and propranolol were prepared by dissolving the drugs in ethanol. Drug-free urine was obtained from healthy volunteers. Urine samples were also collected from healthy volunteers who had received an oral dose of 15 mg of pindolol, 20 mg of oxprenol and 20 mg of propranolol. Procedure. A l-ml aliquot of urine was passed through a column containing 1 g of GDX-502 resin (Tianjin Second Factory of Chemical Reagents). The loaded column was washed with 5 ml of distilled water, and then with 1 ml of methanol. The drug was eluted again with 1 ml of methanol. This solution was evaporated to dryness with a current of nitrogen at room temperature. The resulting residue was dissolved in 150 ~1 of methanol, and a 5-~1 aliquot was injected into the h.p.1.c. column. Three calibration graphs were prepared by treating urine samples containing known amounts of pindolol, oxprenolol and propranolol in the same way. A
Bpcl fd
Id! I-L Pi
Pr
A_-l-.-l 5
1 IO
0
1 5
I
I_
IO
0
4
8
12 16
0
4
8
12 16
RETENTION TIME (mid Fig. 1. Chromatograms and spectra of extracts from human urine: (A) blank urine; (B) blank urine spiked with 3.4 fig ml-’ pindolol; (C) urine from a volunteer, 7 h after an oral dose of 15 mg of pindolol (Pi) giving 4.2 pg ml-*; (D) blank urine spiked with 5.0 rg ml-’ of oxprenolol; (E) urine from a volunteer, 8 h after an oral dose of 20 mg of oxprenolo1 (0) giving 6.1 pg ml-‘; (F) blank urine spiked with 3.0 rg ml-’ propranolol; (G) urine from a volunteer, 6 h after an oral dose of 20 mg of propranolol (Pr) giving 4.3 rg ml-‘. Chromatographic conditions as in the text. The inset spectra are for the wavelength range 208-400 nm at 5-nm intervals.
257
Results and discussion Under the chromatographic conditions described above, the retention times of pindolol, oxprenolol and propranolol were 5.01, 7.13 and 8.71 min, respectively. Figure 1 shows chromatograms of the extracts of blank human urine (A), blank human urine spiked with pindolol (B), oxprenolol (D) and propranolol (F) and urine collected from volunteers receiving an oral dose of pindolol (C), oxprenolol (E) or propranolol (G). Figure 1 also shows the spectra recorded at the drug peaks. These spectra can be used for the identification of the drugs. The calibration data and detection limits are shown in Table 1. The graphs showed good linearity. Table 2 shows the reproducibility of the method and the recovery of pindolol, oxprenolol and propranolol spiked in human urine. These results indicate that the method is rapid and simple, and will be suitable for use in pharmacokinetic studies and drug monitoring in patients. The TABLE 1 Calibration data and detection limits Compound
Pindolol Oxprenolol Propranolol ‘Signal/noise
Linear range (pg ml-‘)
;n=s,
Detection limit8 (ng)
0.2-10.0 1.5-15.0 0.2-10.0
0.997 0.999 0.999
2 12 2
= 2.
TABLE 2 Reproducibility
and recovery of pindolol, oxprenolol and propranolol in human urine
Compound
Concentration (ccg ml-‘)
Relative standard deviation ( %)a
Mean recovery (%)b
Pindolol
0.2 1.0 5.0 10.0
5.3 3.2 2.8 3.4
95.1
Oxprenolol
1.5 6.0 10.0 15.0
4.5 3.6 2.3 3.1
93.2
Propmnolol
0.2 1.0 5.0 10.0
3.3 2.5 1.3 2.7
98.4
aFor 6 measurements at all concentrations.
bFor all 24 experiments.
