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Anal Bioanal Chem (2008) 391:11191127 DOI 10.



Determination of anticarcinogenic and rescue therapy drugs in urine by photoinduced spectrofluorimetry using multivariate calibration: comparison of several second-order methods
M. I. Rodrguez Cceres & I. Durn Mers & N. E. Ornelas Soto & P. L. Lpez de Alba & L. Lpez Martinez

Received: 20 December 2007 / Accepted: 10 March 2008 / Published online: 18 April 2008 # Springer-Verlag 2008

Abstract This paper shows the potential of excitation emission fluorescence spectroscopy and several secondorder methods, such as parallel factor analysis (PARAFAC), multiway partial least-squares (N-PLS) or bilinear leastsquares (BLLS), as a multicalibration technique for the analysis of leucovorin (LV) and irinotecan (CPT-11). Although CPT-11 presents native fluorescence, leucovorin has little native fluorescence; however, under irradiation with short-wavelength UV light in the presence of traces of hydrogen peroxide, leucovorin was converted into a highly fluorescent compound. This reaction has been used for the sensitive and selective determination of both compounds. The convenience of analysing the total luminescence spectrum information when using multivariate calibration methods on fluorescence data is demonstrated. Direct determination of mixtures of both drugs in urine was accomplished on the basis of excitationemission matrices (EEMs) and the three-way multivariate methods. Keywords Irinotecan . Leucovorin . Photoinduced fluorescence . Multivariate calibration . N-PLS/RBL . PARAFAC . BLLS

Introduction Irinotecan (CPT-11) is a water-soluble camptothecin derivative currently used for the treatment of colorectal tumours. It is a selective inhibitor of topoisomerase I which is an enzyme capable of replication and transcription of DNA. CPT-11 is hydrolysed into the active metabolite SN-38, which is 1001,000 times more potent in damaging DNA by inhibition of nuclear topoisomerase I [1]. CPT-11 is used in several combinations in the treatment of metastatic colorectal cancer [2]. In April 2000, it was approved in association with leucovorin (LV) and fluorouracil (5FU) as first line in the treatment of metastatic colorectal cancer [3]. The initial doses for the Saltz regimen [3] are 125 mg m2 of CPT-11, 500 mg m2 of 5FU and 20 mg m2 of LV. All drugs are administered intravenously. As with other camptothecin analogues, CPT-11 contains an extremely labile -hydroxy lactone ring (Scheme 1), which can undergo a reversible, pH-dependent hydrolysis during which the closed lactone form is converted to an open carboxylate form. It has been shown that the lactone ring is absolutely necessary for biological activity [1]. Leucovorin (LV or folinic acid) (Scheme 1) is used as a rescue agent and also as a modulator of the pyrimidine antagonist 5-fluorouracil. The biological activity of LV is apparently restricted to the (6S)-LV diastereoisomer, which is rapidly converted to its active metabolite (6S)-5-methyltetrahydrofolate [4]. A survey of the literature reveals that no fluorescence methods for the determination of CPT-11 have been published to date; however, there are only a few for its parent compound, camptothecin [59]. On the other hand, most of the methods for the analysis are chromatographic.

M. I. Rodrguez Cceres (*) : I. Durn Mers Department of Analytical Chemistry, University of Extremadura, 06071 Badajoz, Spain e-mail: N. E. Ornelas Soto : P. L. Lpez de Alba : L. Lpez Martinez Instituto de Investigaciones Cientficas, University of Guanajuato, Guanajuato, Gto. 36000, Mexico

1120 Scheme 1 Structures of CPT-11 and leucovorin


Anal Bioanal Chem (2008) 391:11191127





H 2N N








Among them, the majority of the methods used fluorescence detection [1012], whereas only a few used photometric [13] or mass detection [14, 15]. Our research group, in a recent study, has established the fluorescence characteristics of CPT-11 and has developed several fluorescence methods in the presence of oxidants (e.g. cerium and iodine) and metal ions (e.g. Fe(II)) [16]. Leucovorin has been analysed preferentially by use of separation techniques, such as HPLC and CE [1720]; however, several photometric and fluorimetric methods for its determination appear in the literature. Thus, the kinetic fluorimetric study of the oxidation reaction of LV with potassium permanganate has been performed [21] in alkaline medium. The proposed method was applied to human urine samples. Also, using the spectrophotometric data, the mixture of triamterene and leucovorin has been resolved by use of PLS-1 without pretreatment of the sample [22]. Moreover, the mixture of metotrexate and leucovorin has been resolved with the aid of chemometric methods, specifically PLS-1 and net analyte signal [23, 24]. In recent years, multiway chemometric techniques have been introduced for the analysis of complex samples. The advantage of using data involving high-dimensional structured information is the higher stability towards interferents and matrix effects compared with first-order methodologies. There are several algorithms for analysing second-order

