capillary electrophoretic determination of triamterene, methotrexate, and creatinine in human urine

J. Sep. Sci. 2005, 28, 658–664 www.jss-journal.de i 2005WILEY-VCH Verlag GmbH&Co. KGaA,Weinheim

Juana Rodr�guez Flores1

Juan J. Berzas Nevado1

Isabel Dur�nMer�s2

Maria J. Rodr�guez G�mez2

1Department of AnalyticalChemistry and FoodsTechnology, University ofCastilla-La Mancha,13071 Ciudad Real, Spain

2 Department of AnalyticalChemistry, University ofExtremadura, 06071 Badajoz,Spain

Capillary electrophoretic determination oftriamterene, methotrexate, and creatinine in humanurine

A capillary zone electrophoresis (CZE) method using a fused-silica capillary(60.2 cm675 lm ID) was investigated for the determination of triamterene (TRI),methotrexate (MTX), and creatinine (CREA) in human urine. The separation was per-formed using a hydrodynamic injection time of 7 s (0.5 psi), a voltage of 25 kV, acapillary temperature of 308C, and 40 mM phosphoric acid adjusted to pH 2.25 byaddition of triethanolamine as separation electrolyte. Under these conditions, analysistakes about 15 min. A linear response over the 0.5–15.0 mg L– 1 concentration rangewas found for TRI and MTX, and 0.5–80.0 mg L–1 for CREA. Dilution of the sample(water:urine, 1 :1 for TRI and MTX, and 1:25 for CREA determination) was the onlystep necessary prior to analysis by electrophoresis. The developed method is easy,rapid, and sensitive and has been applied to determine triamterene, methotrexate,and creatinine in urine samples with satisfactory results.

KeyWords:CZE; Triamterene; Methotrexate; Creatinine; Human urine;

Received: October 22, 2004; revised: February 2, 2005; accepted: February 3, 2005

DOI 10.1002/jssc.200400059

658 Rodr�guez Flores, Berzas Nevado, Dur�nMer�s, Rodr�guez G�mez

1 Introduction

Triamterene (2,4,7-triamino-6-phenylpteridine) (TRI)(Figure 1), is a natriuretic agent which is much used in thetreatment of several diseases. It can also be applied asdoping substance. In sports, this diuretic is abused mainlyfor two reason: to obtain a rapid reduction of body weight,important in sports which are divided into different weightcategories, and to reduce the concentration of medicaldrugs in urine by dilution by means of a rapid production ofan elevated quantity of urine. Triamterene (pK values 2.05and 6.30) is rapidly, but incompletely, absorbed after oraladministration. Once the drug is in the body, between 30and 70% of an oral dose is excreted in the urine [1]. Vari-able amounts are excreted in the bile.

Methotrexate (MTX), an antimetabolic agent, is an antifo-late in a class of folic acid analogs (Figure 1) that havedemonstrated effective antineoplastic activity in the treat-ment of disorders of cell proliferation. It works by interfer-ing with the growth of cancer cells. These cells are theneventually destroyed. Unfortunately, it also interferes withnormal cell growth and, as a result, may cause problemsfor the person taking this medication. MTX (pK values of3.36, 4.70, and 5.71) is used in the treatment of psoriasis,certain forms of cancer, and certain connective tissuesdiseases such as rheumatoid arthritis, lupus, and sclero-

derma [2]. The efficacy of antifolate drugs is related to theextent of intracellular polyglutamation [3,4]. In most cells,polyglutamation does not occur until the cell is exposed to10–6 MMTX for at least 6 h [5].

TRI and MTX can be present in biological fluids due to thediuretic activity of TRI, and their simultaneous determina-tion can be necessary to optimize MTX treatment.

Creatinine (CREA) (Figure 1) is the means by which crea-tine and phosphocreatine are excreted from the body.Over 90% of bodily creatine is present in muscle, with the

Correspondence: Juana Rodr�guez Flores, Department of Ana-lytical Chemistry and Foods Technology, University of Castilla-La Mancha, 13071 Ciudad Real, Spain. Phone: +34 926 295300.Fax: +34 926 295318. E-mail: [email protected].

Figure 1. Structures of the molecules (A) triamterene, (B)methotrexate, and (C) creatinine.