258
assay can be used for the detection of pindolol, oxprenolol and propranolol in doping checks. REFERENCES 1 T. Suzuki, Y. Saitoh, S. Isozaki and R. Ishida, Chem. Pharm. Bull., 20 (1972) 2731. 2 P. S. Rao, L. C. Quesada and H. S. Mueller, Clin. Chim. Acta, 88 (1978) 355. 3M. Schafer, H. E. Geissler and E. Mutschler, J. Chromatogr., 143 (1977) 607. 4 Y. Garceau, I. Davis and J. Hasegawa, J. Pharm. Sci., 67 (1978) 826. 5 M. Guerret, D. Lavenne and F. R. Kiechel, J. Pharm. Sci., 69 (1980) 1191. 6 M. Guerret, J. Chromatogr., 221 (1980) 387. 7 D. B. Jack and W. Riess, J. Chromatogr., 88 (1974) 173. 8 P. H. Degen and W. Riess, J. Chromatogr., 121 (1976) 72. 9T. Walle, J. Pharm. Sci., 63 (1974) 1885. 10 D. S. Saelens, T. Walle and P. J. Privitera, J. Chromatogr., 123 (1976) 185. 11 J. F. Pritchard, D. W. Schneck, W. J. Racz and A. H. Hayes Jr., Clin. Biochem., 11 (1978) 121. 12 D. E. Easterling, T. Walle, E. C. Conradi and T. E. Gaffney, J. Chromatogr., 162 (1979) 439. 13T. Walle, J. Morrison, K. Walle and E. Conradi, J. Chromatogr., 114 (1975) 351. 14T. WaIle, U. K. Walle, D. R. Bridges, E. C. Conradi and T. E. Gaffney, Clin. Chem., 24 (1978) 991. 15 V. T. Vu and F. P. Abramson, Biomed. Mass Spectrom., 5 (1978) 686. 16 M. Bangah, G. Jackman and A. Bobik, J. Chromatogr., 183 (1980) 255. 17 B. Diquet, J. J. Nguyen-Huu and H. Boutron, J. Chromatogr., 311 (1984) 430. 18 S. E. Tsuei and J. Thomas, J. Chromatogr., 181(1980) 135. 19 J. F. Pritchard, D. W. Schneck and A. H. Hayes Jr., J. Chromatogr., 162 (1979) 47. 20 W. D. Mason, E. N. Amick and 0. H. Weddle, Anal. Lett., 10 (1977) 515. 21 R. L. Nation, G. W. Peng and W. L. Chiou, J. Chromatogr., 145 (1978) 429. 22 A. M. Taburet, A. A. Taylor, J. R. Mitchell, D. E. Rollins and J. L. Pool. Life Sci., 24 (1979) 209. 23F. Pritchard, D. Schneck and A. Hayes Jr., Res. Commun. Chem. Pathol. Pharmacol., 23 (1979) 279. 24D. W. Schneck, J. F. Pritchard and A. H. Hayes Jr., Res. Commun. Chem. Pathol. Pharmacol., 24 (1979) 3. 25 M. Lo and S. Riegelman, J. Chromatogr., 183 (1980) 213. 26 H. Takei, H. Ocata and A. Ejima, Chem. Pharm. Bull., 31 (1983) 1392.
Short Communication
DETERMINATION OF P-ADRENORECEPTOR ANTAGONISTS IN URINE BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE-ARRAY SPECTROPHOTOMETRIC DETECTION
LI YE and ZHANG XIANGXI*
Labomtory ofAnalytical Chemistry, Institute of Forensic Sciences, lUinistry of Public Security, P.O. Box 2808-13, Beijing (China) (Received 31st July 1986)
The determination of pindolol, oxprenolol and propranolol in human urine is described. The drugs are isolated with a GDX-502 resin-packed column, separated on a C,, (5-Nrn) reversed-phase column with methanol/aqueous acetic acid as mobile phase and quantified with diode-array spectrophotometric detector. The recovery was >93%, and detection limits were 2 ng for pindolol, 12 ng for oxprenolol and 2 ng for propranolol. Results are given for urine from healthy volunteers who had received the drugs orally.
