Download - Determination of Xipamide metabolite in human urine by high-performance liquid chromatography/diode-array detection, high-performance liquid chromatography/electrospray ionization

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2004; 18: 2505–2512

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1636

To the Editor-in-Chief

Sir,

Determination of Xipamide metabo-

lite in human urine by high-perfor-

mance liquid chromatography/diode-

array detection, high-performance

liquid chromatography/electrospray

ionization mass spectrometry and gas

chromatography/mass spectrometry

Diuretic agents are drugs that increase

renal excretion of water and solutes

(mainly sodium salts). The major pur-

poses of diuretic therapy are to

decrease fluid volume of the body and

to adjust the water and electrolyte bal-

ance. Diuretics are drugs widely used

in clinical practice mainly in the treat-

ment of hypertension and in different

kinds of edema.1–3

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.4 As part of their

efforts to fight drug abuse in sports, the

Medical Commission of the Interna-

tional Olympic Committee has banned

diuretics since the 1988 Seoul Olympic

Games.

Diuretics may be classified accord-

ing to their chemical structure, mecha-

nism, primary site of action in the

nephron and their diuretic potency.5

These diuretics exert their effects by

inhibiting tubular sodium and water

reabsorption by epithelial cells lining

the renal tubule system. Certain diure-

tics (such as carbonic anhydrase inhi-

bitors, loop diuretics, thiazide-like

diuretics and potassium-sparing diure-

tics) suppress sodium and water reab-

sorption by inhibiting the function of

specific proteins that are responsible

Copyright # 2004 John Wiley & Sons, Ltd.

RCM

Letter to the Editor

Table 1. HPLC/DAD operating conditions

HPLC conditionsFlow rate: 1.0 mL/minMobile phase solvent A: Phosphate buffer (pH 6.8)

solvent B: AcetonitrileGradient timetable:Time (min) 0 10 15 18Solvent A (%) 96 70 55 50Solvent B (%) 4 30 45 50Detection wavelength: 232 nmInjection volume: 5mLColumn temperature: 408CColumn: Hypersil-ODS C18 column (4.6 mmi.d.� 100 mm, particle size 5mm)

Table 2. HPLC/ESI-MS operating conditions

HPLC conditions

Flow Rate: 0.2 mL/minMobile Phase solvent A: Acetonitrile

solvent B: 20 mM Ammonium formate (pH 4)Gradient timetable:Time (min) 0 5 18Solvent A (%) 20 50 50Solvent B (%) 80 50 50Injection Volume: 5mLColumn Temperature: 408CColumn: Capcell Pak C18 column (MG type, 2.0 mmi.d.� 150 mm�particle size 5mm)MS conditionsIonization: ESI (electrospray ionization)Mode: Positive ionMass range: m/z 50–420Nebulizing Gas Pressure: 30 psi (N2)Drying Gas Temp: 3508CDrying Gas Flow: 8.0 L/minCapillary Exit Voltage: 60.0 eVMS/MS conditions

Collision gas: He (70 psi)Collision energy: 0.8 eVIsolation width of parent ion: 1.5 Da

Table 3. GC/MS operating conditions

GC conditionsCarrier gas: He at 1.0 mL/minOven temp. program:

Initial temp.(8C)

Initialtime (min)

Rate(8C/min)

Final temp.(8C)

Final time(min)

180 1 15 300 5

Injection volume: 2mLSplit mode: Split (1:10)Injection port temp.: 2808CTransfer line temp.: 2808CColumn: Ultra-2 (cross-linked 5 %phenylmethylsiloxane, 0.2 mmi.d� 17 m length� 0.33mmfilm thickness)MS conditions

Ionization: EISource temp.: 2008CAcquisition mode: scanMass range: m/z 50–600

for (or participate in) the transportation

of electrolytes across the epithelial

membrane; osmotic diuretics inhibit

water and sodium and water reabsorp-

tion by increasing intratubular osmotic

pressure. Different types of diuretics

may inhibit different transporters in dif-

ferent segments of the tubular system.6,7

Xipamide (4-chloro-5-sulfamoyl-20,60-

salicyloxylidide) is a diuretic drug

used for the treatment of high blood

pressure and a edema of cardiac,

hepatic or renal origin. It is a non-

thiazide diuretic with a greater anti-

hypertensive effect and may cause a

lower potassium loss relative to

sodium excretion than these drugs.

