Journal of Analytical Toxicology, Vol. 14, March/April 1990
Analysisof Bumetanide in Human Urine by HighPerformance Liquid Chromatographywith Fluorescence Detection and Gas ChromatographylMass Spectrometry Claire Y. G r a d e e n , D e b b i e M. Billay, and Siu C. C h a n *
Department of Laboratory Medicine, Foothills Hospital, 1403, 29th Street N.W., Calga~ Alberta, T2N 279 Canada
JA b s t r a c t J Bumetanide is a potent diuretic. Its concentration in urine after therapeutic doses is in the low ng/mL range. A procedure using reversed-phase HPLC with fluorescence detection is described. Using this procedure, the authors have been able to measure bumetanide in a urine sample at 10 ng/mL with a signal-to-noise ratio of 7. Therefore, the detection limit is lower than this concentration. Confirmation is by a GC/MS procedure after methylating the diuretic. DerivaUzation is by extractive alkylation with tetrabutylammonium hydrogen sulfate as the phase transfer reagent and iodomethane as the methylating agent.
Introduction Diuretics are misused in sports for two reasons: to reduce body weight so that the athlete can compete in a lower weight class and to dilute the urine so that it is more difficult for the laboratory to detect the presence of other drug substances. The use of this class of drugs is banned by the International Olympic Committee (I.O.C.). The 1988 XVth Olympic Winter Games in Calgary was the first Olympic Games in which this ban was enforced. The list of diuretics on the I.O.C. ban list is shown at the bottom of the page. There are several different types of diuretics on this list: mercurial diuretics, carbonic anhydrase inhibitors, potassium sparing diuretics, benzothiazides, aldosterone antagonists, and loop diuretics. Most potent of these are the loop diuretics, which are represented by bumetanide, etacrynic acid, and furosemide. Intense diuresis could be caused by a therapeutic dose. Bumetanide is 40 to 60 times more potent than furosemide on a weight basis. The therapeutic dose is only 0.5 mg, and its urinary concentration after single dose administration is very low, around 100 n g / m L in the first few hours. High-performance liquid chromatography (HPLC) with UV detection has been shown to be a useful technique for measuring diuretics in urine (1,2). Many thiazides are amenable to detection. However, bumetanide detection is difficult because the urinary concentration after a therapeutic dose is low. Several techniques have been reported for quantitating bumetanide in biological fluids, but they lack either sensitivity or
specificity. These include fluorometry (3), gas chromatography with flame ionization detection (4), and radiometry (5). Radioimmunoassay has been used and proves to be very sensitive, with a detection limit of 3 n g / m L (6). However, the kit is not commercially available. It has been demonstrated that HPLC with fluorescence detection is a very sensitive technique for measuring bumetanide in biological fluids (7-10). Plasma and urine levels of bumetanide were determined by this technique in pharmacokinetic studies. This report demonstrates that HPLC with fluorescence detection is a sensitive and specific technique, and in conjunction with a UV detector linked in tandem, most of the banned diuretics in sports can be detected simultaneously. According to the rules of the I.O.C. Medical Commission, banned substances must be confirmed by mass spectrometry. Our procedure calls for the methylation of bumetanide prior to mass spectrometric analysis. Extractive methylation of bendroflumethiazide was demonstrated useful for the analysis of the diuretic in blood (l 1). On-column methylation of bumetanide with 0.2M trimethylanilinium hydroxide in methanol is also effective (8). The derivative formed was a tetramethytated analog. We use extractive methylation which results in a trimethylated derivative. Examples of Diuretics Banned by the Medical Commission of the International Olympic Committee* Acetazolamide Amiloride Bendroflumethiazide Benzthiazide Bumetanide Canrenone Chlormerodrin Chlortalidone Diclofenamide Etacrynic acid Furosemide Hydrochlorothiazide Mersalyi Spironolactone Triamterene 9 Related compounds are also banned.
