determination of tripamide in human urine by high-performance liquid chromatography and...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2003; 17: 301–306

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.913

Determination of Tripamide in human urine by

high-performance liquid chromatography and

high-performance liquid chromatography/

electrospray ionization tandem mass spectrometry

Yunje Kim*, Joungboon Hwang, Myungsoo Kim and Won LeeDoping Control Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul, Korea2Research Institute of Basic Science and Department of Chemistry, KyungHee University, Seoul, Korea

Received 31 July 2002; Revised 28 November 2002; Accepted 5 December 2002

Tripamide is a drug widely used in clinical practice for the treatment of hypertension and edema.

This work evaluated a screening method for Tripamide and its urinary metabolites in human urine,

using high-performance liquid chromatography diode-array detection (HPLC/DAD). Identification

of these metabolites was investigated by high-performance liquid chromatography/electrospray

ionization tandem mass spectrometry (HPLC/ESI-MS/MS) after dosing with 15 mg Tripamide.

Acid hydrolysis showed that Tripamide is conjugated in the body. Two suspected metabolites

were detected by HPLC/DAD. HPLC/ESI-MS/MS analysis suggested that these metabolites were

probably hydroxylated together with loss of the -NH2 group and dehydrogenation. These results

will be useful in confirmation methods for Tripamide in doping control. Copyright # 2003 John

Wiley & Sons, Ltd.

Diuretic agents are drugs that increase renal excretion of

water and solutes (mainly sodium salts). The major purposes

of diuretic therapy are to decrease fluid volume in the body,

and to adjust the water and electrolyte balance. Diuretics are

often used in the management of pathological conditions

such as edema (e.g. in congestive heart failure and certain

renal disorders) and hypertension.1–4 Most diuretics exert

their effects by inhibiting tubular sodium and water reab-

sorption by epithelial cells lining the renal tubule system.

Certain diuretics (such as carbonic anhydrase inhibitors,

loop diuretics, thiazide-like diuretics and potassium-sparing

diuretics) suppress sodium and water reabsorption by inhi-

biting the function of specific proteins that are responsible

for (or participate in) the transportation of electrolytes across

the epithelial membrane; osmotic diuretics inhibit water and

sodium reabsorption by increasing intratubular osmotic

pressure. Different types of diuretics may inhibit different

transporters in different segments of the tubular system.5,6

Recently, diuretics have been abused in sports with weight

classes, such as weightlifting, wrestling and boxing. Athletes

try to reduce their body weight in order to qualify for lower

weight classes. It is also reported that athletes use diuretics to

avoid detection of doping agents by reducing their urine

concentration.7 As a part of their effort to fight drug abuse in

sports (doping), the Medical Commission of the International

Olympic Committee has banned diuretics since the 1988

Olympic Games. Tripamide is a sulfonamide-derived anti-

hypertensive agent that has been shown to possess diuretic

properties more consistent with the action of the loop

diuretics than with those of the thiazides.8 Although

Tripamide, like thiazide diuretics, is not listed as yet, it is

possible that will be put on the list.

Metabolite monitoring is important for the determination

of any medication by drugs in doping tests. Several

chromatographic methods have been reported for the

separation, detection, and quantitative measurement of

individual diuretic agents in biological fluids. Published

methods included those based on thin-layer chromatography

(TLC),9 gas-liquid chromatography (GLC),10 gas chromato-

graphy/mass spectrometry (GC/MS),10,11 high-performance

liquid chromatography (HPLC),12–16 and high-performance

liquid chromatography/mass spectrometry (HPLC/MS).17

Generally, metabolites in urine are polar so it is not easy to use

GC/MS without derivatization.

Recently, HPLC with detection by tandem mass spectro-

metry (HPLC/MS/MS) has been used for trace level

bioanalysis. This technique allows highly sensitive determi-

nation without the need for derivatization as required for

GC/MS. It is also possible to devise methods specific for

metabolites and structurally similar compounds.

This paper describes an HPLC/DAD screening proce-

dure and an HPLC/MS/MS confirmation procedure for

Tripamide in human urine.