Xipamide offers a suitable alternative

to other diuretics in the treatment of

patients with mild to moderate hyper-

tension and of patients with edema due

to a variety of causes.8

Metabolite monitoring is important

for the determination of any medica-

tion by drugs. Several chromatogra-

phic methods have been reported for

the separation, detection, and quanti-

tative measurement of individual

diuretic agents in biological fluids. Pub-

lished methods include those based

on thin-layer chromatography (TLC),9

gas-liquid chromatography (GLC),10

gas chromatography/mass spectro-

metry (GC/MS),10,11 high-performance

liquid chromatography (HPLC),12–16

and high-performance liquid chroma-

tography/mass spectrometry (HPLC/

MS).17 Generally, metabolites in urine

are polar, so it is not easy to use GC/MS

without derivatization. More recently,

HPLC with detection by tandem mass

spectrometry (HPLC/MS/MS) has

been used for trace level bioanalysis.18

This technique allows highly sensitive

determinations 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 the HPLC/

diode-array detection (DAD) screening

procedure and HPLC/MS confirma-

tion method of Xipamide in human

urine for doping control tests. The

metabolite of Xipamide was also stu-

died by GC/MS after methylation for

characterization.

Xipamide tablets were purchased

from Bukwang Pharm. Co., Ltd. (Seoul,

Korea). Anhydrous sodium sulfate was

Scheme 1. Structure of Xipamide.

Figure 1. HPLC/DAD chromatograms of Xipamide in (a) spiked urine, (b) blank

urine, and (c) dosed urine.

Copyright # 2004 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 2505–2512

2506 Letter to the Editor

purchased from Sigma Chemical Co.

(MO, USA) and HCl was from Merck

(Darmstadt, Germany). Diethyl ether,

ethyl acetate and acetone were HPLC

grade from J. T. Baker (Phillipsburg,

NJ, USA). Acetonitrile and methanol

from J. T. Baker and distilled water

were used after filtering through Milli-

pore filters (0.5 and 0.45 mm, respec-

tively) and sonication for 20 min. The

internal standard (ethyltheophylline)

was prepared at the Doping Control

Center (KIST, Korea)

A Hewlett-Packard (Palo Alto, CA,

USA) HP 1100 series liquid chromato-

graph, coupled with a HP 1100 series

G1315A diode-array detector, was

used to screen Xipamide and its meta-

bolite. The column for HPLC was a

Hypersil-ODS C18 column (4.6 mm

i.d.� 100 mm, particle size 5mm, Ther-

mohypersil). The HPLC/ESI-MS sys-

tem consisted of an HP 1100 series

binary pump HPLC system (Agilent,

Palo Alto, CA, USA) and an LC/MSD

ion trap equipped with an electrospray

ionization (ESI) source. It was used for

confirmation of Xipamide and charac-

terization of suspected metabolites.

The column for HPLC/ESI-MS was a

Capcell Pak C18 column (MG type,

2.0 mm i.d.� 150 mm�particle size

5 mm, Shiseido). A Trace GC-Polaris Q

(Finnigan Inc., San Jose, CA, USA) was

used for reconfirmation of the suspec-

ted metabolite. The column for GC/

MS was an Ultra-2 capillary column

(0.2 mm i.d.� 17 m length� 0.33 mm

film thickness, Agilent Technologies).

A Lauda (Lauda-Konigshofen, Ger-

many) Ecoline RE112 freezer was used

to freeze the aqueous layer. A Turbo-

vap1 LV evaporator supplied by

Zymark Corporation (Hopkinton,

MA, USA) was used to evaporate the

extracted organic solvents.

A healthy male volunteer (age 29,

weight 70 kg) was dosed with one

Diurexan tablet (Xipamide, 20 mg/

tab). Urine samples were collected for

48 h and subjected to the screening

procedure; these urine samples were

kept at 2–38C.

Anhydrous sodium sulfate (1 g) was

added to the urine (pH 5 adjusted with

phosphate buffer). After vortexing,

5 mL distilled diethyl ether were

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 trans-

ferred to another tube and evaporated

to dryness at 408C under a gentle

stream of nitrogen. The residue was

then reconstituted with 200 mL metha-

nol, of which 5 mL were injected on the

HPLC/DAD and LC/MS system.

Then, 150mL of the residue were dried

under a nitrogen gas stream, and 50mL

of methyl iodide and 150 mL of acetone

were added and then vortex-mixed.

The mixture was heated at 808C for 1 h

and then cooled to room temperature.

Finally, 2 mL of this solution were

analyzed by the GC/MS system.

Operating conditions for HPLC/

DAD and HPLC/ESI-MS, used for the

detection of Xipamide and the char-

acterization of its metabolite, are

described in Tables 1 and 2. Mass

spectrometry (MS) and tandem mass

spectrometry (MS/MS) analyses were

performed on a LC/MSD ion trap mass

spectrometer for confirmation. The

entire column eluent was directly

introduced into an ESI interface

through a 50 cm long PEEK tubing

(0.13 mm i.d.). Operating conditions

for GC/MS are described in Table 3.