data, such as parallel factor analysis (PARAFAC), bilinear least-squares (BLLS) and multiway partial least-squares (N-PLS). PARAFAC is a decomposition method, which conceptually can be compared to bilinear PCA. The decomposition is made into triads or trilinear components, and each component consists of one score vector and two loading vectors. An advantage of PARAFAC is the uniqueness of the solution. This means that the true underlying spectra will be found if the right number of components is used and the signal-to-noise ratio is appropriate [25]. The decomposition of excitationemission matrices (EEMs) data into three-dimensional arrays, by PARAFAC, exploits the second-order advantage [26]. This advantage allows direct extraction of the spectral profiles as well as the relative concentrations of individual sample components [27]. BLLS is a technique recently implemented based on a direct least-squares procedure [28]. It starts with a calibration step in which approximations to pure analyte matrices at unit concentration are found by direct least-squares. To estimate the pure analyte matrices, the calibration data matrices are first vectorised and grouped. A procedure analogous to classical least-squares is then performed [29]. Concentration estimation is usually carried out through the least-squares predictor [28]. A promising alternative is NPLS, which is a genuine multiway method. But these

Anal Bioanal Chem (2008) 391:11191127


algorithms do no present the second-order advantage, unless they are coupled with a procedure known as residual bilinearisation (RBL) [3032]. Recently, two reviews about the algorithms and applications of second- and third-order multivariate calibration have been published [33, 34]. Several of those chemometric tools, as is the case of trilinear least-squares (TLLS), unfolded-PLS coupled with residual trilinearisation (U-PLS/RTL) and PARAFAC, have been used with fluorescence data for the determination of leucovorin [35, 36]. In this paper, an analytical methodology has been compared with different three-way multivariate calibration methods for the resolution of the mixture of LV and CPT-11 in urine samples. Excitationemission matrices (EEMs) were recorded in order to apply second-order calibration methods. Although the results obtained with the three proposed methods are similar, PARAFAC is demonstrated to perform better than N-PLS/RBL or BLLS.

Stock standard solution of CPT-11 was prepared by dissolving 0.0100 g of this compound in ethanol and stock standard solution of LV was prepared by dissolving 0.0100 g in ultra-pure water and both solutions were kept at 4 C in the dark. Working CPT-11 and LV solutions of different concentrations were prepared by dilution of stock solution with ultrapure water. Buffer solution (pH 4.0, Ct = 0.5 M) was prepared from acetic acid and sodium hydroxide. Fresh urine samples were collected from different healthy people (2532 years old). General procedure for the individual fluorescent determination of CPT-11 and LV via previous photoirradiation A suitable aliquot containing 15 g of CPT-11 or 540 g of LV was transferred into a 10-mL calibrated flask, 0.2 mL of 0.5 M solution of acetate buffer, pH 4.0, and 0.7 mL of 0.25% hydrogen peroxide were added, and the mixture was dilute to the mark with deionised water. An aliquot of these solutions was transferred to the cell and the cell was placed in an optical bench 30 cm from the mercury lamp for 8 min. The solution was magnetically stirred during the UV irradiation. The fluorescent intensity was measured at 440 nm with excitation at 270 nm for the determination of leucovorin and at 426 nm with excitation at 254 nm for the determination of CPT-11. The temperature was maintained constant at 20 C. Procedure for the simultaneous determination of CPT-11 and LV in human urine by second-order calibration methods A 100-L aliquot of urine sample spiked with appropriate amounts of CPT-11 and LV (in the range that can be found in urine) was placed in a 10-mL volumetric flask. Acetate buffer (0.2 mL, 0.5 M, pH 4.0) and 0.7 mL of 0.25% hydrogen peroxide were added and the mixture was diluted to the mark with deionised water. Then, 3 mL of these solutions was transferred into the quartz cell and the cell was placed in an optical bench 30 cm from the mercury lamp for 12 min. EEMs were recorded in the range 245 385 nm, at 5-nm intervals for excitation, and 390485 nm at 5-nm intervals for emission.