9Author to whom correspondence should be addressed.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
123
Journal of Analytical Toxicology, Vol. 14, March/April 1990
Experimental
Chemicals. Pure drug substances were gifts from pharmaceutical companies. Iodomethane and tetrabutylammonium hydrogen sulfate were obtained from Sigma. Other chemicals and solvents were of the best commercial grades available. Instruments. Screening and quantitation of urinary bumetanide was performed with a Hewlett-Packard 1090 HPLC equipped with an HP 1046A programmable fluorescence detector: excitation wavelength 231 nm and emission wavelength 426 nm for bumetanide and 223 nm and 415 nm respectively for bendroflumethiazide. A reversed-phase column, Hypersil ODS 5 #m, 100 mm x 2.1 mm was used. A diode array detector was also connected to the system behind the fluorescence detector. The monitoring wavelengths were 230, 240, 270, and 290 rim. For confirmation, an HP 5890A GC linked to an HP 5970 series mass selective detector (MSD) was used. The column w a s a 12.5-m x 0.25-mm i.d. DB-I polymethyl siloxane fusedsilica capillary column with a film thickness of 0.25 #m, from J&W Scientific. Volunteer's study. A 0.5-rag bumetanide tablet (Bumex, Roche Laboratories) was taken by a healthy female volunteer. Cumulative urine samples were collected at 0, l, 2, 3, 6, 8, and 12 h post-dose. The samples were stored at 4~ prior to analysis. Extraction. Acidify 2 mL of each of the calibration standards (concentrations 0, 10, 50, 100, 500, and 1,000 n g / m L of bumetanide in drug-free urine respectively) and excretion study urine samples with l mL of 0.01M hydrochloric acid. Add 2,000 ng of bendroflumethiazide as internal standard. Extract with 5 mL of ethyl acetate. After centrifugation at 3,000 rpm for 5 min, separate the organic layer and dry it under a stream of nitrogen at 40~ HPLC analysis. Reconstitute the extract with 100 #L of methanol. Inject 2 #L into the HPLC. The mobile phases are (A) 0.05M ammonium acetate and (B) acetonitrile. A gradient elution mode is used with the initial percentage of A set at 90, changed linearly to 40 in 10 rain, and run isocratically for another l0 rain. The flow rate is 0.3 mL/min. The column oven temperature is 40~ Under these conditions, the retention time of bumetanide is 6.2 min, and that of the internal standard, bendroflumethiazide, is 8.6 min in the fluorescence detector. Recovery. Spike a 2-mL urine sample with 200 ng bumetanide. Extract diuretic according to the procedure described above, except that the internal standard is not added initially. To the dried extract, add 2,000 ng of bendroflumethiazide before reconstituting with 100 #L of methanol. For comparison, dissolve 200 ng of bumetanide and 2,000 ng of bendroflumethiazide in 100 #L of methanol. Analyze these two solutions by HPLC. Derivatization. For GC analysis, bumetanide is converted to its methylated derivative. Dissolve the dried extract in 2 mL of dichloromethane. Add 1 mL of phase transfer reagent (0.1M tetrabutylammonium hydrogen sulfate in 0.2M sodium hydroxide) and 200 #L of iodomethane. Shake the mixture vigorously on an Eberbach shaker at room temperature for 30 min. After centrifugation for 5 rain at 3,000 rpm, remove the organic layer and dry it under a stream of nitrogen at 40~ GC/MS analysis. Reconstitute the dried derivative with 200 #L of ethyl acetate. Inject 2 #L into the GC. The analysis is performed in the splitless mode. The temperature program is as follows: the initial temperature is 80~ then the temperature is raised to 200~ at a rate of 30~ and to 300~ at a rate of 10~ and finally it is raised to 310~ at a rate of 20~ and held at that temperature for 5.5 min. The 124
linear velocity of the helium carrier gas is 42.5 cm/s at 200~ The temperature of the injection port is 280~ and that of the transfer line is 300~ Under these conditions, the retention times of bumetanide and bendroflumethiazide are I 1.5 and 13.6 min respectively.