The molecular mass of Xipamide is

354.04 Da; its structure is shown in

Scheme 1. It is a sulfonamide diuretic

with a high boiling point and polarity.

HPLC/DAD chromatograms of stan-

dard spiked urine, blank urine, and

the urine sample from the dosed

volunteer are shown in Fig. 1. We

found unmetabolized Xipamide and

one unknown peak, designated Xipa-

mide-M1, at a shorter retention time

(6.23 min) than Xipamide (8.91 min), in

the dosed urine. Figure 2 shows the

ultraviolet spectra of the unknown

peak (Xipamide-M1) and of Xipamide

itself; these spectra are very similar.

From these results, the metabolite of

Xipamide was suspected to be a

compound of similar structure but

substituted by a polar functional

group such as hydroxyl to account

for the reduced retention in reversed-

phase HPLC.

[MþH]þ ions were mainly produced

in positive ESI mode, and [MþNa]þ

and/or [MþK]þ ions were also pro-

duced by impurities in the solvent.

Figure 3 shows the total ion chromato-

gram (TIC), extracted ion chromato-

gram (EIC), mass spectrum, and the

MS/MS spectrum of the [MþH]þ

Figure 2. UV spectra of (a) Xipamide and (b) Xipamide-M1 from dosed urine

obtained by HPLC/DAD.

Letter to the Editor 2507


Figure 3. (a) Total ion chromatogram, (b) extracted ion chromatogram for [MþH]þ and

(c) MS and (d) MS/MS spectra of Xipamide standard, obtained by HPLC/ESI-MS.



Figure 4. (a) Total ion chromatogram, (b) extracted ion chromatogram for [MþH]þ and

(c) MS and (d) MS/MS spectra of Xipamide-M1, obtained by HPLC/ESI-MS from dosed

urine.



ion, of the Xipamide standard. The

spectrum of the standard (Fig. 3(b))

includesm/z 355 ([MþH]þ) andm/z 377

([MþNa]þ), and shows the isotope

pattern characteristic of the Cl atom

(m/z 357). Figure 3(d) shows the

MS/MS spectrum of m/z 355 from

Xipamide as the precursor ion, and

major fragment ions atm/z 338, 274 and

290 were detected in addition to less

abundant fragments at m/z 210 and

122.

If the metabolism of Xipamide leads

to hydroxylation (insertion of an O-

atom), the molecular mass of Xipa-

mide-M1 is expected to be 370.04 Da.

We detected Xipamide-M1 in urine

from the dosed volunteer by HPLC/

ESI-MS. Figure 4 shows the TIC, EIC for

the [MþH]þ ion at m/z 371, the mass

spectrum and MS/MS spectrum. The

mass spectrum of Xipamide-M1 shows

the expected [MþH]þ ion at m/z 371

and the sodium adduct ion [MþNa]þ at

m/z 393 (Fig. 4(b)). We could not

determine from this information where

the additional hydroxyl group was

Figure 5. GC/MS chromatograms of methylated Xipamide in (a) spiked urine, (b) blank

urine, and (c) dosed urine.



substituted, so MS/MS was applied to

obtain information about the position

of the hydroxyl group. Figure 4(c)

shows the MS/MS spectrum of m/z

371 from Xipamide-M1. The main frag-

ment ions were observed at m/z 354

([MH–H2O]þ), 290 ([MH–SO2NH2]þ),

275, and 137. Xipamide and Xipamide-

M1 are much alike in fragmentation

pattern. These results suggested that

Xipamide-M1 contained one additional

hydroxyl group, but did not provide

any information concerning the posi-

tion of the hydroxyl group.

We also used GC/MS for character-

ization of the metabolite of Xipamide.

Figure 5 shows the GC/MS chro-

matograms obtained of methylated

Xipamide in spiked urine, balnk urine

and dosed urine. The derivatization

(methylation) method was applied to

the dosed human urine, and both

Xipamide and its metabolite were

detected. Figure 6 shows the EIC and

mass spectrum of the methylated

Xipamide standard obtained by GC/

MS.

The mass spectrum (Fig. 6(b)) of

methylated Xipamide shows the mole-

cular ion [M]þ at m/z 410 and the base

peak at m/z 289 ([M–CH3, -C8H10]þ).

Thus the [M]þ (m/z 410) ion shows

that four methyl groups were substi-

tuted in the Xipamide standard (4Me-

Xipamide). The main fragment ions

were observed at m/z 303 ([M–

SO2N(CH3)2]þ), 276 ([M–C9H12N]þ)

and 233 ([M–C9H12N–N(CH3)]þ).

