Talanta 54 (2001) 1121–1133

Experimental design methodologies to optimise thespectrofluorimetric determination of Losartan and Valsartan

in human urine

E. Cagigal, L. Gonzalez, R.M. Alonso *, R.M. JimenezDpto. Quımica Analıtica, Facultad de Ciencias, Uni�ersidad del Paıs Vasco, Apdo. 644, E-48080 Bilbao, Spain

Received 29 November 2000; received in revised form 16 February 2001; accepted 27 February 2001

Abstract

A spectrofluorimetric method has been developed for the determination of two angiotensin II receptor antagonists(ARA II): Losartan and Valsartan. A fractional factorial design and a central composite design were used. The keyfactors considered in the optimization process were pH, temperature and emission slit width. Maximum fluorescentintensity was established as response for each experiment. The response surfaces confirmed the robustness of themethod. A clean-up procedure was used for urine samples that consisted of a solid-phase extraction using C8cartridges. The total analysis time was lower than 30 min. This method proved to be accurate (RE, 8%), precise (intra-and inter-day coefficients of variation were lower than 8% and sensitive enough (LOQ c.a. 0.5 �g ml−1) to be appliedto the determination of Losartan and Valsartan in urine samples. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: ARA II; Experimental design; Human urine; Spectrofluorimetry

www.elsevier.com/locate/talanta

1. Introduction

Angiotensin II receptor antagonists (ARA II)are a new family of antihypertensive drugs char-acterised by their high selectivity, specificity andlong-lasting effect on the renin–angiotensin sys-tem [1]. The therapy with these drugs offers anexcellent quality of life for hypertensive patientsdue to the absence of important side-effects [2].The World Health Organisation (WHO) Guideli-

nes (1999) have recommended the prescription ofthese drugs as a first-line treatment [3].

This family of antagonists are a chemicallyheterogenous group of compounds, but they haveas a common moiety the imidazole ring. Theexistence of several fluorescent functional groupsin the molecular structure of ARA II such asbiphenyl, tetrazole, imidazole and benzimidazole[4,5] makes possible the development of a spec-trofluorimetric method for the determination ofARA II in biological fluids such as urine andplasma.

The determination methods developed for theseantagonists are mainly chromatographic methods

* Corresponding author. Tel.: +34-4-6012686; fax: +34-4-4648500.

E-mail address: qapalror@lg.ehu.es (R.M. Alonso).

0039-9140/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S0 039 -9140 (01 )00379 -4

E. Cagigal et al. / Talanta 54 (2001) 1121–11331122

with photometric [6–12], fluorimetric [13–19] ormass-spectrometry detection [20–23]. In the liter-ature, there have been no studies dealing with theuse of spectrophotometry or spectrofluorimetryfor direct determination of these ARA II.

The compounds studied in this work wereLosartan, 2-butyl-4-chloro-1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl] imidazole-5-methanol monopot-assium salt, an heterocyclic biphenyltetrazole andValsartan, S-N-valeryl-N-{[2�-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl} valine, a non-heterocyclicderivative.

Both Losartan and Valsartan are affected byfirst-pass metabolism. The Losartan parent com-pound is rapidly converted (14% of initial dose) inits main active metabolite EXP 3174, via oxida-tion of the imidazole 5-hydroxymethyl group.Valsartan is slightly transformed into an inactivemetabolite, valeryl-4-hydroxyvalsartan.

With the development of a fluorescent methodfor the determination of Losartan and Valsartan,the influence of certain parameters such as pH,temperature and chemical surroundings on theirintrinsic fluorescence needs to be considered inorder to obtain the maximum sensitivity to allowtheir determination. Renal excretion is a minorpathway for the elimination of Losartan and Val-sartan. Thus, the small amounts of the dose ex-creted in the urine as unchanged parentcompound (3–5% for Losartan and 7–13% forValsartan) led us to develop a fluorescent methodwith sufficient sensitivity so as to determine themin urine at the expected concentration levels (�gml−1 levels). Experimental design was used tooptimise the measurement conditions for the spec-trofluorimetric method.