CZE of triamterene, methotrexate, and creatinine in urine 659

remainder being in the plasma (usually a 5 mg/L) andbrain [6]. The main dietary source of creatine is red meat,which suggests why vegetarians have been shown tohave lower levels of CREA in serum [7].

The 24-h CREA content of urine remains roughly constantfor a given individual [8], and it is often used as a normal-ization factor when other urinary components are meas-ured. Changes in urinary CREA content can indicate renalproblems. A typical 24-h sample of urine (600 to 1600 mL)contains 1–3.3 g CREA. Actual concentrations fluctuatewith the time of day and the intake of water, and thus it isusual to analyze 24-h totals rather than measuring actualconcentrations at any time.

Initial measurements of triamterene have been directlymade in pharmaceutical preparations by perchloric acidtitrations [9,10]. Mixtures of triamterene and amiloridehydrochloride in pure and pharmaceutical preparationshave been analysed by fluorimetry [11]. Also a polaro-graphic method [12] has been described for triamtereneand its analogues in biological fluids. Usually, however,the specific determination of this natriuretic agent inbiological fluids involves liquid chromatography [13–16].Capillary zone electrophoresis has been used for determi-nation of triamterene [17,18] with other drugs in urinesamples.

Many analytical methods have been reported for analysisof MTX in biological fluids, using enzyme multiplied immu-noassay [19], fluorescence polarization immunoas-say [20], radioimmunoassay [21], capillary zone electro-phoresis [22,23], and HPLC [24–26].

Reversed-phase HPLC has been used to analyze urinarycreatinine alone [27] and in conjunction with a variety ofother species, including iothalmic acid [28], estriol [29],amino acids and organic acids [30], and pteridines [31].Enzymatic detection methods have been shown to be analternative to UV detection, where coeluting compoundscan be a problem [32]. Determination of CREA [33], andsimultaneous determinations of serum CREA and uricacid [34], pseudouridine [35] or amino acids, bioactiveamines and nucleic acid bases [36] have also been car-ried out using ion-pair chromatography with sodium dode-cyl sulfate as the ion-pairing reagent. Some analyseshave involved the use of several columns [37].

Gatti et al. [38] analyzed CREA in urine in 3.5 min, bycapillary zone electrophoresis (CZE) using a 0.1 M-acet-ate, pH 4.4 running buffer and Kong et al. [39] proposed amethod for determining CREA, creatine, urea, and uricacid at pH 3.50.This separation takes about 15 min. Inorder to separate creatinine from other UV absorbing neu-tral species, micellar electrokinetic capillary chromatogra-phy (MEKC) is employed. Burke et al. [40] analyzed crea-tine and CREA in urine by capillary micellar electrophor-esis in 5.7 min, using a 30 mM-phosphate buffer of pH 6

containing 0.15 M-SDS. Pobozy et al. [41] analyzed dialy-sate creatinine by MEKC in urine in 7.0 min, using 30 mMborate-100 mM sodium dodecyl sulphate backgroundelectrolyte at pH 9. Zuppi et al. [42] proposed amethod forthe simultaneous determination of hippuric acid and crea-tinine in urine samples based on MEKC. The creatininepeak was resolved in a migration time of 4.12 min using a20 mM sodium phosphate, pH 7.2, 25 mM sodium dode-cyl sulfate, 5% (v/v) acetonitrile.

Recently Paroni et al. [43] has proposed a method forcreatinine determination in serum. The serum was depro-teinized with acetonitrile and, after dilution with water, thesupernatant was injected in pressure mode into the capil-lary electrophoresis equipment using a phosphoric bufferof pH 2.5 as separation electrolyte.

The development of analytical methods for the simulta-neous determination of TRI, MTX, and CREA appears toprovide a convenient method of acquiring informationabout renal function in the MTX administration necessaryin the treatment of some forms of cancer. To the best ofour knowledge, there has been no report on the paralleldetermination of triamterene, methotrexate, and creati-nine.

2 Experimental

2.1 Chemical

All chemicals and solvents were of analytical reagentgrade. Triamterene (TRI) and creatinine (CREA) wereobtained from Aldrich, and methotrexate (MTX) fromSigma; triethanolamine was also purchased from Aldrich.Stock standard solutions of TRI, MTX, and CREA wereprepared by dissolving appropriate amounts of the corre-sponding analyte in deionized water and stored underrefrigeration at 48C.