Results and Discussion
Excretion study. Although the dosage was only 0.5 mg, the effect of the diuretic was felt very quickly, and intense diuresis resulted. Urine output was significant, and the volume in the first three hours was 1.5 L. The s'pecific gravity and creatinine content of the urine also dropped drastically. There was a feeling of dehydration, and a large quantity of fluid was consumed to replenish the loss. The effect started to dissipate after 12 hours. This is summarized in Table I. Recovery. The absolute recovery of bumetanide in this procedure is calculated by the following equation: (Peak Area)e.b./(Peak Area)i.s.l Recovery = (Peak Area)u.b./(Peak Area)i.s.2 where e.b. denotes extracted bumetanide in the spiked urine, i.s.l refers to the internal standard in the extract, u.b. denotes unextracted bumetanide in the reference solution, i.s.2 refers to the internal standard therein, and Peak Area refers to the respective peaks in the liquid chromatograms. Recovery at the 100-ng/mL level was found to be 88.7~ + 6.3~ (mean +_ standard deviation), N = 5. Quantitation. The calibration, using peak area ratios, is linear up to 1,000 ng/mL, r = 0.9999, with calibration points at 0, 10, 50, 100, 500, and 1,000 ng/mL. The technique of fluorescence detection proves to be very sensitive, as shown in Figure 1, a chromatogram of the 10-ng/mL calibration standard. The amount injected onto the column was 0.35 ng. The signal-to-noise ratio was 7. Table II compiles the concentration of bumetanide in the urine samples collected at different times. The concentration of the diuretic is in the low n g / m L range at all times. The data indicates that excretion of unchanged bumetanide is highest in the second hour after drug administration. About 40% of the dose is excreted unchanged in the urine within 12 hours.
Table I. Symptoms and Effects after Oral Administration of 0.5 mg of Bumetanide Time Specific Creatlnine Volume (h) gravity (mmollL) (mL) 0
1.023
10.6
-
Symptomsand effects
1
1.012
2
1.005
5.5 0.8
300 660
slightly dehydrated, generally feeling good
3
1.004
1.4
540
6
1.015
9.2
160
slight headache, weakness, dizziness, dehydration
8
1.007
4.4
120
dranklarge quantity of fluid
12
1.024
14.4
100
effectsstarting to disappear
Journal of Analytical Toxicology, Vol. 14, March/April 1990
-100 JJ~
O~
UV Z90 nm
....l
J
-loo
sa7~
UV 270 ~m
-soo
,II?~ 750
~ZS *lOO I
_,= ~L.. 3
1
S
5
T
O
1~
11
I~
II
19
It
15
17
13 RET[NI f0N Till[
Figure 1. Fluorescenceliquid chromatogramof the 10-ng/mL calibration standard. Retentiontimes are in minutes.
(fllintlte~)
Figure 3. UV and fluorescenceliquidchromatogramsof several diuretics: (A) furosemide, (B) piretanide, (C) bumetanide, (D) cyclopenthiazide, (E) etozolin, and (F) canrenone.
Table II. Urinary Excretion of Bumetanide after Administration of a 0.5-mg Dose Time (h)
Volume (mL)
0 1 2 3
Concentration (ng/mt) 0 113 106 106 238 24 24
300 660 540 160 120 100
6 8 12
Scan S98 ( 1 1 , 4 5 4 m l n ) I ~88~
9~o]
|
,~176176 6~8~'
o~
"~176176 38OO"
or
0 33.9 70.0 57.2 38.1 2.9 2.4
I~RTR:HS2CR24R.D
SCRLE.
f-- 3rs
8~8'
Bumetanlde ~g)
s3
"~,o6
254 SO2N(CH:~) 2
/
; eO N CH2 CH~CH2CH2
3 18
]
/
....... t
/
77
I00
2~176 Mass/Chor~e
300
40~
Figure 2. Mass spectrum of trimethylbumetanide.