Copyright # 2003 John Wiley & Sons, Ltd.

*Correspondence to: Y. Kim, Doping Control Center, Korea Insti-tute of Science and Technology, PO Box 131, Cheongryang,Seoul, Korea.E-mail: [email protected]

EXPERIMENTAL

ReagentsTripamide (N-aza-endo-tricyclo[5,2,1,0]decan-4-yl)-4-chloro-

3-sulfamoyl benzamide) was purchased from Eisai Co., Ltd.

(Tokyo, Japan); its purity was 98.5%. Tripamol tablets were

purchased from Hanil Pharm. Ind. Co. Ltd. (Suwon, Korea).

The internal standard (ISTD, ethyl theophylline) was pre-

pared in the Doping Control Center, Korea Institute of

Science and Technology, Seoul. Anhydrous sodium sulfate

was purchased from Sigma Chemical Co. (MO, USA) and

HCl was from Merck (Darmstadt, Germany). b-Glucuroni-

dase and b-glucuronidase/arylsulfatase were purchased

from Roche Mannheim (Indianapolis, IN, USA). Diethyl

ether, methanol, ethyl acetate, methylene chloride, and

chloroform were analytical grade from J. T. Baker (Phillips-

burg, NJ, USA). Phosphate buffer solution (0.2 M), used as

mobile phase for HPLC, was made from KH2PO4 and

K3PO4 purchased from Sigma Chemical Co. Acetonitrile

from J. T. Baker and distilled water were used after filtering

through a Millipore filter (0.5 and 0.45 mm, respectively) and

sonication for 20 min.

Instrument and equipmentA Hewlett-Packard (Palo Alto, CA, USA) HP 1100 series liquid

chromatograph, coupled with a HP 1100 series G1315A diode-

array detector, was used to screen for Tripamide. The column

for HPLC was a Capcell Pack C18 column (2.0 mm F�150 mm, particle size 5 mm). The HPLC/ESI-MS/MS system

consisted of an HP 1100 series binary pump HPLC system

(Agilent, Palo Alto, CA, USA) with an LC/MSD ion trap

equipped with an electrospray ionization source (Agilent,

Palo Alto, CA, USA). It was used for confirmation of Tripa-

mide and characterization of suspected metabolites. A Lauda

(Lauda-Konigshofen, Germany) Ecoline RE112 freezer was

used to freeze the aqueous layer. A Turbovap1 LV evaporator

supplied by Zymark Corporation (Hopkinton, MA, USA) was

used to evaporate the extracted organic solvents to dryness.

HydrolysisHydrolysis of glucuronides in urine was evaluated using b-

glucuronidase (from E. coli), b-glucuronidase/arylsulfatase

(from Helix pomatia), and chemical hydrolysis using 6 M

HCl. Hydrolysis by b-glucuronidase (from E. coli) was per-

formed as follows: 5 mL urine were adjusted to pH 7 with

KH2PO4/K3PO4 after adding 15 mL ISTD (50 mg/mL); then

50 mL b-glucuronidase (from E. coli) were added and the mix-

ture was incubated at 558C for 1 h. Hydrolysis with b-glucur-

onidase/arylsulfatase (from Helix pomatia) was performed as

follows: 5 mL urine were adjusted to pH 5.2 with 1 mL 1 N

acetate buffer after adding 15 mL ISTD (50 mg/mL), and

then incubated at 378C for 24 h. NaH2PO4 and Na2HPO4

were then added to adjust the pH to 7. Hydrolysis with 6 M

HCl was performed as follows: to 5 mL urine was added 1 mL

6 M HCl after adding 15 mL ISTD (50 mg/mL); then the mix-

ture was heated at 1058C for 30 min, after which 5 M KOH was

added to adjust the pH to 7.

