Determination of chemotherapeutic drugs in human urine by capillary electrophoresis with UV and fluorimetric detection using solid-supported liquid-liquid extraction for sample clean-up.

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Journal of Separation Science

Determination of chemotherapeutic drugs in human urine by capillary electrophoresis with UV and fluorimetric detection using solid-supported liquid–liquid extraction for sample clean-up María del Carmen Hurtado-Sánchez, María Isabel Acedo-Valenzuela, Isabel Durán-Merás, María Isabel Rodríguez-Cáceres* Department of Analytical Chemistry, University of Extremadura, 06006, Badajoz, Spain

Running title: CE-UV-FD method to analyze chemotherapeutic agents

*

Corresponding author: María Isabel Rodríguez-Cáceres

Fax number: +34 924 27 42 44 E-mail address: [email protected]

Abbreviations: CPT, camptothecin, CPT-11, irinotecan; CRC, colorectal cancer; DAD, diode array detector; FD, fluorescence detector; LV, leucovorin; 5-FU, 5-fluorouracil; SN-38, 7ethyl-10-hydroxycamptothecin; SS-LLE, solid-supported liquid-liquid extraction; TF, tegafur. Keywords: capillary electrophoresis, chemotherapeutic agents, colorectal cancer, human urine.

Received: 08-Jan-2015; Revised: 11-Mar-2015; Accepted: 14-Mar-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201401443. This article is protected by copyright. All rights reserved.

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Abstract Capillary electrophoresis was used for the rapid determination of three chemotherapeutic drugs employed to treat colorectal cancer: irinotecan, tegafur and leucovorin, and their main metabolites (7-ethyl-10-hydroxycamptothecin and 5-fluorouracil), in human urine samples. A phosphate buffer (pH 11.34; 20 mM) was selected as the background electrolyte. A hydrodynamic injection (9 s, 30 mbar) was applied and the separation was carried out using a separation temperature and voltage of 25°C and 25 kV, respectively. A capillary with two detection windows for serial online ultraviolet and fluorescence detection was satisfactorily employed. A solid-supported liquid–liquid extraction procedure was optimized for the cleanup of the urine samples and the extraction of the analytes. Matrix effects were assessed and suppression signal was observed for three of the analytes, thus, matrix-matched calibration was used for compensating residual matrix effects on these analytes. The proposed method allows the separation and quantification of the chemotherapeutics in less than 6 min. Detection limits range between 0.01 to 0.30 mg L-1. The method was satisfactorily applied to the determination of the target compounds in human urine samples, with recoveries of 92.4– 107.7%.

This article is protected by copyright. All rights reserved.

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1 Introduction Colorectal cancer (CRC) is the third most common cause of cancer death and the second most lethal cancer overall [1]. According to the United States National Institute, the highest levels of success of first-line combination therapy on overall survival are achieved with the combination of irinotecan (CPT-11) and bolus and/or infusional 5-fluorouracil/leucovorin (5FU/LV) [2]. Many anticancer drugs are sparingly water soluble and they show a low permeability at the intestinal level. The administration of a pro-drug represents one approach to overcome these obstacles [3]. CPT-11 is a pro-drug water soluble camptothecin derivative, and in vivo it is converted to its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38). As a single agent, or in combined modality regimens, has been investigated in gastric cancer [4], non-small cells lung cancer [5], advancer solid malignancy tumor and others [6].

On the other hand, 5-fluorouracil (5-FU) has been used in cancer therapy [7] since its discovery in 1957. This molecule is active as a single agent in colorectal cancer, and as part of combination regimens for breast, head and/or neck, and upper gastrointestinal malignancies [3]. Its direct administration has several disadvantages and, to avoid them, the use of the pro-drug tegafur (TF) was introduced.

Leucovorin (LV) is used as a rescue agent and also as a modulator of the pyrimidine antagonist 5-FU. The biological activity of LV is apparently restricted to the (6S)-LV diastereoisomer,

which

is

rapidly

converted

methyltetrahydrofolate [8].


to

its

active

metabolite

(6S)-5-

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In April 2000, CPT-11 was approved in association with 5-FU and LV as first line in the treatment of advanced colorectal cancer [9]. This combination is known as IFL schedule when the drugs are administered as a bolus formulation, whereas is called FOLFIRI regimen when the drugs are administered intravenously [10]. There are many treatment schemes, among others, the Saltz regimen [9]: 125 mg m-2 of CPT-11, 500 mg m-2 of 5-FU and 20 mg m-2 of LV (IV bolus), and the FOLFIRI regimen [10]: CPT-11 (180 mg m-2 IV over 90 min), concurrently with LV (400 mg m-2 [or 2 x 250 mg m-2] IV over 120 min); followed by 5-FU (400–500 mg m-2 IV bolus) then 5-FU (2400–3000 mg m-2 intravenous infusion over 46 h).