Summary.
fi-adrenoreceptor antagonists such as pinodolol, oxprenolol and propran0101 are widely used in the treatment of cardiac arrhythmias, hypertension, angina and thyrotoxicosis, but are also sometimes used as doping agents in sport. Various methods are available for determining these drugs in urine or other biological liquids involving either spectrofluorimetry [ 1,2], thin-layer chromatography [ 3, 41, gas chromatography [ 5-121, gas chromatography/ mass spectrometry [13-l 51 or high-performance liquid chromatography (h.p.1.c.) [16-261. These methods require a time-consuming extraction precedure and are for determination of a single drug and its metabolites. ln this communication, a simple h.p.1.c. method is described for determining pindolol, oxprenolol and propranolol in human urine, Experimental Reagents. All reagents were of analytical grade. Methanol, ethanol and acetic acid were purchased from Beijing Chemical Works (China). The water was twice-distilled from glass. Oxprenolol and propranolol were obtained from Beijing Second Pharmaceutic Works (China). Pindolol was a gift from Shijiazhuang Pharmaceutic Works (Hebei, China). Chromatogmphic system. The Hewlett-Packard HP1090 liquid chromatograph used was equipped with a DR5 solvent delivery system, variablevolume auto-injector, autosampler, HP3392A integrator, HP7470A plotter and HP1040A diode-array detector. The column (150 X 2.1 mm i.d.) was packed with 5 pm GYQGC18 (Beijing Factory of Chemical Reagents, China). Other conditions were as follows: mobile phase, methanol/2% acetic 0003-2670/87/$03.60
0 1987 Elsevier Science Publishers B.V.
256
acid (9:1, v/v); flow rate, 0.5 ml min”; column temperature, 30°C; detector wavelength, 260 nm (band-width 80 nm). Standard solution and urine samples. Standard stock solutions of pindolol, oxprenolol and propranolol were prepared by dissolving the drugs in ethanol. Drug-free urine was obtained from healthy volunteers. Urine samples were also collected from healthy volunteers who had received an oral dose of 15 mg of pindolol, 20 mg of oxprenol and 20 mg of propranolol. Procedure. A l-ml aliquot of urine was passed through a column containing 1 g of GDX-502 resin (Tianjin Second Factory of Chemical Reagents). The loaded column was washed with 5 ml of distilled water, and then with 1 ml of methanol. The drug was eluted again with 1 ml of methanol. This solution was evaporated to dryness with a current of nitrogen at room temperature. The resulting residue was dissolved in 150 ~1 of methanol, and a 5-~1 aliquot was injected into the h.p.1.c. column. Three calibration graphs were prepared by treating urine samples containing known amounts of pindolol, oxprenolol and propranolol in the same way. A
Bpcl fd
Id! I-L Pi
Pr
A_-l-.-l 5
1 IO
0
1 5
I
I_
IO
0
4
8
12 16
0
4
8
12 16
RETENTION TIME (mid Fig. 1. Chromatograms and spectra of extracts from human urine: (A) blank urine; (B) blank urine spiked with 3.4 fig ml-’ pindolol; (C) urine from a volunteer, 7 h after an oral dose of 15 mg of pindolol (Pi) giving 4.2 pg ml-*; (D) blank urine spiked with 5.0 rg ml-’ of oxprenolol; (E) urine from a volunteer, 8 h after an oral dose of 20 mg of oxprenolo1 (0) giving 6.1 pg ml-‘; (F) blank urine spiked with 3.0 rg ml-’ propranolol; (G) urine from a volunteer, 6 h after an oral dose of 20 mg of propranolol (Pr) giving 4.3 rg ml-‘. Chromatographic conditions as in the text. The inset spectra are for the wavelength range 208-400 nm at 5-nm intervals.
257
Results and discussion Under the chromatographic conditions described above, the retention times of pindolol, oxprenolol and propranolol were 5.01, 7.13 and 8.71 min, respectively. Figure 1 shows chromatograms of the extracts of blank human urine (A), blank human urine spiked with pindolol (B), oxprenolol (D) and propranolol (F) and urine collected from volunteers receiving an oral dose of pindolol (C), oxprenolol (E) or propranolol (G). Figure 1 also shows the spectra recorded at the drug peaks. These spectra can be used for the identification of the drugs. The calibration data and detection limits are shown in Table 1. The graphs showed good linearity. Table 2 shows the reproducibility of the method and the recovery of pindolol, oxprenolol and propranolol spiked in human urine. These results indicate that the method is rapid and simple, and will be suitable for use in pharmacokinetic studies and drug monitoring in patients. The TABLE 1 Calibration data and detection limits Compound
Pindolol Oxprenolol Propranolol ‘Signal/noise
Linear range (pg ml-‘)
;n=s,
Detection limit8 (ng)
0.2-10.0 1.5-15.0 0.2-10.0
0.997 0.999 0.999
2 12 2
= 2.