G C / M S confirmation. The mass spectrum of the trimethylated derivative of bumetanide is shown in Figure 2. The molecular ion is m / z 406. The ion m/z 298 results from the elimination of the dimethylated aminosulfonyl group from M § while m / z 363 originates from the molecular ion by loss of a propyl group in the butylamino side chain. The m / z 375 ion derives from M § with the loss of OCH, in the methylated carboxylic function. Further loss from m/z 363 of HSO2N(CH3)2 gives rise to m / z 254. The m / z 318 ion is the result of losing HN(CH3)2 from m / z 363. Alternatively, m / z 318 can also be derived from m / z 375 with the loss of the butyl group in the
butylamino side chain. It is of interest to note that when bumetanide is derivatized by on-column methylation (8), it forms a tetramethylated derivative in which the butylamino function is also methylated. Screening of diuretics. We have demonstrated that bumetanide can be screened for by HPLC with fluorescence detection; however, not all diuretics have native fluorescence and consequently are not amenable to this mode of detection. However, if a diode array detector is linked to the same system, many diuretics can be detected. This is illustrated in Figure 3, which shows a set of chromatograms from a urine sample spiked with 10/xg/mL of each of the following diuretics: bumetanide, canrenone, cyclopenthiazide, etozolin, furosemide, and piretanide. The sample was extracted as described and analyzed by HPLC. The lower four are UV chromatograms with monitoring wavelengths at 230, 240, 270, and 290 nm respectively, and the uppermost chromatogram is with fluorescence detection. The retention times of these diuretics with UV detection are as follows: furosemide, 5.1 min; piretanide, 6.1 min; bumetanide, 6.6 rain; cyclopenthiazide, 9.1 rain; etozolin, 10.1 min; and canrenone, 10.5 min. Because the concentration of bumetanide is quite high in this extract, it is detected conveniently with both fluorescence and UV detection. With fluorescence detection, its retention time is 6.3 min, shorter than that with UV detection, because the fluorescence detector is placed upstream of the diode array detector.
Acknowledgment We would like to thank the following pharmaceutical companies for their generous gifts of pure drug substances: Bard, England (furosemide); Berk, England (bendroflumethiazide and furosemide); Boots, England (bendroflumethiazide); CibaGeigy, Canada (chlortalidone and cyclopenthiazide); Godecke, West Germany (etozolin); Hoechst, Canada (furosemide and piretanide); Roche, Canada (bumetanide); Searle, Canada (canrenone); and Squibb, Canada (bendroflumethiazide).
125
Journal of AnalyticalToxicology,Vol. 14, March/April 1990
References 1. P.A. Tisdall, T.P. Moyer, and J.P. Anhalt. Liquid chromatographic detection of thiazide diuretics in urine. C/in. Chem. 26:702-706 (1980). 2. R.O. Fullinfaw, R.W. Bury, and R.F.W. Moulds. Liquid chromatographic screening of diuretics in urine. J. Chromatogr. 415: 347-56 (1987). 3. E.H Ostergaard, M.P. Magnussen, C.K. Nielsen, E. Eilertsen, and H.H. Frey. Pharmacological properties of 3-n-butylamino-4phenoxy-5-sulfamylbenzoic acid (bumetanide), a new potent diuretic. Arzneim. Forsch. 22:66-72 (1972). 4. P.W. Felt, K, Roholt, and H. Sorensen. GLC determination and urinary recovery of bumetanide in healthy volunteers. J. Pharm. Sci. 62:375-79 (1973). 5. S.C. Halladay, D.E. Carter, I.G. Sipes, B.B. Brodie, and R. Bressler. Evidence for the metabolism of bumetanide in man. Life Sci. 17: 1003-1009 (1975). 6. A.A. Holazo, W.A. Colburn, J.H. Gustafson, R.L. Young, and M. Parsonnet. Pharmacokinetics of bumetanide following intra-
126
venous, intramuscular, and oral administrations to normal subjects. J. Pharm. ScL 73:1108-13 (1984). 7. L.A. Marcantonio and W.H.R. Auld. Determination of the diuretic bumetanide in biological fluids by high-performance liquid chromatography. J. Chromatogr. 183:118-23 (1980). 8. L.M. Walmsley and L.F. Chasseaud. Determination of bumetanide in the plasma of non-human primates by high-performance liquid chromatography. J. Chromatogr. 226:441-49 (1981). 9. D.E. Smith. High-performance liquid chromatographic assay for bumetanide in plasma and urine. J. Pharm. ScL 71:520-23 (1982). 10. B. Ameer and MB. Burlingame. Determination of bumetanide in human plasma and urine by high-performance liquid chromatography with fluorescence detection. Anal. Lett. 21:1589-1601 (1988). 11. M.A. Gay. Bendrofluazide assay including extractive alkylation for GC. In Blood Drugs and Other Analytical Challenges, E. Reid, Ed., Ellis Horwood, Chichester, England, 1978,pp. 133-39. Manuscript received April 14, 1989; revision received October 12, 1989.