The traditional step-by-step approach, althoughwidely used, involves a large number of indepen-dent runs and does not enable us to establish themultiple interacting parameters. Experimental de-sign allows a large number of factors to bescreened simultaneously to determine which ofthem has a significant effect on the fluorescentsignal. A response model that shows the relation-ship of each factor towards the response as well asthe interactions between factors is obtained, andthe factors can be optimized to give the maximumresponse (fluorescent signal) with a relatively low

number of experiments. Factorial and central-composite (full factorial design plus six starpoints) designs were mainly used for approachingthe response surface with regard to the maximumfluorescent signal [24,25]. The multivariate optimi-sation was carried out sequentially. First of all,screening designs (i.e. factorial design) were usedto determine (i) which parameters had an effecton studied response, (ii) which parameters had aninteraction effect, (iii) whether these effects werepositive or negative and (iv) whether they weresignificant. Afterwards, optimisation designs (i.e.central composite design) had to be carried out, atleast at three levels for each significant factor.These designs are used to compute a non-linearcorrelation between significant factors and re-sponse, the so-called response surface model. Thenon-linear model was succesfully used to deter-mine optima (maxima or minima), steepest as-cents, and so on.

2. Experimental

2.1. Apparatus

The spectrofluorimeter used was a ShimadzuRF-540 (Kyoto, Japan) equipped with a Xenonshort arc lamp (Ushio, Japan) controlled by asignal integrator Shimazdu DR-3 (Kyoto, Japan).All operations with the fluorimeter were con-trolled by means of the program FLUORIM [26].Quartz cuvettes with a pathlength of 1.0 cm havebeen used. Fluorescent measurements were madeat constant temperature using a thermostatic bathHaake D8 (Karlsruhe, Germany).

pH of solutions was measured with a Radiome-ter Copenhagen PHM84 pH-meter (Bargsvaerd,Denmark) using a Crison glass-combined elec-trode model 5209 (Barcelona, Spain) with a refer-ence system Ag/AgCl and electrolyte KCl 3M sat.AgCl.

Urine samples were centrifuged at constanttemperature (4°C) in a Eppendorf model 5804Rcentrifuge (Hamburg, Germany), prior to thesolid-extraction step. The extraction cartridgeswere placed in a vacuum manifold from Supelco(Bellefonte, PA) to perform the solid-phase ex-

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traction step. The extracted urine samples wereevaporated to dryness under a nitrogen streamusing a Zymack Turbovap LV Evaporator(Barcelona, Spain).

2.2. Reagents and chemicals

The angiotensin II receptor antagonists werekindly supplied by the manufacturers: Losartan(Merck, NJ) and Valsartan (Novartis Pharma,Basel, Switzerland).

Acetonitrile and methanol were Lab-ScanHPLC grade (Dublin). All reagents used wereMerck p.a. (Darmstadt, Germany). The waterused in all the experiments was obtained fromMilli-RO and Milli-Q systems (Millipore, Bed-ford, MA).

Solid-phase extraction was performed usingVarian Bond Elut C8 columns non-endcapped 1ml/100 mg (Barcelona, Spain).

The buffer solutions were H3PO4/KH2PO4 (pH2), CH3COOH/CH3COONa (pH 4), KH2PO4/K2HPO4 (pH 7) and H3BO3/NaH2BO3 (pH 9). Inorder to achieve the desired pH value, volumes of1 M HCl and KOH or NaOH solutions wereadded.

Stock solutions of these antagonists (1000 �gml−1) were prepared in both methanol and aceto-nitrile and were stored at 4°C in the dark. Work-ing solutions were prepared daily by dilution fromstock solution.

2.3. Experimental design optimisation solutions

Solutions of Losartan and Valsartan (1 or 5 �gml−1) were prepared daily from the stock solu-tions of 1000 �g ml−1 in the desired conditionsfor the whole range of experiments. Their relativefluorescent intensity were measured on the spec-trofluorimeter at the predetermined conditions foreach experimental design. The optimised excita-tion and emission wavelength for both com-pounds were used.