Dilute urine solutions were prepared daily by diluting freshhuman urine with purified water (1 :1 for MTX and TRI,and 1:25 v/v for CRE determination).

Buffer solution was prepared by diluting the appropriatevolume of H3PO4 in deionized water and then adjusting tothe required pH with triethanolamine. This was prepareddaily and the set of separation vials was changed after sixruns.

2.2 Instrumentation

Analysis was performed with a Beckman P/ACE System5510 capillary electrophoresis instrument with diode-arraydetection (DAD) and controlled by means of a Dell Dimen-sion P133V with P/ACE station software. This equipmenthas a 75 lm ID fused silica separation capillary of 60.2 cmtotal length (50 cm to the detector) mounted in a userassembled cartridge (Beckman) with a 1006800 lmdetection window.


Original

Pap

er


The use of a photodiode detector allowed us to confirmthe identity of the peaks, not only by their migration time,but also by the overlay of the UV-Vis spectra of the sam-ples with a standard.

2.3 Treatment of the urine samples

Fresh human urine samples were obtained from differentvolunteers. These samples were used after a simple dilu-tion (1 :1 for TRI and MTX or 1:25 for CREA, urine:waterdue to the high concentration of CREA in urine). Urinesamples spiked with the MTX and TRI compounds understudy (CRE was not necessary due to its presence inurine) were immediately injected into the capillary electro-phoresis equipment. The stability of the solutions waschecked for 2 hours by ensuring that they gave satisfac-tory electropherograms (the area and the migration timeswere constant).

2.4 Operating conditions

Before the first use, the capillary was conditioned by rins-ing with 0.1 MNaOH for 20 min, with water for 15 min, andlastly with the background electrolyte solution for 10 min.At the start of each sequence of analyses, the capillarywas washed for 2 min with 0.1 M NaOH, and then for4 min with electrolyte separation buffer. This was suffi-cient to restore and reequilibrate the capillary wall surfacebetween sample injections

The running buffer was a 40 mM phosphate buffer(pH = 2.25 adjusted by addition of triethanolamine solu-tion 99.5%). It was prepared by dissolving the appropriatequantity of H3PO4 in deionised water and adjusting to therequired pH by addition of triethanolamine. All separationswere conducted using 2-mL vials for rinsing, washing, andbiological samples. Urine samples were preserved at308C inside the capillary electrophoresis equipment.

The injection of the samples was performed hydrodynami-cally for 7 s (0.5 psi). Separations of MTX and TRI wereperformed in 15 min and the CREA determination wasobtained at 5 min, performed in parallel both determina-tion at 25 kV and 308C as optimal conditions of separa-tion. Under these conditions the current was approxi-mately 61 lA. The best value of the wavelength for a par-allel determination of the three compounds was 210 nm.

Duplicate injections of the biological samples were per-formed and averages of corrected peak area (area/migra-tion time) and height peak signals were used for quantita-tive analysis.

3 Results and discussionWith the goal of studying the influence of the urine matrixin the electrophoretic separation and quantification oftriamterene, methotrexate, and creatinine, the influence

of all variables was investigated with diluted solutions ofurine. For the determination of triamterene and metho-trexate it is sufficient to dilute the urine with water in a ratioof 1 :1. On the other hand, since creatinine is present inurine in high concentration, it was necessary to use a larg-er dilution factor of 1 :25 (urine:water).

3.1 Optimization of chemical and instrumentalvariables for the TRI, MTX, and CREAdetermination

3.1.1 Optimisation of pH and ionic strength ofelectrolyte

The pH of the running electrolyte had a significant impactboth on the ionization of the acidic silanols on the capillarywall and on the electrophoretic mobilities of the com-pounds studied. Because of the structures of the analytes(Figure 1), acid buffers could be used to promote theirionization. The influence of pH was examined in urinesamples diluted 1:1 and with buffer solutions prepared bydissolving the appropriate quantity of H3PO4 in deionisedwater and adjusting the pH to the required value by addi-tion of triethanolamine (range of pH between 2.0 to 3.5).The best results were obtained at pH 2.25, with H3PO4

40 mM. At these low pH values, the migration velocities ofTRI and MTX were less than the electroosmotic flow(EOF), due to the cationic forms of the two compounds.Under these conditions, the order of elution was TRI –MTX and themigration times are 9.2 and 15.8 min respec-tively.