Experimental Instrumentation and software Fluorescence spectral measurements were performed on a Varian Cary Eclipse fluorescence spectrophotometer, equipped with two CzernyTurner monochromators and a xenon flash lamp, and connected to a PC via an IEEE 488 (GPIB) serial interface. The Cary Eclipse software was used for data acquisition. Fluorescence measurements were recorded in a 10-mm quartz cell at 20 C, by use of a thermostatically controlled cell holder and a Selecta Model 382 thermostatically controlled water bath. An Osram 200-W HBO high-pressure mercury lamp with an Oriel Model 8500 power supply was used for the photoreaction. The photochemical setup includes a light box consisting of an electric fan, a mercury lamp, and a quartz lens. A 10-mm quartz cell was used in the irradiation process. The cell was placed in an optical bench 30 cm from the mercury lamp. The solutions were magnetically stirred during the UV irradiation. All calculations were done using MatLab 5.3, using different routines and the graphical interface MVC2, developed by A. C. Olivieri [36], which allows secondorder calibration. Reagents

Results and discussion All experiments were performed with analytical reagent grade chemicals. CPT-11 was obtained from Sigma-Aldrich and LV was obtained from Acros Organics. Hydrogen peroxide, sodium hydroxide, acetic acid and hydrochloric acid were obtained from Panreac. Spectral characteristics of LV LV is a weakly fluorescent compound, but when a solution of LV is irradiated it generates a strongly fluorescent


Anal Bioanal Chem (2008) 391:11191127

photoproduct. The combination of UV radiation and hydrogen peroxide has been applied successfully in advanced oxidation processes [37]. This combination was checked and it could be observed that the kinetics of the photoreaction and the photoproduct stability were highly influenced by the presence of traces of hydrogen peroxide. For this reason, the effects of chemical variables such as H2O2 concentration, pH of the medium, irradiation time and temperature were studied with the object of determining the optimal conditions for the photooxidation of both analytes. Figure 1 shows the excitation and emission spectra of LV before (a, a) and after 12 min of irradiation (b, b) at pH 4.0 and at 20 C, with 0.018% of H2O2. As can be seen, the excitation spectrum of LV photoproduct has two maxima located at 270 and 344 nm, whereas the emission spectrum shows only a maximum located 440 nm.

Influence of chemical variables on the photooxidation process of LV These studies were carried out at different irradiation times with the object of analysing the influence of each variable on the fluorescence intensity of the photoproduct, as in the irradiation time necessary to obtain the photoproduct. Influence of H2O2 concentration A large increase in the velocity of formation of the photoproduct is observed in the presence of traces of H2O2. The influence of H2O2 percentage was studied in the range from 0 and 0.025% and at different irradiation times. The kinetics of the photooxidation reaction increased with H2O2 concentration but at the same time decreased the stability of the photoproduct. A concentration of 0.018% H2O2 was selected because this afforded rapid kinetics and the intensity of fluorescence of LV photoproduct is maintained at the higher values and constant. Influence of pH A study of the influence of acidity of the medium on the kinetics of formation of the photoproduct has been carried out. The pH of the solutions was adjusted over the range 2 6 by the addition of trace amounts of hydrochloric acid or sodium hydroxide. The H2O2 concentration was maintained at 0.018%. The photoreaction rate and the fluorescence of the photoproduct increase in acidic medium up to pH 3.6. For pH values higher than 4.10, the fluorescence emission decreases. A pH of 4.0 was therefore chosen as optimum and it was fixed with acetic acid/sodium acetate buffer

120 b'

Fluorescence Intensity (a.u.)



a 0 300 400




a 600 a' b' b

Table 1 Analytical and statistical parameters for the fluorimetric determination of CPT-11 and LV via previous photoirradiation in the presence of H2O2 CPT-11 Dynamic range (g mL1) Equation Standard deviation of slope (Sm) Standard deviation of intercept (Sb) Correlation coefficient (R) RSD (n=11) LOD (ng mL1)a LOD (ng mL1)b LOQ (ng mL1)b
a b

Fluorescence Intensity (a.u.)