2.4. Urine samples collection

Drug-free urine was obtained from severalhealthy female and male volunteers aged 20–50years old.

Urine samples of Losartan and Valsartan wereobtained from hypertensive patients under treat-ment with Diovan and Vals (Valsartan — 80 mg)and Cozaar (Losartan — 50 mg). All sampleswere collected for 24 h at different time intervals(0–2, 2–4, 4–8 and 8–24 h after their daily oraldose).

All urine samples were collected inpolypropilene tubes and frozen at −20°C untilanalysis.

2.5. Solid-phase extraction procedure for thetreatment of urine samples

Once thawed and vortex-mixed, 5 ml of humanurine were acidified with 2.5 ml of 0.1 M phos-phate buffer, pH 2. The mixture was shaken andcentrifuged for 5 min at 5000 rpm (16.1×g).

The Bond Elut C8 cartridge (1 ml/100 mg) wasconditioned with 2 ml of methanol and 1 ml 0.1M phosphate buffer, pH 2. The cartridge was notallowed to dry before the application of 5 ml ofthe sample previously acidified. The application ofthe sample was carried out in a very low vacuum(P�5 mmHg), allowing the sample to drawslowly through the cartridge. The column wasthus washed with 5 ml volume of a mixture ofmethanol–0.1 M phosphate buffer at pH 2 (40:60,v/v) and dried in a full vacuum (P�20 mm Hg)for 20 min. The analytes of interest were elutedwith 5 ml of methanol. To prevent adsorption ofthe drugs on the tube walls, 0.2 ml of 10% (v/v)ethylenglycol in methanol was added to the eluate[18].

Afterwards, the eluate was evaporated to dry-ness at 40°C under a gentle stream of nitrogen.The remaining residue was dissolved in 3 ml of amixture of 0.05 M phosphate buffer at pH 2 and5% of acetonitrile (optimal conditions extractedfrom the experimental design). The relativefluorescent intensity of this solution was measuredin the spectrofluorimeter under the optimised con-ditions: T= (20�0.5)°C, excitation and emissionslit width=5 nm, Losartan (�exc=247 nm, �em=387 nm) and Valsartan (�exc=259 nm, �em=399nm).

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2.6. Assay �alidation

The validation was performed in accordance tothe summary report of the conference on ‘‘Analyt-ical Methods Validation: Bioavailabilty, Bioequiv-alence and Pharmacokinetics Studies’’, which hasprovided guidelines for pharmacokinetics studiesin humans and animals [27].

Calibration data were generated by spikingblank urine samples with the appropriate volumeof stock solution of these ARA II yielding con-centrations of 0.5, 1.0,1.5, 2, 2.5, 3.0, 4.0 and 5.0�g ml−1. Calibration curves resulted from therepresentation of the relative fluorescent intensity(IF,rel.) versus concentration and were fitted to thelinear regression y=a+bx.

The limit of quantification (LOQ) was definedas the lowest concentration of the calibrationcurve that can be measured with accuracy andprecision enough, and so a maximum coefficientof variation of �20% is allowed for this point.The limit of detection was defined as the concen-tration that differs easily from background levels.As spectrofluorimetry does not allow us to havethe noise value, the limit of detection (LOD) isdefined with the IUPAC criteria and calculated asthe analyte concentration giving rise to a signalvalue equal to the blank signal plus threefold theregresion standard deviation [28].

The intra- and inter-day precision and accuracywere determined using six samples of differentconcentration levels, at the limit of quantification,as well as the medium and highest concentrations(0.5, 2.5 and 5.0 �g ml−1 were the concentrationlevels for precision, and 1.0, 2.0, 3.0 and 4.0 �gml−1 were the concentration levels for accuracy).The inter-day precision and accuracy were estab-lished over a period of 1 month. Precision werecharacterised by the relative standard deviation(RSD, %), whereas the accuracy was expressed asa percentage error of the nominal versus foundconcentration (RE, %). For both, the limits ofacceptable variance were set at 15% except for thelimit of quantification concentration level havinga limit of 20%.

The specificity of the assay was established withsix independent sources of the same matrix.