3.1.2 Optimisation of pH and ionic strength ofelectrolyte

For determination of CREA, the influence of pH wasexamined in urine samples diluted 1:25 with buffer solu-tions prepared by dissolving the appropriate quantity ofH3PO4 in deionised water and adjusting the pH to therequired value with triethanolamine (range between 2.0 to3.5). The best result was obtained at pH 2.25, with H3PO4

40 mM. Under these conditions, the elution time of CREAis approximately 5.0 min.

The influence of the H3PO4 concentration on the mobilityof TRI and MTX was studied by increasing the phosphoricacid concentration from 20 to 50 mM at 25 kV and 258C,and in all solutions the pH was fixed at 2.25 with triethanol-amine. When the concentration of buffer was increasedthe migration times also increased. A phosphoric acidsolution concentration of 40 mMwas selected as optimumand, under these conditions, the migration time was9.2 min for triamterene and 15.8 min for methotrexate.For CREA, the effect of buffer solution concentration (30–60 mM) on the migration time of the compound was stud-ied on a urine:water 1:25 solution. A buffer concentration



of 40 mM was selected as optimum, on the basis of cur-rent efficiency and lowmigration time.

3.1.3 Effect of applied voltage

The effect of the voltage applied from 5 to 25 kV wasinvestigated. When the voltage increases, the migrationtimes of TRI and MTX decrease. An applied potential of25 kV yielded the best compromise in terms of run timeand current generated (61.0 lA). This voltage was used insubsequent stages of method development.

This instrumental variable was also optimal for CREAdetermination.

3.1.4 Effect of temperature

The effect of the capillary temperature on the separationwas tested in the range of 20 to 358C. Solute migrationtimes increased because of changes in the viscosity of themedium. A temperature of 308C was selected as optimumvalue giving the best compromise between run time andlevel of baseline noise.

3.1.5 Optimization of injection time

To improve detection limits in human urine, the time ofhydrodynamic injection (by pressurizing the sample vial)was varied between 3 and 9 s. The corrected peak areaincreased with increasing injection time. The optimumvalue chosen was 7 s. The pressure used for injectionwas always 0.5 psi.

3.1.6 Optimization of the washing step

Sample components from the diluted urine can beadsorbed onto the capillary surface and change the effec-tive charge on the wall. Therefore, the capillary is flushedbetween injections for 2 min with 0.1 M NaOH and for4 min with electrolyte in order to obtain an unchangingelectropherogram.

3.1.7 Optimal conditions

An electropherogram of urine diluted 1:1 for determina-tion of TRI and MTX obtained under the optimized condi-tions is presented in Figure 2.

An electropherogram of urine diluted 1:25 for determina-tion of CREA obtained under the optimized conditions ispresented in Figure 3.

Due to the absence of other compound in the urine diluted1:25 for the CREA determination, the injection from theshort end of the capillary (7.6 cm as effective length to thediode-array detector) was also studied. In Figure 4 wecan see an electropherogram recorded under optimalselected conditions for the determination of CREA. Theshort time of 0.85 minutes for CREA analysis was trulyremarkable.

4 Validation of the electrophoreticprocedure

4.1 Stability of the solutions

The stability of urine samples diluted 1 :1 and 1:25 andspiked with the compounds was evaluated by comparingcorrected peaks areas obtained at different time intervals.


Figure 2. Electropherogram obtained from a urine samplewith dilution 1 :1, spiked with 2 mg L– 1 of TRI and MTX; oper-ating conditions: pH = 2.25; 40 mM phosphoric acid withtriethanolamine; 25 kV; 308C

Figure 3. Electropherogram obtained from a urine samplewith dilution 1 :25 for determination of CREA; operating con-ditions: pH = 2.25; 40 mM phosphoric acid with triethanol-amine; 25 kV; 308C. Creatinine in urine 520 mg L– 1.


They were stable for at least 2 hours and 3 hours fordiluted urine samples 1:1 and 1:25, respectively.