Figure 7 shows the EIC of the [M]þ

ion atm/z 440 and the mass spectrum of

Xipamide-M1, obtained by GC/MS

from dosed human urine. The m/z 440

ion, which was 30 Da more than the

molecular ion of 4Me-Xipamide, indi-

cates both hydroxylation and methyla-

tion of Xipamide. Thus Xipamide-M1

has one additional hydroxyl group

compared with Xipamide, even though

its position could not be determined.

However, we could expect the addi-

tional hydroxyl group to appear in the

dimethylbenzene group, based on

observation of m/z 289 in the mass

spectrum of the Xipamide standard

(Fig. 6(b)) and of dosed urine (Fig. 7(b)).

In conclusion, we have evaluated a

screening method and a confirmation

method for Xipamide in human

urine. When we compared the chro-

matograms of the blank urine, spiked

urine and dosed urine by HPLC/DAD,

we found an unknown metabolite that

was shown to be 16 Da heavier than

Xipamide by HPLC/MS and GC/MS.

These results suggested that one

hydroxyl group substitutes to Xipa-

mide as the metabolite. Even though

the exact position of hydroxylation

could not be determined, we can apply

these observations to confirm Xipa-

mide in human urine, and these results

will assist doping control.

Figure 6. (a) Extracted ion chromatogram for [MþH]þ and (b) MS spectrum of

methylated Xipamide standard obtained in dosed urine by GC/MS.



AcknowledgementThis research was supported in 2004 by theKyung Hee University Research Fund(KHU-20031102).

Yunje Kim1*, Kyungjin Han1,2

and Ki-Jung Paeng2

1Doping Control Center,Korea Institute of Science and

Technology, P.O. Box 131,Cheongryang, Seoul, Korea

2Department of Chemistry,College of Science,

Yonsei University, Seoul, Korea

*Correspondence to: Y. Kim, DopingControl Center, Korea Institute ofScience and Technology, P.O. Box 131,Cheongryang, Seoul, Korea.E-mail: [email protected]

REFERENCES

1. Bridges JW, Chasseaud LF. Progressin Drug Metabolism, vol. 7. JohnWiley: New York, 1983; 58–171.

2. Weiner IM, Gilman A, RallTW, Nies AS, Taylor P (eds).Goodman and Gilman’s Pharmaco-logical Basis of Therapeutics. Perga-mon Press: New York, 1990;708.

3. Dirks JH, Sutton RAL (eds). Diure-tics, Physiology, Pharmacology andClinical Use. W. B. Saunders Co.:Philadelphia, PA, 1986.

4. Schanzer W. The problems of diure-tics in doping control. Institut furBiochemie der Deutschen Sport-hochschule: Koln, 1988, 1, privatecommunication.

5. Ventura R, Segura J. J. Chromatogr. B1996; 687: 127.

6. International Olympic Committee,Medical commission, InternationalOlympic Charter against Doping inSport, IOC: Lausanne, 1990.

7. Kawasaki K, Fukiyama K. Xenobio-tica 1981; 11: 197.

8. Legorburu MJ, Alonso RM. Bio. Bioe-nergetics 1993; 32: 57.

9. Hadzija BW, Mattocks AM. J. Chro-matogr. 1982; 229: 425.

10. Fleuren HLJM, Van Rossum JM. J.Chromatogr. 1978; 152: 41.

11. Vandenheuvel WJA, Gruber VF,Walker RW, Wolf WJ. J. Pharm. Sci.1975; 64: 1309.

12. Swart KJ, Botha H. J. Chromatogr.1987; 413: 315.

13. Hessey GA II, Constanzer ML,Bayne WF. J. Chromatogr. 1986; 380:450.

14. Sherry JH, O’Donnell JP, Colby HD.J. Chromatogr. 1986; 374: 183.

15. Snedden W, Sharma JN, FernandezPG. Ther. Drug Monit. 1982; 4: 381.

16. Rapaka RS, Roth J, Viswanathan C,Goehl TJ, Prasad VK, Cabana BE.J. Chromatogr. 1982; 227: 463.

17. McCue BA, Cason MM, Curtis MA,Faulkner RD, Dahlin DC. J. Pharm.Biomed. Anal. 2002; 28: 199.

18. Lindstrom B. J. Chromatogr. 1974;100: 189.

Received 20 April 2004Revised 12 August 2004

Accepted 13 August 2004

Figure 7. (a) Extracted ion chromatogram for [MþH]þ and (b) MS spectrum of

methylated Xipamide-M1 in dosed urine obtained by GC/MS.



Download - Determination of Xipamide metabolite in human urine by high-performance liquid chromatography/diode-array detection, high-performance liquid chromatography/electrospray ionization

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