3. Results and discussion

3.1. Factors affecting fluorescent intensity.Experimental design

Chemical and instrumental variables (excitationand emission wavelength, percentage and natureof organic solvent, pH, temperature, excitationand emission slit width and buffer concentration)were studied to obtain the best measurement con-ditions and maximum relative fluorescent inten-sity. The optimisation was developed byexperimental design, including two kinds of de-signs: the fractional factorial design to evaluatewhich of the variables were significant factors andthe central composite design, to obtain the re-sponse surface from which the optimal factorsthat give a maximum relative fluorescent responsecan be deduced.

Some studies were carried out prior to theoptimisation by experimental design. Thus, excita-tion and emission wavelengths were optimisedsearching the maximum relative fluorescent inten-sity by means of the recorded spectra. The excita-tion and emission optimal wavelengths were 247and 387 nm for Losartan and 259 and 399 nm forValsartan. Knowing the acidity constants forthese antagonists (pKa Losartan 3.15, pKa Valsar-tan 4.90) [29], a general study of pH was made soas to verify if both, acidic and basic, species werefluorescent. As acid species of Losartan and Val-sartan proved to be fluorescent, pH values lowerthan 5.0 were only assayed. Several solvents weretested for the fluorescent measurements (water,acetonitrile and methanol), and the maximum rel-ative fluorescent intensity was obtained with ace-tonitrile–water mixtures, so they were the startpoint for the optimisation.

3.1.1. Fractional factorial designThe variables considered in the fractional facto-

rial design for Losartan and Valsartan were pH,buffer concentration, percentage of acetonitrile,temperature and excitation and emission slitwidth. The aim of this design was to evaluatewhich of the variables had an influence on therelative fluorescent signal and which did not. Ow-ing to the high number of experiments for a full

E. Cagigal et al. / Talanta 54 (2001) 1121–1133 1125

two-level factorial design (26), we decided first torun a 26–3 factorial design in order to perform alower number of experiments, associating threefactors to two second-order interacting columnsand to the third-order interaction column of thematrix. Thus, the temperature and the excitationand emission slit width were defined as a combina-tion of the other three variables (pH, buffer concen-tration and percentage of acetonitrile) as shown inthe following equations:

Temperature�pH×buffer concentration

×percentage of acetonitrile (1)

Excitation slit width�pH

×buffer concentration(2)

Emission slit width�pH

×percentage of acetonitrile.

(3)

The temperature and the excitation and emissionslit width were chosen for the fractionality sincethey were a priori the variables that least interactedwith the other three (pH, buffer concentration andpercentage of acetonitrile). A three-level fractionalfactorial design involving eight runs plus threecentral points was chosen. The design matrix andthe results for the experiments are given in Table1 for both ARA II.

Some of the initially proposed experiments hadto be rejected (experiment no. 1) or modified(experiment nos 8, 9, 10 and 11) because they hadsuch different relative fluorescent signals that couldnot be measured with the same sensitivity parame-ter value, and in order to establish reliable conclu-sions, all the runs must be of equal sensitivity.

The data analysis of the results for both ARA IIgiven in Table 1 was performed using the non-linearregression analysis program NLREG [30]. Re-sponse surfaces (Y) were taken as a function of theconsidered variables (xi) using polynomials of dif-ferent degrees depending on the experimental de-sign followed. The general polynomial function is

Y=�0+�i

�i xi+�ij

�ij xi xj (4)

where Y is the relative fluorescent intensity, xi

denotes the variables considered for the optimisa-tion of the fluorescent response, and �i and �ij arethe parameters to be calculated. The estimation ofthe parameters (�i and �ij) was achieved by theminimisation of the square sum of errors (U) asgiven by the equation

U=�n

i

(Yexp−Ycalc)2 (5)

where n is the number of experiments, Yexp is therelative fluorescent signal measured, and Ycalc is theresponse calculated by the program based on theregresion model considered.

The analysis of the output was based on theevaluation of the prob(t) parameter associated witheach �i parameter, since prob(t) indicates theprobability of �i being zero. Those parameterswhose probability of being zero was greater than10%, i.e. prob(t)�0.1, were systematicallyeliminated.