4.2 Precision

The precision of this method for the determination oftriamterene, methotrexate, and creatinine is expressed in

terms of relative standard deviation (RSD). Eleven injec-tions of 1 :1 diluted urine spiked with 12 mg L– 1 of TRI andMTX, and eleven injections of 1 :25 diluted urine spikedwith 10 mg L–1 of CREA were performed sequentially.The precision of peak areas, peak height, and migrationtime as RSD (%) were 2.76, 2.76, and 0.56%, respec-tively, for triamterene, 2.64, 5.56, and 0.67, respectively,for methotrexate and 2.68, 2.45, and 0.50%, respectively,for creatinine.

4.3 Linearity

In order to evaluate the matrix effect of the urine, for thequantification of TRI and MTX, two different calibrationcurves were plotted, one with urine from one person andthe other with a pool of urine from three different persons.In both cases the detector responses (corrected peakareas and height peaks), were measured for triamtereneand for methotrexate and they were linearly dependent onthe compound concentration over a range of 0.5 to 15 and1 to 15 mg L– 1, respectively.

The results obtained for the equations relating correctedpeak areas and height peaks to concentration wereacceptable for biological applications for the two analyseddrugs. The linear regression equations obtained underoptimal conditions are summarized in Table 1.

For CREA, different calibration curves in the presenceand in the absence of urine were plotted. In the first, a cali-bration curve using six different standard solutions ofCREA was drawn. In the second, a calibration curve was


Figure 4. Electropherogram of creatinine operating with theshort end of the capillary (10 cm as effective length); operat-ing conditions: pH 2.25; 40 mM phosphoric acid with trietha-nolamine; 25 kV; 308C. Creatinine in urine 520 mg L– 1.

Table 1. Statistical data for determination of TRI and MTX in urine (1:1) in terms of CPA and height.

Triamterene Methotrexate

CPA Height peak CPA Height peak

Urine from one person

Slope (l rS) 8.146102 l2.366101 1.636103 l 4.296101 3.486102 l 1.266101 5.516102 l 2.666101

Intercept (l rI) 1.946102 l 1.896102 7.026102 l3.446102 2.716102l 1.006102 5.076102 l 2.146102

Regression Coeff. 0.9962 0.9969 0.9942 0.9897

LODa) (mg L– 1) 0.70 0.63 0.87 1.17

LODb) (mg L– 1) 1.63 1.48 2.06 2.72

Linear range (mg L– 1) 0–15 0–15

Urine from three persons

Slope (lrS) 6.226102 l 3.356101 6.876102 l 2.796101 3.426102ll 1.186101 3.186102l1.486101

Intercept (l rI) 1.746103l 2.826102 1.706103 l 2.356102 2.946102l9.966101 4.306102l1.246102

Regression Coeff. 0.9886 0.9935 0.9953 0.9915

LODa) (mg L– 1) 1.43 1.07 0.88 1.19

LODb) (mg L– 1) 2.81 2.12 1.81 2.43

Linear range (mg L– 1) 1–15 1–15

a) Winefordner and Long criterion. (G. L. Long, J. S. Winefordner, Anal. Chem. 1983, 55, 712.)b) Clayton criterion. (C. A. Clayton, J. W. Hines, P. D. Etkins, Anal. Chem. 1987, 59, 2506.)


constructed always using a urine sample obtained from a44-year-old healthy woman and spiked with variousamounts of CREA in the range between 0 (without addi-tion) and 12 mg L–1. Also, a third calibration curve wasdrawn using a mixture of urine samples from three differ-ent volunteers, spiked with CREA as was indicated above.Standard addition methodology was applied for the sakeof comparison and to test the possibility of applying theexternal standard method in routine analysis.

The linearity of the electrophoretic response with respectto the CREA concentration was checked in all threecases. The regression lines are summarized in Table 2.The slopes show very similar values in the three cases,from which we can deduced that the biological matrixdoes not interfere in analysis of CREA, may be due to thelarge dilution factor.

As a consequence, a calibration curve using the externalstandard method could be proposed when the analysis isdone with a 1:25 v/v (urine:water) dilution.