LV 0.54.0 Y=41.29X + 4.31 4.3 1.10 0.9989 7.7 (0.5 g mL1) 1.50 (3.0 g mL1) 259 48.2 160.6


0.051.0 Y=295.55X + 4.00 93 2.64 0.9996 1.6 (0.1 g mL1) 1.5 (0.8 g mL1) 28 12.5 41.7


0 300 400 500

Wavelength,nm Fig. 1 Excitation and emission spectra of a LV and b CPT-11 before (a, a) and after (b, b) 12 min of irradiation at pH 4.0 and at 20 C, with a 0.018% of H2O2

Claytons method [37] IUPAC

Anal Bioanal Chem (2008) 391:11191127 Fig. 2 Contour plot of an excitationemission matrix (EEM) for an aqueous solution containing CPT-11 and LV


Emission wavelength, nm











Excitation wavelength, nm

0.1 M. At this pH, 8 min is enough to obtain the maximum fluorescence of the photoproduct. Influence of temperature The effect of the temperature on the fluorescence intensity of the photoproduct was examined between 10 and 45 C. The temperature does not have an effect on the rate of the kinetics; however, the fluorescence intensity of the photoproduct decreases at high temperatures. A constant temperature of 20 C was selected as optimum. Spectral characteristic of CPT-11 CPT-11 presents native fluorescence in acidic and basic media and its fluorescence emission is practically unaffected by the irradiation. Figure 1b shows the excitation and emission spectra of CPT-11 at pH 4.0 before (a, a) and after (b, b) 12 min of irradiation in the presence of 0.018% of H2O2. As can be seen, the excitation spectrum has two maxima located at 255 and 368 nm, whereas the emission spectrum has only a maximum located at 426 nm. When CPT-11 was irradiated in the presence of H2O2, the intensity of the fluorescence decreases slightly. The effect of the chemical variables such as H2O2 percentage and irradiation time on the fluorescence intensity of CPT-11 is negligible. Analytical figures of merit Under the optimal physicochemical conditions, regression analysis of the fluorescence intensity, against concentration of each analyte, was performed after irradiation for 8 min. Calibration regressions up to 4 g mL1 and up to 1 g mL1 were established from solutions of LV and CPT-11, respectively. Table 1 summarises analytical and

statistical parameters of each compound. This table also includes the detection limits calculated from the standard deviation values of slope and origin intercept, and choosing
Table 2 Composition of calibration (Cal1Cal17) and validation (Test1Test11) sets Sample Theoretical concentration (g mL1) CPT-11 Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10 Cal11 Cal12 Cal13 Cal14 Cal15 Cal16 Cal17 Test1 Test2 Test3 Test4 Test5 Test6 Test7 Test8 Test9 Test10 Test11 0.00 0.00 0.00 0.25 0.05 0.25 0.50 0.27 0.27 0.08 0.46 0.08 0.46 0.27 0.27 0.27 0.27 0.25 0.40 0.35 0.40 0.45 0.05 0.10 0.15 0.00 0.25 0.07 LV 0.00 0.00 1.50 0.00 1.70 0.00 1.70 0.50 3.00 0.80 0.80 2.60 2.60 1.70 1.70 1.70 1.70 2.40 2.10 1.50 2.40 1.50 2.80 1.00 1.20 0.80 0.00 3.00

130 CPT-11 120 120 130 LV

Anal Bioanal Chem (2008) 391:11191127

% Recovery

% Recovery









PARAFAC NPLS/RBL BLLS PARAFAC NPLS/RBL BLLS Fig. 3 Boxwhisker plot of the mean recovery values calculated by application of PARAFAC, N-PLS and BLLS in the analysis of 11 synthetic mixtures of CPT-11 and LV