The most general function allowed for the frac-tional experimental design of Losartan and Valsar-tan taking into account the combination ofparameters assumed to fraction the full factorialdesign is

Y=�0+�1x1 +�2x2+�3x3+�4x4+�5x5

+�6x6+�12x1x2 (6)

where Y is the relative fluorescent intensity, x1 is thepH value, x2 is the buffer concentration, x3 is thepercentage of acetonitrile, x4 is the temperature, x5

is the excitation slit width and x6 is the emission slitwidth. The rest of the parameters were not consid-ered because of their correspondence with somevariables by means of linear combinations.

Different regressions were assayed, correspond-ing with the most general function allowed for thisdesign. The choice criterion was the best fit to theregression (percentage of variance explained). Thefinal equations obtained are Eq. (7) for Losartanand Eq. (8) for Valsartan.

Y= −1.40x1+0.22x4+0.35x6 (7)

Y=51.41−9.98x1−229.31x2−0.54x4+2.12x6

+74.55x1x2 (8)

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Table 1Design matrix and relative fluorescent intensity values in the fractional factorial design for Losartan and Valsartan

TemperaturepHRun no. Excitation slit widthAcetonitrileBuffer concentration Emission slit width (nm) IF,rel.

(nm)(°C)(M) percentage

Losartan Valsartan

2 0.01 0.2405 22 3.5 0.0520 14.5 27.63 1.5 0.25 5 40 2

2 2.9 16.820204 50.253.540 20 2 7.0 12.61.55 0.05 20

20 4.0 49.72200.05 206 3.521.5 2 0.1 0.30.25 20 207

40 10 10 9.7 13.98 3.5 0.25 209 52.5 6.7 14.70.15 12.5 30 5

5 6.6 15.4510 12.52.5 0.15 3011 52.5 6.7 16.50.15 12.5 30 5

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It could be concluded that only the pH value,temperature and emission slit width affect therelative fluorescent intensity of Losartan (Eq. (7)),whereas in the case of Valsartan, another parame-ter has to be considered, the buffer concentration(Eq. (8)).

From the values obtained for the parameters inthe fractional factorial design, it could be estab-lished that, on the one hand, the relative fluorescentresponse for both ARA II was negatively affectedby the increase of pH values (�1�0) as the previousqualitative studies confirmed, and on the otherhand, it was positively affected by the increase ofemission slit width (�6�0) as was expected accord-ing to fluorescence theory. The temperature inter-action was not thoroughly explained by means ofthis fractional factorial design, for the Valsartanrelative fluorescent signal decreases as temperaturevalues are increased, as fluorescent theory predictsdue to the loss of energy by non-radiant mecha-nisms, but in Losartan’s case, this relationship isnot fulfilled. Besides, the increase in buffer concen-tration has a negative effect on the relative fluores-cent response (�2�0) for Valsartan.

3.1.2. Central composite designAs factors affecting the fluorescent behaviour of

Losartan and Valsartan obtained from the frac-tional factorial design were different, the basis ofcentral composite design was also different for bothARA II.

3.1.2.1. Losartan. From the factorial design, threeof the variables could be excluded because they donot seem to have an influence on the fluorescentresponse. A central composite design was builtusing the other three variables: pH, temperatureand emission slit width. A two-level factorial designplus star orthogonal composite design (−�3 and+�3) involving 14 runs plus three central pointswas chosen. Table 2 gives the design matrix for thisexperiment and the relative fluorescent signal ob-tained in each run.

As it can be seen, there is not the same propor-tional relationship between the star points levels forpH (−�3 and +�3, correspond to −0.5 and+0.5), temperature (−�3 and +�3, correspondto −7 and +7) and emission slit width (−�3 and+�3, correspond to −3 and +10). For this lastparameter, the spectrofluorimeter model does notallow the choice of any value for slit width, so theoperator is forced to choose among certain criticalvalues.