Urine extractionAfter hydrolysis, anhydrous sodium sulfate (2 g) was added

to the urine. After vortexing, distilled diethyl ether was

added. After shaking for 20 min and centrifugation at

2500 rpm for 5 min, the solution was frozen in the freezer

(�308C). The organic phase was transferred to another tube

and evaporated to dryness at 408C under a gentle stream of

nitrogen. The residue was then reconstituted with 200 mL

methanol, of which 5 mL were injected onto the HPLC/

DAD and HPLC/ESI-MS/MS system.

Volunteer sampleOne healthy man (age 29; weight 75 kg) was given one Tripa-

mol tablet (Tripamide, 15 mg/tablet). Urine samples were

collected for 48 h post-dosing and subjected to the screening

procedure. These urine samples were kept at 2–38C.

Analysis of Tripamide and its metabolites byHPLC/DAD and HPLC/ESI-MS/MSHPLC/DAD operating conditions are described in Table 1.

Operating conditions for HPLC/ESI-MS/MS, used for the

confirmation of Tripamide and for the characterization of

suspected metabolites, are described in Table 2. Mass spec-

trometry and tandem mass spectrometry analyses were per-

formed using a LC/MSD ion trap mass spectrometer. The

entire column eluent was directly introduced into an electro-

spray ionization (ESI) interface through a 50 cm long PEEK

tubing (0.13 mm i.d.).

Table 1. HPLC/DAD operating conditions

Flow rate: 0.3 mL/minMobile phase

Solvent A: phosphatebuffer (pH 6.8)Solvent B: acetonitrile

Gradient time table:Time (min) 0 5 10 20 30A solvent (%) 85 85 80 55 50B solvent (%) 15 15 20 45 50

Column temperature: 458CDetection wavelength: 230 nmInjection volume: 5 mLColumn:

Capcell Pak, Type UG120, C18(Particle size 5 mm, 2.0 mm x 150 mm)

Table 2. HPLC/ESI-MS/MS operating conditions

HPLC conditionsMobile phase

Solvent A: H2OSolvent B: acetonitrile

Gradient time table:Time (min) 0 5 10 20 30Solvent A (%) 85 85 80 55 50Solvent B (%) 15 15 20 45 50

MS conditionsIonization: ESI (electrospray ionization)Mode: positive ionMass range: m/z 50–700Nebulizing gas pressure: 40 psi (N2)Drying gas temp.: 3508CDrying gas flow: 9.00 L/minCapillary exit voltage: 70.9 eVIsolation mass: 370, 386Amplitude: 0.95, 0.9Mass range: m/z 100–500Collision gas: He (70 psi)

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 301–306

302 Y. Kim et al.

RESULTS AND DISCUSSION

Detection of Tripamide by HPLC/DADThe molecular weight of tripamide is 369.09 Da and its struc-

ture, together with that of the internal standard (ethyl theo-

phylline), is shown in Fig. 1. This internal standard is

commonly used in doping control for caffeine and diuretics

at this laboratory. It was used here to check the retention

time in HPLC. Tripamide is a sulfonamide diuretic and has

a high boiling point and polarity. A chromatogram of a Tripa-

mide standard and its UV spectrum are shown in Fig. 2.

HPLC/DAD chromatograms of standard spiked urine, of

blank urine, and of a urine sample from the dosed volunteer

after acid hydrolysis are shown in Fig. 3. We investigated

whether or not the Tripamide metabolites were conjugated

by hydrolysis using b-glucuronidase, b-glucuronidase/

Figure 1. Structures of Tripamide and ISTD (ethyl theo-

phylline). Figure 2. HPLC/DAD chromatogram and spectrum of Tripa-

mide spiked in human urine.

Figure 3. Chromatograms of hydrolyzed Tripamide-spiked urine, blank

urine and dosed urine by HPLC/DAD.

Detection of Tripamide in human urine 303


arylsulfatase and 6 M HCl. As shown in Fig. 4, we could not

find any metabolites of Tripamide by hydrolysis with b-glu-

curonidase or b-glucuronidase/arylsulfatase. Only by using

HCl hydrolysis could we find the suspected metabolites M1

and M2, for which UV spectra are shown in Fig. 5. We could

not detect the parent compound Tripamide in any dosed

urine after acid hydrolysis.