a false positive and a false negative probability value of 0.05 [38]. For a series of 11 measurements on 0.5 and 3 g mL1 of LV and on 0.1 and 0.3 g mL1 of CPT-11, relative standard deviations of 7.7 and 1.5% and 1.6 and 1.5%, respectively, were obtained (95% confidence level). Optimisation of the procedure for the simultaneous determination of CPT-11 and LV by multivariate method Although spectra of both analytes are overlapped, the resolution of the mixture by use of univariate methods was attempted. Unsatisfactory results were obtained. Also, the application of PLS-1 method to first-order data was performed. Once again, unsatisfactory results were obtained in the spiked urine samples. Owing to the inability of the first-order calibration method, second-order multivariate methods such as PARAFAC, N-PLS/RBL and BLLS/RBL were assayed in order to evaluate if an improvement of the results could be obtained. Second-order data calibration methods The experimental three-way data have been obtained by recording the fluorescence excitationemission matrices of

the binary mixtures after irradiation with the external lamp. Figure 2 shows the contour plot corresponding to the EEM for a sample containing LV and CPT-11, irradiated for 12 min in the presence of 0.018% of H2O2. The three-way data used were obtained by recording excitationemission matrices and the MVC2 interface, PARAFAC, N-PLS/RBL and BLLS/RBL were applied. A calibration set of 17 samples was constructed by using a central composite design [39, 40] and two blanks were also included (Table 2). EEMs were recorded in the ranges 245385 nm at 5-nm intervals for excitation and 390485 nm at 5-nm intervals for emission. For calibration and prediction purposes, the whole wavelength range was used. The set of calibration samples was investigated with the aid of PARAFAC, N-PLS and BLLS. As expected from the sample composition, the number of factors found in all cases was two, calculated according to the Haaland and Thomas criterion [41, 42] for N-PLS and according with the core consistency analysis (CONCORDIA criterion) [43] for PARAFAC and BLLS. The statistical parameters and the results obtained in the analysis of the test set of synthetic samples (11 samples, Table 2), are summarised in Table 3. The results obtained for the two analytes are satisfactory for all the methods employed. The mean

Table 3 Statistical parameters obtained in the analysis of the test of synthetic samples by the different second-order calibration methods PARAFAC CPT-11 REP (%)a Mean recovery (%)b
a b

N-PLS LV 9 102 (8) CPT-11 5 104 (9) LV 8 102 (8)

BLLS CPT-11 5 104 (9) LV 10 103 (8)

5 101 (6)

Relative error of prediction Values between parentheses correspond to the standard deviations computed for the recoveries of the 11 test samples

Anal Bioanal Chem (2008) 391:11191127


Fluorescence Intensity (normalized)

Fluorescence Intensity (normalized)


A CPT-11


B CPT-11 Urine LV



0.2 LV 0.1 Urine



0 240 280 320 Wavelength, nm 360 400

0 400 420

440 460 480 Wavelength, nm Fig. 4 Profiles extracted by the parallel factor analysis model for urine sample spiked with the analytes. a Emission spectral profiles; b excitation spectral profiles

recovery values for each analyte are plotted in Fig. 3, using a boxwhisker plot. Recoveries around 100% are found for both analytes with the three multivariate methods used, but better standard deviation values of the recoveries are obtained when PARAFAC is applied. Analysis of urine samples The analysis of urine samples has to been carried out with multivariate methods which achieve the second-order advan-

tage. Because of this, N-PLS and BLLS were combined with residual bilinearisation (RBL). In all cases, urine samples were spiked with different amounts of LV and CPT-11. When applying PARAFAC, core consistency analysis [43] was applied for each newly analysed sample, since there is no guarantee that each unknown urine sample will behave in the same manner as the previously studied ones. The result was that all urine samples required the consideration of three factors: two for the analytes and the remaining one for the urine background.

Table 4 Results obtained when applying second-order methods in the analysis of urine samples Actual PARAFAC Predicted CPT-11 0.25 0.00 0.25 0.37 0.40 0.08 0.00 0.25 Average recoverya REP (%)b LV 2.20 0.80 0.50 1.50 0.80 1.80 1.00 Average recovery REP (%)
a b

NPLS/RBL Recovery (%) Predicted Recovery (%)

BLLS/RBL Predicted Recovery (%)

0.22 0.21 0.33 0.37 0.07 0.25 90 ( 5) 9 1.80 0.68 0.42 1.10 0.62 1.40 0.85 82 ( 7) 16