Three rejected variables from the fractional fac-torial design were fixed to carry out the central

Table 2Design matrix and relative fluorescent intensity values in the central composite design for Losartan

IF,relRun no. Emission slit width (nm)pH Temperature (°C)

15.31 5202.53.5 202 5 4.3

3 2.5 40 5 8.52.35404 3.5

2.5 205 20 217.26 3.5 20 20 64.0

407 202.5 134.08 3.5 40 20 35.6

309 104 8.310 60.310302

1047 16.631113 10 48.812 3

3 3013 30 183.414 3 30 2 0.6

29.4103015 33 3016 10 29.8317 30 10 29.8

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composite design. The buffer concentration wasfixed at the lowest level, 0.05 M. The percentage ofacetonitrile was 5%, enough to dissolve the analyteat the working concentration. The chosen value forthe excitation slit width was 5 nm, an intermediatevalue that gives an acceptable sensitivity.

The most general function for central compositedesign for Losartan was

Y=�0+�1x1+�2x2+�3x3+�12x1x2+�13x1x3

+�23x2x3+�11x12+�22x2

2+�33x32

+�123x1x2x3 (9)

where Y is the relative fluorescent intensity, x1 is thepH value, x2 is the temperature and x3 is theemission slit width.

The data collected in Table 2 were analysed byNLREG following the methodology mentionedabove. From the output data, the following equa-tion can be deduced

Y= (−5.7�1.6)x2+ (31.9�3.8)x3

+ (1.5�0.5)x1x2+ (−8.2�1.3)x1x3 (10)

which explains 95.4% of the total variance, whichrepresents an excellent adjustment of the experi-mental values to this theoretical model. The relativefluorescent intensity decreases as the temperatureincreases, as theory assumes due to the losses ofenergy by non-radiant emission processes. Theemission slit width has a great positive effect on thefluorescent response. Besides, there are interactionparameters that count for pH and temperature andpH and emission slit width.

Three-dimensional representations keeping oneof the variables at the central point value arepresented in Fig. 1 for Losartan. From these plots,optimal conditions for the determination of Losar-tan can be deduced and the function shown in Eq.(10) can be derived. The obtained response showsthe absence of critical points, maximum or mini-mum, as fluorescent responses represented by in-creasing plans. Therefore, the optimal conditionswere those far enough from the absolute minimumobserved in Fig. 1(a). Instrumental variables madeus introduce some constraints in the method devel-oped, so we had to choose a non-maximal emissionslit width (5 nm) in order to work at the samesensibility levels in all cases.

3.1.2.2. Valsartan. For this ARA II, only two of thetotal six variables could be excluded because theydid not seem to have an influence on the fluorescentresponse. A central composite design with fourvariables, pH, buffer concentration, temperatureand emission slit width, was presented. This designinvolved 24 runs plus three central points. At firstsight, they can seem an excessive number of exper-iments, but in fact, several factors, such as temper-ature and emission slit width, are instrumentalvariables and so easily controlled. Table 3 gives thedesign matrix for these experiments and the fluores-cent response obtained.

The two rejected variables in the fractionalfactorial design were fixed in this central compositedesign. The percentage of acetonitrile was 5%, andthe excitation slit width was 5 nm, for reasonsexplained above.

The most general function for central compositedesign for Valsartan was

Y=�0+�1x1 + �2x2+�3x3+�4x4+�12x1x2

+�13x1x3+�14x1x4+�23x2x3+�24x2x4

+�34x3x4+�11x12+�22x2

2+�33x32+�44x4

2

+�123x1x2x3 +�124x1x2x4+�134x1x3x4

+�234x2x3x4+�1234x1x2x3x4 (11)

where Y is the relative fluorescent intensity, x1 is thepH value, x2 is the buffer concentration, x3 is thetemperature, and x4 is the emission slit width.

Tha data analysed by NLREG led us to thefollowing equation

Y= (0.18�0.02)x1x4+ (−0.009�0.001)x3x4

+ (0.036�0.002)x42 (12)

which explains 98.9% of total variance. Emissionslit width has an important favourable effect overthe fluorescent response, quadratic term and inter-actions with pH and temperature. However, thebuffer concentration (x2) seemed to have no rele-vant interaction, as in the case of Losartan. Thus,for both drugs, the main factors for fluorescentresponse are the same: pH, temperature and emis-sion slit width.