4.4 Study of interferences

Different interferences were studied in the triamterene,methotrexate, and creatinine determinations. Differenturines from persons that have taken: antibiotic (amoxici-line and clavulanic acid), mucolytic (carbocysteine), andantipyretic and anti-inflammatory (ibuprofen), were ana-lyzed. The peak purity was analyzed by scanning andcomparison between the absorption spectra obtaining atdifferent migration times in each electrophoretic peak. Nointerference in the determination of the three compoundswas observed in any of the cases.

Creatine, as bioprecursor of CREA, was also studied as apossible interference. No interference was seen in CREAanalysis.

4.5 Applications

To demonstrate the usefulness of the proposed method,several aliquots of TRI and MTX solutions were added tospiked urine previously diluted 1:1 (Table 3). In all cases

the standard addition method was used for the determina-tion of drugs in the real samples due to the matrix effect ofthe urine. To determine the recoveries of TRI and MTX inhuman urine the corrected peak area and the height ofelectrophoretic peaks were used for the drug determina-tion. Good recoveries were obtained for both analyzedcompounds. Table 3 shows the obtained recoveries.

In the case of CREA, determinations were performed on agroup of nine persons of different ages (from 4 to 75 yearsold), three of which were pregnant women and one was adiabetic male (who had been treated with insulin for 20years and therefore needed very frequent control of renalfunction). Analyses were also undertaken at differenttimes of day (in the morning, at mid-day, or in the after-noon), with the object of establishing a relation betweenthem. The results are listed in Table 4.

For pregnant women a considerable drop in CREA levelswas observed because urinary excretion is greater duringpregnancy. The CREA excretion is not influenced by ageof subject or time of day, whether urine is collected in themorning (called sanguinus urine), afternoon, or night.

CREA control in diabetic persons is very importantbecause prolonged treatment with insulin can lead to dia-betic nephropathy. The CREA level of the diabetic manlies in the normal range, as was also confirmed bymedicalexamination.


Table 2. Statistical data for determination of CREA in terms of corrected peak area (CPA).

Standard calibration Urine calibration 1c) Urine calibration 2d)

Intercept (CPA) (l rI) 1.626102 l 6.136101 4.606103 l 3.706101 1.216104 l 9.536101

Slope (l rS) 3.006102 l 1.63 3.066102 l 5.39 3.146102 l 8.32

Regres. Coef. 0.9998 0.9991 0.9979

LOD (mg L – 1) a) 0.612 0.872 3.20

LOD (mg L – 1) b) 1.57 0.741 1.86

Linear range (mg L – 1) 0.5 – 80 0 – 12 (more endogenous CREA) 0 – 20 (more endogenous CREA)

a) Winefordner and Long criterion.b) Clayton criterion.c) Calibration obtained in the presence of woman’s urine (1 :25 dilution)d) Calibration obtained in the presence of a urine mixture (1 :25 dilution) of three healthy persons

Table 3. Recoveries obtained by each compound in urinesamples.

Initial con-centration(mg L – 1)

Found con-centrationa)

(mg L – 1)

%Recov-ery

(CPA)

Found con-centration b)

(mg L – 1)

%Recov-ery

(Height)

TRI 4.00 4.44 111.0 3.87 96.7

6.00 6.05 100.8 5.48 91.4

MTX 4.00 4.27 106.8 3.96 99.0

6.00 5.57 92.8 5.57 92.8

a) Analytical signal: CPA.b) Analytical signal: Height peak.


Acknowledgements

We are indebted to the DGIS of the Ministerio de Ciencia yTecnolog�a (Spain) for financial support BQU 2001-1190.The author M.J.R.G. wishes to thank the Ministerio deEducaci�n, Cultura y Deporte for financial support of thestay at the University of Castilla La Mancha (Spain).

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Table 4. Determination of creatinine in different urine sam-ples and at different times of the day.

Person(age in years)

Creatinine, mg L-1

Morning Midday Afternoon

Boy a 5 852.3 1349.9 427.9

Girl a 7 604.5 1866.6 953.2

Man A 40 2970.0 992.3 1089.9

Woman A 40 547.8 293.9 547.6

Woman A 70 1351.3 561.8 853.3

Pregnant(twomonths)

374.8 141.3 371.1

Pregnant(six months)

– 215.8 –

Pregnant(eight months)

– 235.2 –

Diabetic mana) – 1455.8 –

capillary electrophoretic determination of triamterene, methotrexate, and creatinine in human urine

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