88 84 89 93 88 100

0.21 0.21 0.32 0.36 0.06 0.24 86 ( 7) 14 1.49 0.58 0.32 1.05 0.60 1.30 0.61 69 ( 5) 36

84 84 86 90 75 96

0.21 0.21 0.32 0.36 0.06 0.24 86 ( 7) 10 1.50 0.63 0.30 1.03 0.56 1.30 0.67 69 ( 5) 33

84 84 86 90 75 96

82 85 84 73 77 78 85

68 73 64 70 75 72 61

68 79 60 69 70 72 67

Values in parentheses correspond to the standard deviation computed for the recoveries of the eight urine samples Relative error of prediction


Anal Bioanal Chem (2008) 391:11191127

When applying N-PLS/RBL and BLLS/RBL it was necessary to assess the number of unexpected components. This was done by analysing the residuals and the necessary number of factors for interferences reaches one, in both cases. Figure 4 shows the loading profiles for excitation and emission, provided by PARAFAC when processing a urine sample spiked with 0.25 g mL1 of CPT-11 and 0.50 g mL1 of LV, together with the calibration set, where components have been labelled according to the order assigned by the model in the specific data array under investigation. They appear in the order of their contribution to the overall variance; in this particular case, the interference appears in second place, indicating that it is the second source of fluorescence intensity across this particular data array. In all cases, analyte profiles were identified by comparison with standards, with the remaining ones corresponding to the urine background. The prediction results for the spiked urine set when analysing data by the PARAFAC, N-PLS/RBL and BLLS/ RBL models are presented in Table 4. All predictions are seen to be reasonable for samples of human urine. Adequate recoveries for CPT-11 with the three models and nonsignificant differences in the REP values were found. On the other hand, for LV analysis, adequate recoveries were obtained when PARAFAC was used and recoveries around 70% were found with N-PLS/RBL and BLLS/RBL. In this particular case, the calibration with BLLS and with N-PLS, including latent variables in the model, in combination in both cases with RBL, achieved the second-order advantage without improvement of analytical recoveries; however, in most recent literature, better results are found when the second-order advantage is obtained when the second-order methods are coupled with RBL, because this combination has more flexible internal structure, which makes a more appropriate approach for processing second-order data [34].

methods used give similar results for the determination of CPT-11; in the case of LV determination, PARAFAC performs somewhat better than N-PLS and BLLS, both coupled with RBL.

Acknowledgement Financial support from the Ministerio de Educacin y Ciencia of Spain (Project CTQ2005-02389) was obtained.