TABLE 2 Reproducibility
and recovery of pindolol, oxprenolol and propranolol in human urine
Compound
Concentration (ccg ml-‘)
Relative standard deviation ( %)a
Mean recovery (%)b
Pindolol
0.2 1.0 5.0 10.0
5.3 3.2 2.8 3.4
95.1
Oxprenolol
1.5 6.0 10.0 15.0
4.5 3.6 2.3 3.1
93.2
Propmnolol
0.2 1.0 5.0 10.0
3.3 2.5 1.3 2.7
98.4
aFor 6 measurements at all concentrations.
bFor all 24 experiments.
258
assay can be used for the detection of pindolol, oxprenolol and propranolol in doping checks. REFERENCES 1 T. Suzuki, Y. Saitoh, S. Isozaki and R. Ishida, Chem. Pharm. Bull., 20 (1972) 2731. 2 P. S. Rao, L. C. Quesada and H. S. Mueller, Clin. Chim. Acta, 88 (1978) 355. 3M. Schafer, H. E. Geissler and E. Mutschler, J. Chromatogr., 143 (1977) 607. 4 Y. Garceau, I. Davis and J. Hasegawa, J. Pharm. Sci., 67 (1978) 826. 5 M. Guerret, D. Lavenne and F. R. Kiechel, J. Pharm. Sci., 69 (1980) 1191. 6 M. Guerret, J. Chromatogr., 221 (1980) 387. 7 D. B. Jack and W. Riess, J. Chromatogr., 88 (1974) 173. 8 P. H. Degen and W. Riess, J. Chromatogr., 121 (1976) 72. 9T. Walle, J. Pharm. Sci., 63 (1974) 1885. 10 D. S. Saelens, T. Walle and P. J. Privitera, J. Chromatogr., 123 (1976) 185. 11 J. F. Pritchard, D. W. Schneck, W. J. Racz and A. H. Hayes Jr., Clin. Biochem., 11 (1978) 121. 12 D. E. Easterling, T. Walle, E. C. Conradi and T. E. Gaffney, J. Chromatogr., 162 (1979) 439. 13T. Walle, J. Morrison, K. Walle and E. Conradi, J. Chromatogr., 114 (1975) 351. 14T. WaIle, U. K. Walle, D. R. Bridges, E. C. Conradi and T. E. Gaffney, Clin. Chem., 24 (1978) 991. 15 V. T. Vu and F. P. Abramson, Biomed. Mass Spectrom., 5 (1978) 686. 16 M. Bangah, G. Jackman and A. Bobik, J. Chromatogr., 183 (1980) 255. 17 B. Diquet, J. J. Nguyen-Huu and H. Boutron, J. Chromatogr., 311 (1984) 430. 18 S. E. Tsuei and J. Thomas, J. Chromatogr., 181(1980) 135. 19 J. F. Pritchard, D. W. Schneck and A. H. Hayes Jr., J. Chromatogr., 162 (1979) 47. 20 W. D. Mason, E. N. Amick and 0. H. Weddle, Anal. Lett., 10 (1977) 515. 21 R. L. Nation, G. W. Peng and W. L. Chiou, J. Chromatogr., 145 (1978) 429. 22 A. M. Taburet, A. A. Taylor, J. R. Mitchell, D. E. Rollins and J. L. Pool. Life Sci., 24 (1979) 209. 23F. Pritchard, D. Schneck and A. Hayes Jr., Res. Commun. Chem. Pathol. Pharmacol., 23 (1979) 279. 24D. W. Schneck, J. F. Pritchard and A. H. Hayes Jr., Res. Commun. Chem. Pathol. Pharmacol., 24 (1979) 3. 25 M. Lo and S. Riegelman, J. Chromatogr., 183 (1980) 213. 26 H. Takei, H. Ocata and A. Ejima, Chem. Pharm. Bull., 31 (1983) 1392.