Analysisof Bumetanide in Human Urine by HighPerformance Liquid Chromatographywith Fluorescence Detection and Gas ChromatographylMass Spectrometry Claire Y. G r a d e e n , D e b b i e M. Billay, and Siu C. C h a n *
Department of Laboratory Medicine, Foothills Hospital, 1403, 29th Street N.W., Calga~ Alberta, T2N 279 Canada
JA b s t r a c t J Bumetanide is a potent diuretic. Its concentration in urine after therapeutic doses is in the low ng/mL range. A procedure using reversed-phase HPLC with fluorescence detection is described. Using this procedure, the authors have been able to measure bumetanide in a urine sample at 10 ng/mL with a signal-to-noise ratio of 7. Therefore, the detection limit is lower than this concentration. Confirmation is by a GC/MS procedure after methylating the diuretic. DerivaUzation is by extractive alkylation with tetrabutylammonium hydrogen sulfate as the phase transfer reagent and iodomethane as the methylating agent.
Introduction Diuretics are misused in sports for two reasons: to reduce body weight so that the athlete can compete in a lower weight class and to dilute the urine so that it is more difficult for the laboratory to detect the presence of other drug substances. The use of this class of drugs is banned by the International Olympic Committee (I.O.C.). The 1988 XVth Olympic Winter Games in Calgary was the first Olympic Games in which this ban was enforced. The list of diuretics on the I.O.C. ban list is shown at the bottom of the page. There are several different types of diuretics on this list: mercurial diuretics, carbonic anhydrase inhibitors, potassium sparing diuretics, benzothiazides, aldosterone antagonists, and loop diuretics. Most potent of these are the loop diuretics, which are represented by bumetanide, etacrynic acid, and furosemide. Intense diuresis could be caused by a therapeutic dose. Bumetanide is 40 to 60 times more potent than furosemide on a weight basis. The therapeutic dose is only 0.5 mg, and its urinary concentration after single dose administration is very low, around 100 n g / m L in the first few hours. High-performance liquid chromatography (HPLC) with UV detection has been shown to be a useful technique for measuring diuretics in urine (1,2). Many thiazides are amenable to detection. However, bumetanide detection is difficult because the urinary concentration after a therapeutic dose is low. Several techniques have been reported for quantitating bumetanide in biological fluids, but they lack either sensitivity or
specificity. These include fluorometry (3), gas chromatography with flame ionization detection (4), and radiometry (5). Radioimmunoassay has been used and proves to be very sensitive, with a detection limit of 3 n g / m L (6). However, the kit is not commercially available. It has been demonstrated that HPLC with fluorescence detection is a very sensitive technique for measuring bumetanide in biological fluids (7-10). Plasma and urine levels of bumetanide were determined by this technique in pharmacokinetic studies. This report demonstrates that HPLC with fluorescence detection is a sensitive and specific technique, and in conjunction with a UV detector linked in tandem, most of the banned diuretics in sports can be detected simultaneously. According to the rules of the I.O.C. Medical Commission, banned substances must be confirmed by mass spectrometry. Our procedure calls for the methylation of bumetanide prior to mass spectrometric analysis. Extractive methylation of bendroflumethiazide was demonstrated useful for the analysis of the diuretic in blood (l 1). On-column methylation of bumetanide with 0.2M trimethylanilinium hydroxide in methanol is also effective (8). The derivative formed was a tetramethytated analog. We use extractive methylation which results in a trimethylated derivative. Examples of Diuretics Banned by the Medical Commission of the International Olympic Committee* Acetazolamide Amiloride Bendroflumethiazide Benzthiazide Bumetanide Canrenone Chlormerodrin Chlortalidone Diclofenamide Etacrynic acid Furosemide Hydrochlorothiazide Mersalyi Spironolactone Triamterene 9 Related compounds are also banned.