1. Chabot GG (1997) Clin Pharmacokinet 33:345359 2. Vanhoefer U, Rougier P, Borner M, Muoz A, Van Laethem JL, Sobrero A (2004) EJC Supplements 2(7):1420 3. Saltz LB, Cox JV, Blanke C, Rosen LS, Fehrenbacher L, Moore MJ et al (2000) N Engl J Med 343(13):905914 4. Straw JA, Newman EM, Dorowshow JH (1987) NCI Monogr 5:4145 5. Dey J, Warner IM (1996) J Photochem Photobiol A 101:2127 6. Dey J, Warner IM (1997) J Luminiscence 71:105114 7. Chourpa I, Millot JM, Sockalingum GD, Riou JF, Manfait M (1998) Biochem Biophys Acta 1379:353366 8. Posokhov Y, Biner H, Ili S (2003) J Photochem Photobiol A 158:1320 9. Dvoranov D, Brezov V, Valko M, Stasko A (2007) J Photochem Photobiol A 185:172180 10. Gravel E, Bourget P, Mercier L, Paci A (2005) J Pharm Biomed Anal 39:581586 11. Poujol S, Pinguet F, Malosse F, Astre C, Ychou M, Culine S, Bressolle F (2003) Clin Chem 49:19001908 12. Sparreboom A, de-Brujin P, de-Jonge MJA, Loos WJ, Stoter G, Verweij J, Nooter K (1998) J Chromatogr B Biomed Appl 712:225235 13. Di B, Fang F, Li YY, Yu LJ, Song HL, Zhang ZX (1999) YaowuFenxi-Zazhi 19:293296 14. Bardin S, Guo W, Johnson JL, Khan S, Ahmad A, Duggan JX, Ayoub J, Ahmad I (2005) J Chromatogr A 1073:249255 15. Ragot S, Marquet P, Lachatre F, Rousseau A, Lacassie E, Gaulier JM, Dupuy JL, Lachatre G (1999) J Chromatogr B Biomed Appl 736:175184 16. Rodrguez Cceres MI, Durn-Mers I, Ornelas Soto NE, Lpez de Alba PL, Lpez Martnez L (2008) Talanta 74:14841491 17. Duan GL, Zheng LX, Chen J, Cheng WB, Li D (2002) Biomed Chromatogr 16(4):282286 18. Chladek J, Sispera L, Martinkova J (2000) J Chromatogr B Biomed Appl 744(2):307313 19. Rodrguez Flores J, Cataeda Pealvo G, Espinosa Mansilla A, Rodrguez Gmez MJ (2005) J Chromatogr B Anal Technol Biomed Life Science 819(1):141147 20. Hadley MR, Camilleri P, Hutt AJ (2000) Electrophoresis 21 (10):19531976 21. Durn-Mers I, Espinosa Mansilla A, Rodrguez Gmez MJ, Salinas Lpez F (2001) Talanta 55(3):623630 22. Durn-Mers I, Espinosa Mansilla A, Salinas Lpez F, Rodrguez Gmez MJ (2002) J Pharm Biomed Anal 27(12):8190 23. Durn-Mers I, Espinosa Mansilla A, Salinas Lpez F, Rodrguez Gmez MJ (2002) Anal Bional Chem 373(45):251258 24. Espinosa Mansilla A, Durn-Mers I, Rodrguez Gmez MJ, Muoz de la Pea A, Salinas Lpez F (2002) Talanta 58(2):255263 25. Bro R (1997) Chemom Intell Lab Syst 38:149171 26. Culzoni MJ, Goicoechea HC, Pagani AP, Cabezon MA, Olivieri AC (2006) Analyst 131:718732

Conclusions Different multiway calibration methods were employed in order to determine LV and CPT-11 mixtures in a complex biological sample (in this case urine), for which the fluorescence spectra of the components of the mixture overlap greatly. First-order multivariate methods, such PLS-1, do not allow the resolution of the mixture. Due to the complexity of the problem to be solved, second-order calibration methods were necessary in this work and the determination of these analytes in urine samples was assayed, and satisfactory results were obtained. In comparing the residual error of prediction (REP) values provided by the three algorithms, the three second-order

Anal Bioanal Chem (2008) 391:11191127 27. Muoz de la Pea A, Espinosa Mansilla A, Mora Dez N, Bohoyo Gil D, Olivieri AC, Escndar GM (2006) Appl Spectrosc 60:330338 28. Linder M, Sundberg R (1998) Chemom Intell Lab Syst 42:159 178 29. Faber NM, Ferr J, Boqu R, Salivas JH (2002) Chemom Intell Lab Syst 63:107116 30. hman J, Geladis P (1990) J Chemometrics 4:7990 31. Olivieri AC (2005) J Chemometrics 19:253265 32. Linder M, Sundberg R (2002) J Chemometrics 16:1227 33. Bro R (2006) Crit Rev Anal Chem 36:279293 34. Escandar GM, Faber NM, Goicoechea HC, Muoz de la Pea A, Olivieri AC, Poppi RJ (2007) Trends Anal Chem 26:752 765

1127 35. Arancibia JA, Olivieri AC, Gil DB, Mansilla AE, Durn-Mers I, de la Pea AM (2006) Chemom Intell Lab Syst 80(1):7786 36. Olivieri AC, Arancibia JA, de la Pea AM, Durn-Mers I, Mansilla AE (2004) Anal Chem 76(19):56575666 37. Colonna GM, Caronna T, Marcandalli B (1999) Dyes Pigments 41:211220 38. Clayton LC, Wines JW, Elkins PD (1987) Anal Chem 59:2506 2514 39. Box GEP, Wilson KB, Stat JR (1951) Soc Ser B Stat Methodol 13:145 40. Box GEP, Hunter JS (1957) Ann Math Stat 28:195241 41. Thomas EV, Haaland DM (1988) Anal Chem 60:11931202 42. Thomas EV, Haaland DM (1990) Anal Chem 62:10911099 43. Bro R, Kiers HAL (2003) J Chemometrics 17:274286