Three-dimensional plots for Valsartan are pre-sented in Fig. 2 keeping one of the variables at the

E. Cagigal et al. / Talanta 54 (2001) 1121–1133 1129

Fig. 1. Response surface for the central levels of (a) emission slit width, (b) temperature and (c) pH for the central composite designof Losartan.

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Table 3Design matrix and relative fluorescent intensity values in the central composite design for Valsartan

Buffer concentration (M) Temperature (°C) Emission slit width (nm)Run no. IF,relpH

0.05 201 52 1.32 3 0.05 20 5 1.7

23 0.15 20 5 1.20.15 203 54 1.6

25 0.05 40 5 136 0.05 40 5 1.2

0.15 402 57 0.938 0.15 40 5 1.3

0.05 202 209 19.9310 0.05 20 20 22.2211 0.15 20 20 19.1

0.15 203 2012 22.70.05 4013 202 15.60.05 403 2014 20.8

215 0.15 40 20 14.1316 0.15 40 20 17.2

0.1 303.5 1017 6.11.518 0.1 30 10 4.6

0.19 302.5 1019 5.10.01 3020 102.5 6.20.1 472.5 1021 4.3

2.522 0.1 13 10 7.10.1 302.5 3023 36.1

2.524 0.1 30 2 0.10.125 302.5 10 5.60.1 30 10 5.626 2.5

2.5 0.1 30 10 5.927

central point value. The second-grade polynomialfunction obtained for fluorescent response (Eq.(12)) shows the presence of a critical point. Opti-mal conditions for the determination of Valsartancan be deduced, deriving the function showed inthis equation. As well as in Losartan’s case, thevalue chosen for emission slit width (5 nm) didnot give a maximum fluorescent response, butinstrumental constraints were superimposed with-out discarding a good value of response for theother factors. These conditions are shown as wellas Losartan’s conditions in Table 4.

3.2. Determination of Losartan and Valsartan inhuman urine

Once optimised the variables that affect therelative fluorescent signal, the spectrofluorimetric

method was applied to the determination ofLosartan and Valsartan in human urine.

The solid-phase extraction procedure was previ-ously optimised in our laboratory for the screen-ing of several ARA II in human urine by

Table 4Experimental conditions for the spectrofluorimetric determina-tion of Losartan and Valsartan

Losartan Valsartan

pH 2.0 2.00.05 mol l−1Buffer concentration 0.05 mol l−1

Acetonitrile percentage 5%5%(20�0.5)°CTemperature (20�0.5)°C

Excitation slit width 5 nm 5 nm5 nmEmission slit width 5 nm

247 nm 259 nm�ex

387 nm�em 399 nm

E. Cagigal et al. / Talanta 54 (2001) 1121–1133 1131

Fig. 2. Response surface for the central levels of (a) emission slit width, (b) temperature and (c) pH for the central composite designof Valsartan.

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Table 5Validation assay of spectrofluorimetric determination ofLosartan and Valsartan in spiked human urine

Losartan Valsartan

0.5–5Linear concentration 0.5–5range (�g ml−1)

Precision (% RSD)a

Intra-day �6.7 �7.7�6.0�7.1Inter-day−5.66 to 7.25Accuracy (% RE)b −3.69 to 8.32

0.40.3L.O.D. (�g ml−1)L.O.Q. (�g ml−1) 0.5 0.5

a Precision values were studied at four concentration levels:0.5, 2.5 and 5 �g ml−1. Data presented in this table are themaximum values found.

b Accuracy values were studied at four concentration levels:1, 2, 3 and 4 �g ml−1. Data presented in this table aresummarised for all concentration values.

inter-day value found was within 8% of the nomi-nal value for Losartan and Valsartan. The accu-racy was consequently at least 92% in both cases.The intra- and inter-day precision did not exceedthe 8% coefficient of variation (CV).