9Author to whom correspondence should be addressed.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
123
Journal of Analytical Toxicology, Vol. 14, March/April 1990
Experimental
Chemicals. Pure drug substances were gifts from pharmaceutical companies. Iodomethane and tetrabutylammonium hydrogen sulfate were obtained from Sigma. Other chemicals and solvents were of the best commercial grades available. Instruments. Screening and quantitation of urinary bumetanide was performed with a Hewlett-Packard 1090 HPLC equipped with an HP 1046A programmable fluorescence detector: excitation wavelength 231 nm and emission wavelength 426 nm for bumetanide and 223 nm and 415 nm respectively for bendroflumethiazide. A reversed-phase column, Hypersil ODS 5 #m, 100 mm x 2.1 mm was used. A diode array detector was also connected to the system behind the fluorescence detector. The monitoring wavelengths were 230, 240, 270, and 290 rim. For confirmation, an HP 5890A GC linked to an HP 5970 series mass selective detector (MSD) was used. The column w a s a 12.5-m x 0.25-mm i.d. DB-I polymethyl siloxane fusedsilica capillary column with a film thickness of 0.25 #m, from J&W Scientific. Volunteer's study. A 0.5-rag bumetanide tablet (Bumex, Roche Laboratories) was taken by a healthy female volunteer. Cumulative urine samples were collected at 0, l, 2, 3, 6, 8, and 12 h post-dose. The samples were stored at 4~ prior to analysis. Extraction. Acidify 2 mL of each of the calibration standards (concentrations 0, 10, 50, 100, 500, and 1,000 n g / m L of bumetanide in drug-free urine respectively) and excretion study urine samples with l mL of 0.01M hydrochloric acid. Add 2,000 ng of bendroflumethiazide as internal standard. Extract with 5 mL of ethyl acetate. After centrifugation at 3,000 rpm for 5 min, separate the organic layer and dry it under a stream of nitrogen at 40~ HPLC analysis. Reconstitute the extract with 100 #L of methanol. Inject 2 #L into the HPLC. The mobile phases are (A) 0.05M ammonium acetate and (B) acetonitrile. A gradient elution mode is used with the initial percentage of A set at 90, changed linearly to 40 in 10 rain, and run isocratically for another l0 rain. The flow rate is 0.3 mL/min. The column oven temperature is 40~ Under these conditions, the retention time of bumetanide is 6.2 min, and that of the internal standard, bendroflumethiazide, is 8.6 min in the fluorescence detector. Recovery. Spike a 2-mL urine sample with 200 ng bumetanide. Extract diuretic according to the procedure described above, except that the internal standard is not added initially. To the dried extract, add 2,000 ng of bendroflumethiazide before reconstituting with 100 #L of methanol. For comparison, dissolve 200 ng of bumetanide and 2,000 ng of bendroflumethiazide in 100 #L of methanol. Analyze these two solutions by HPLC. Derivatization. For GC analysis, bumetanide is converted to its methylated derivative. Dissolve the dried extract in 2 mL of dichloromethane. Add 1 mL of phase transfer reagent (0.1M tetrabutylammonium hydrogen sulfate in 0.2M sodium hydroxide) and 200 #L of iodomethane. Shake the mixture vigorously on an Eberbach shaker at room temperature for 30 min. After centrifugation for 5 rain at 3,000 rpm, remove the organic layer and dry it under a stream of nitrogen at 40~ GC/MS analysis. Reconstitute the dried derivative with 200 #L of ethyl acetate. Inject 2 #L into the GC. The analysis is performed in the splitless mode. The temperature program is as follows: the initial temperature is 80~ then the temperature is raised to 200~ at a rate of 30~ and to 300~ at a rate of 10~ and finally it is raised to 310~ at a rate of 20~ and held at that temperature for 5.5 min. The 124
linear velocity of the helium carrier gas is 42.5 cm/s at 200~ The temperature of the injection port is 280~ and that of the transfer line is 300~ Under these conditions, the retention times of bumetanide and bendroflumethiazide are I 1.5 and 13.6 min respectively.