Characterization of Tripamide and its metabolitesby HPLC/ESI-MS/MSWe tried to identify Tripamide using HPLC/ESI-MS/MS.

[MþH]þ ions were mainly produced in ESI-positive mode,

but [MþNa]þ and/or [MþK]þ ions were also produced by

impurities in the solvent. Figure 6 shows the total ion chroma-

togram, mass spectrum, and the MS/MS spectrum of the

[MþH]þ ion of Tripamide obtained by HPLC/ESI-MS/MS.

The spectrum of the standard includes m/z 370 ([MþH]þ) and

408 ([MþK]þ), as shown in Fig. 6(b). Figure 6(c) shows the MS/

MS spectrum of m/z 370 from Tripamide as the precursor ion,

and major fragment ions at m/z 289, 262 and 250 were detected

in addition to many others. The peak atm/z 289 is due to loss of

81 Da from the precursor ion, almost certainly SO2þNH3. The

peak at m/z 262 corresponds to an additional loss of 27 Da,

almost certainly HCN or HNC. Figure 7 shows total ion chro-

matograms for spiked urine, blank urine, and dosed (and

hydrolyzed) urine obtained by HPLC/ESI-MS/MS.

We detected two peaks in dosed (and hydrolysed) urine by

acid hydrolysis. We characterized metabolites M1 and M2 in

dosed urine by HPLC/ESI-MS/MS. Figures 8 and 9 show the

total ion chromatograms, mass spectra and MS/MS spectra

for M1 and M2. In Fig. 8(b), the molecular mass of M1 is

indicated to be 384 Da ([MþH]þ ion at m/z 385). The increase

of 15 Da probably corresponds to the replacement of the -NH2

group by OH, another hydroxylation (O-insertion), and

Figure 4. HPLC chromatograms of Tripamide-dosed urine by hydrolysis

with b-glucuronidase, b-glucuronidase/arylsulfatase and 6 M HCl.

Figure 5. UV spectra of M1 and M2 from dosed urine by

HPLC/DAD.

304 Y. Kim et al.


Figure 6. Total ion chromatogram and MS and MS/MS

spectra of a Tripamide standard.

Figure 7. Total ion chromatograms of a Tripamide-spiked

urine, blank urine and dosed urine by HPLC/ESI-MS/MS.


spectra of M1 in dosed urine by HPLC/ESI-MS/MS.


spectra of M2 in dosed urine by HPLC/ESI-MS/MS.

Detection of Tripamide in human urine 305


dehydrogenation. So even though we could not determine

where the additional hydroxyl group was attached, we could

suppose M1 involves two hydroxyl groups. In the MS/MS

spectrum in Fig. 8(c), we could detect m/z 367 ([MH–H2O]þ),

m/z 349 ([MH–HCl]þ) andm/z 323 (probably loss of C2H2 from

m/z 349 as no 37Cl peak is evident). In the spectrum in Fig. 9(b),

m/z 385 for M2 was the major ion, as in Fig. 8(b). The MS/MS

spectrum of M2 in Fig. 9(c) is very close to that in Fig. 8(c). Their

structures of M1 and M2 are only slightly different, probably

with respect to OH substitution.

CONCLUSIONS

We have evaluated a screening method and a confirmation

method for Tripamide in human urine. From the results of

acid hydrolysis, Tripamide was shown to be completely con-

jugated in the body after dosing. We detected two suspected

metabolites of Tripamide by HPLC/DAD and then charac-

terized them using HPLC/ESI-MS/MS. We think these pro-

ducts were probably hydroxylated with loss of the -NH2

group and dehydrogenation. It is not certain whether or not

these are the real metabolites of Tripamide; however, we did

determine that they definitely exist in Tripamide-dosed

urine. Even though these observations need much more

study, we can apply them to confirm Tripamide in human

urine, and these results will assist doping control.

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determination of tripamide in human urine by high-performance liquid chromatography and...

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