The spectrofluorimetric method was applied tothe determination of Losartan and Valsartan inurine samples obtained from hypertensive pa-tients. In Table 6, the concentrations found forboth ARA II in urine of hypertensive patientsafter the intake of a therapeutic dose of the drugwere given. The amount excreted over the wholeperiod (up to 24 h) corresponded to the valuesreported in literature for Losartan (�5% inal-tered-dose 50 mg) and Valsartan (�7% inaltered-dose 80 mg) [31,32]. Besides, the results for urinesamples from hypertensive patients under treat-ment with Valsartan were also corroborated byHPLC-UV, following a screening method devel-oped for some angiotensin II receptor antagonists[12].

4. Conclusions

Fractional factorial designs have their basis onsystematic multivariate optimization schemes in-stead of univariate procedures. Thus, they offeran additional advantage compared to univariateprocedures and allow distinctions between effectscaused by a single variable or by interacting vari-ables to be made. Besides, this fact, together withprevious knowledge of the technique used, allowsthe main effects to be focused on and allows onlya fraction of the complete factorial experiment tobe run.

HPLC-UV [12], and later, it was successfully ap-plied for the individual spectrofluorimetric deter-mination of Losartan and Valsartan. Theoutstanding statistical data of these determina-tions are shown in Table 5. The calibration curveswere linear over the range of 0.5–5 �g ml−1 withmean correlation coefficients of 0.997.

The method proved to be sufficiently sensitivefor the determination of these ARA II in urine,according to the excretion percentages and thetherapeutical doses for Losartan and Valsartan.The limit of quantitation (LOQ) for both ARA IIwas near 0.5 �g ml−1.

It was also sufficiently precise and accurate, tomeet the requirements of ‘‘Analytical MethodsValidation: Bioavailability, Bioequivalence andPharmacokinetic Studies’’ [27]. The intra- and

Table 6Concentrations (�g ml−1) obtained in urine from hypertensive patients under treatment with Cozaar (Losartan 50 mg) and Vals andDiovan (Valsartan 80 mg)a

0–2 h 8–24 h2–4 hLosartan 4–8 h

2.6�0.43.27�0.34 1.1�0.41.1�0.5Patient 1 (Cozaar)Patient 2 (Cozaar) 2.0�0.8 7.1�0.7 7.3�0.8 5.0�0.8

0–2 h 2–4 hValsartan 4–8 h 8–24 h1.8�0.3 5.5�0.28.5�0.23.0�0.4Patient 3 (Diovan)�LOQ 0.5�0.4Patient 4 (Vals) 1.6�0.3 �LOQ

a Error is expressed as � t · s, where the t parameter corresponds to a confidence interval of 95%.

E. Cagigal et al. / Talanta 54 (2001) 1121–1133 1133

By using experimental design, it was possible toobtain a fast and easy way to optimise the exper-imental conditions for the spectrofluorimetric de-termination of Losartan and Valsartan in humanurine. Similar conclusions were deduced for bothdrugs, so the relevant parameters for their spec-trofluorimetric determination were the same (pH,temperature and emission slit width), and theexperimental conditions fixed for both ARA IIwere identical. The response surfaces explainedaround 95% of the total variance.

The spectrofluorimetric method has been shownto be sensitive and precise for the determinationof Losartan and Valsartan. The assay validationwas adequate in terms of linearity, limit of quanti-tation, precision and accuracy regarding the gen-erally accepted guidelines for assay validation inthe bioanalytical laboratory.

The analysis per sample takes less than 30 min,including a solid-phase extraction step. Thismethod can be successfully applied to real urinesamples from hypertensive patients, under treat-ment with Losartan and Valsartan. The resultsobtained were also corroborated by a complemen-tary method (HPLC-UV).

Acknowledgements

The authors thank the Interministerial Commi-sion of Science and Technology (Project PB98-0231) for financial support and Merck Sharp andDohme and Novartis Pharma for their kind sup-ply of these ARA II. L. Gonzalez thanks theBasque Country Government for a FPI grant.They also thank N. Etxebarria for his skilfulassistance.

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