Results and Discussion
Excretion study. Although the dosage was only 0.5 mg, the effect of the diuretic was felt very quickly, and intense diuresis resulted. Urine output was significant, and the volume in the first three hours was 1.5 L. The s'pecific gravity and creatinine content of the urine also dropped drastically. There was a feeling of dehydration, and a large quantity of fluid was consumed to replenish the loss. The effect started to dissipate after 12 hours. This is summarized in Table I. Recovery. The absolute recovery of bumetanide in this procedure is calculated by the following equation: (Peak Area)e.b./(Peak Area)i.s.l Recovery = (Peak Area)u.b./(Peak Area)i.s.2 where e.b. denotes extracted bumetanide in the spiked urine, i.s.l refers to the internal standard in the extract, u.b. denotes unextracted bumetanide in the reference solution, i.s.2 refers to the internal standard therein, and Peak Area refers to the respective peaks in the liquid chromatograms. Recovery at the 100-ng/mL level was found to be 88.7~ + 6.3~ (mean +_ standard deviation), N = 5. Quantitation. The calibration, using peak area ratios, is linear up to 1,000 ng/mL, r = 0.9999, with calibration points at 0, 10, 50, 100, 500, and 1,000 ng/mL. The technique of fluorescence detection proves to be very sensitive, as shown in Figure 1, a chromatogram of the 10-ng/mL calibration standard. The amount injected onto the column was 0.35 ng. The signal-to-noise ratio was 7. Table II compiles the concentration of bumetanide in the urine samples collected at different times. The concentration of the diuretic is in the low n g / m L range at all times. The data indicates that excretion of unchanged bumetanide is highest in the second hour after drug administration. About 40% of the dose is excreted unchanged in the urine within 12 hours.
Table I. Symptoms and Effects after Oral Administration of 0.5 mg of Bumetanide Time Specific Creatlnine Volume (h) gravity (mmollL) (mL) 0
1.023
10.6
-
Symptomsand effects
1
1.012
2
1.005
5.5 0.8
300 660
slightly dehydrated, generally feeling good
3
1.004
1.4
540
6
1.015
9.2
160
slight headache, weakness, dizziness, dehydration
8
1.007
4.4
120
dranklarge quantity of fluid
12
1.024
14.4
100
effectsstarting to disappear
Journal of Analytical Toxicology, Vol. 14, March/April 1990
-100 JJ~
O~
UV Z90 nm
....l
J
-loo
sa7~
UV 270 ~m
-soo
,II?~ 750
~ZS *lOO I
_,= ~L.. 3
1
S
5
T
O
1~
11
I~
II
19
It
15
17
13 RET[NI f0N Till[
Figure 1. Fluorescenceliquid chromatogramof the 10-ng/mL calibration standard. Retentiontimes are in minutes.
(fllintlte~)
Figure 3. UV and fluorescenceliquidchromatogramsof several diuretics: (A) furosemide, (B) piretanide, (C) bumetanide, (D) cyclopenthiazide, (E) etozolin, and (F) canrenone.
Table II. Urinary Excretion of Bumetanide after Administration of a 0.5-mg Dose Time (h)
Volume (mL)
0 1 2 3
Concentration (ng/mt) 0 113 106 106 238 24 24
300 660 540 160 120 100
6 8 12
Scan S98 ( 1 1 , 4 5 4 m l n ) I ~88~
9~o]
|
,~176176 6~8~'
o~
"~176176 38OO"
or
0 33.9 70.0 57.2 38.1 2.9 2.4
I~RTR:HS2CR24R.D
SCRLE.
f-- 3rs
8~8'
Bumetanlde ~g)
s3
"~,o6
254 SO2N(CH:~) 2
/
; eO N CH2 CH~CH2CH2
3 18
]
/
....... t
/
77
I00
2~176 Mass/Chor~e
300
40~
Figure 2. Mass spectrum of trimethylbumetanide.
G C / M S confirmation. The mass spectrum of the trimethylated derivative of bumetanide is shown in Figure 2. The molecular ion is m / z 406. The ion m/z 298 results from the elimination of the dimethylated aminosulfonyl group from M § while m / z 363 originates from the molecular ion by loss of a propyl group in the butylamino side chain. The m / z 375 ion derives from M § with the loss of OCH, in the methylated carboxylic function. Further loss from m/z 363 of HSO2N(CH3)2 gives rise to m / z 254. The m / z 318 ion is the result of losing HN(CH3)2 from m / z 363. Alternatively, m / z 318 can also be derived from m / z 375 with the loss of the butyl group in the
butylamino side chain. It is of interest to note that when bumetanide is derivatized by on-column methylation (8), it forms a tetramethylated derivative in which the butylamino function is also methylated. Screening of diuretics. We have demonstrated that bumetanide can be screened for by HPLC with fluorescence detection; however, not all diuretics have native fluorescence and consequently are not amenable to this mode of detection. However, if a diode array detector is linked to the same system, many diuretics can be detected. This is illustrated in Figure 3, which shows a set of chromatograms from a urine sample spiked with 10/xg/mL of each of the following diuretics: bumetanide, canrenone, cyclopenthiazide, etozolin, furosemide, and piretanide. The sample was extracted as described and analyzed by HPLC. The lower four are UV chromatograms with monitoring wavelengths at 230, 240, 270, and 290 nm respectively, and the uppermost chromatogram is with fluorescence detection. The retention times of these diuretics with UV detection are as follows: furosemide, 5.1 min; piretanide, 6.1 min; bumetanide, 6.6 rain; cyclopenthiazide, 9.1 rain; etozolin, 10.1 min; and canrenone, 10.5 min. Because the concentration of bumetanide is quite high in this extract, it is detected conveniently with both fluorescence and UV detection. With fluorescence detection, its retention time is 6.3 min, shorter than that with UV detection, because the fluorescence detector is placed upstream of the diode array detector.
Acknowledgment We would like to thank the following pharmaceutical companies for their generous gifts of pure drug substances: Bard, England (furosemide); Berk, England (bendroflumethiazide and furosemide); Boots, England (bendroflumethiazide); CibaGeigy, Canada (chlortalidone and cyclopenthiazide); Godecke, West Germany (etozolin); Hoechst, Canada (furosemide and piretanide); Roche, Canada (bumetanide); Searle, Canada (canrenone); and Squibb, Canada (bendroflumethiazide).
125
Journal of AnalyticalToxicology,Vol. 14, March/April 1990
References 1. P.A. Tisdall, T.P. Moyer, and J.P. Anhalt. Liquid chromatographic detection of thiazide diuretics in urine. C/in. Chem. 26:702-706 (1980). 2. R.O. Fullinfaw, R.W. Bury, and R.F.W. Moulds. Liquid chromatographic screening of diuretics in urine. J. Chromatogr. 415: 347-56 (1987). 3. E.H Ostergaard, M.P. Magnussen, C.K. Nielsen, E. Eilertsen, and H.H. Frey. Pharmacological properties of 3-n-butylamino-4phenoxy-5-sulfamylbenzoic acid (bumetanide), a new potent diuretic. Arzneim. Forsch. 22:66-72 (1972). 4. P.W. Felt, K, Roholt, and H. Sorensen. GLC determination and urinary recovery of bumetanide in healthy volunteers. J. Pharm. Sci. 62:375-79 (1973). 5. S.C. Halladay, D.E. Carter, I.G. Sipes, B.B. Brodie, and R. Bressler. Evidence for the metabolism of bumetanide in man. Life Sci. 17: 1003-1009 (1975). 6. A.A. Holazo, W.A. Colburn, J.H. Gustafson, R.L. Young, and M. Parsonnet. Pharmacokinetics of bumetanide following intra-
126
venous, intramuscular, and oral administrations to normal subjects. J. Pharm. ScL 73:1108-13 (1984). 7. L.A. Marcantonio and W.H.R. Auld. Determination of the diuretic bumetanide in biological fluids by high-performance liquid chromatography. J. Chromatogr. 183:118-23 (1980). 8. L.M. Walmsley and L.F. Chasseaud. Determination of bumetanide in the plasma of non-human primates by high-performance liquid chromatography. J. Chromatogr. 226:441-49 (1981). 9. D.E. Smith. High-performance liquid chromatographic assay for bumetanide in plasma and urine. J. Pharm. ScL 71:520-23 (1982). 10. B. Ameer and MB. Burlingame. Determination of bumetanide in human plasma and urine by high-performance liquid chromatography with fluorescence detection. Anal. Lett. 21:1589-1601 (1988). 11. M.A. Gay. Bendrofluazide assay including extractive alkylation for GC. In Blood Drugs and Other Analytical Challenges, E. Reid, Ed., Ellis Horwood, Chichester, England, 1978,pp. 133-39. Manuscript received April 14, 1989; revision received October 12, 1989.