To date, no analytical method about the simultaneous resolution of the five analytes presented in this work has been developed. In the literature, binary mixtures of these drugs have been determined. Thus, without using separation techniques, Durán Merás et al. [11] resolve the binary mixture of CPT-11 and SN-38, using spectrofluorimetry coupled with firstorder multivariate calibration (PLS-1). Also, second-order multivariate methods has been applied to resolve the mixture of CPT-11 and LV using photoinduced spectrofluorimetry and PARAFAC, N-PLS and BLLS [12], and the mixture LV and methotrexate has been resolved using four-way fluorescence excitation-emission-kinetic data [13].

HPLC with UV [14–16], fluorescence (FD) [17–19] or MS detection [14, 20–22] has been the most used technique for the analysis of these compounds. UV detection has been employed for the resolution of TF and 5-FU in human plasma [14], and in beagle dog plasma [15], and for the analysis of LV and 5-FU in human plasma [16], whereas HPLC with fluorimetric detection [17–19] has been mainly used for the resolution of the mixture CPT-11 and SN-38. Recently, this technique coupled with on-line SPE has also been used for the analysis of CPT-11 and its metabolites SN-38 and SN-38 glucuronide in mice plasma after CPT-11 administration and in cultured cancer cells exposed to SN-38 [23]. CPT-11 and other cytostatic drugs have been analyzed in aquatic environments using HPLC/MS previous SPE treatment of the samples with Oasis HLP cartridges [24].


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In the last decades, CE has been one of the separation techniques most employed in the determination of anticancer drugs in complex biological samples due to its high resolution, fast analysis and relative low cost and solvent consumption compared to HPLC [25, 26]. Therefore, CE is a simple, cheap and easily automatable technique that also presents great versatility in terms of modes of operation and applicability, since it allows the separation of a wide variety of compounds, both ionic and molecular. For that reason, CE has become an important technique in pharmaceutical analysis, with even more applicability than HPLC in this field [27]. CE has been employed for the determination active pharmaceutical ingredients (API), drug impurity testing, chiral drug separation, pharmacokinetic/pharmacodynamic studies, determination of drugs and metabolites in biological fluids, and for the study of physicochemical properties such as log P and pKa or of drug-macromolecule binding constants [28,29].

Only one reference about the use of CE for the analysis of CPT-11 and SN-38 in urine and saliva has been found [30]. In this work, a borate buffer solution containing sodium dodecyl sulfate and acetonitrile as BGE, and UV detection was employed.

The determination of 5-FU and tegafur in pharmaceuticals has been improved by using large-volume sample stacking in CE [31]. Also, due that in the last decades 5-FU has been one of the most frequently antineoplastic agents used; it has been regarded for environmental monitoring. For this reason, Mahnik et al. [32] propose the use of CE for its determination in hospital effluents. On the other hand, the determination of 5-FU in plasma samples has also been carried out by CE, using a running buffer composed of TRIS/H3PO4 (pH 7.0; 30 mM) and 5% isopropanol [33]. Rodriguez Flores et al. [34] developed a rapid CZE method for the direct determination of 6-thioguanine, methotrexate and 5-FU in human urine, with a LOD for 5-FU of 2.60 mg L-1. Also, CZE has been applied to the analysis of 5-FU in urine and BSA with electrochemical detection [35]. Finally, the determination of LV together with methotrexate and folic acid, in human urine, has been performed by CZE, using a phosphate buffer (pH 12; 15 mM), in approximately 9.0 min [36]. In this paper, we describe the development of an easy, quick and cheap capillary electrophoretic method with fluorescence and diode array detectors (DAD) in series, CE–FD– DAD, for the analysis of the chemotherapeutic compounds used in the first-line of treatment of colorectal cancer (Fig. S1).


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2 Materials and Methods

2.1 Chemicals and materials For all experiments, analytical reagent grade chemicals and solvents were used. CPT-11, SN-38, LV, TF and the internal standard camptothecin (CPT) were supplied from Sigma– Aldrich (Madrid, Spain), and 5-FU was purchased from Fluka (Madrid, Spain). Stock standard solutions of individual analytes (with concentrations ranging from 156.0 to 331.4 mg L−1) were prepared in methanol, with exception of LV, which was dissolved in ultrapure water, and stored under refrigeration at 4°C. The BGE was prepared from disodium hydrogen phosphate (Panreac, Barcelona, Spain), adjusting the pH to 11.34 with NaOH (Scharlau, Barcelona, Spain). Analytical-grade methanol and acetonitrile (Sigma–Aldrich, Madrid, Spain) were used. Chloroform and ethyl acetate were purchased from Panreac (Barcelona, Spain). Ultrapure water was obtained from a Millipore Milli-QA10 System (Millipore S.A.S., Molsheim, France).

2.2 Instrumentation and software All CE experiments were conducted using a CE system Hewlett Packard 3DCE (Agilent Technologies, Waldbronn, Germany), equipped with temperature control in the sample tray (by a thermostatic bath) and in the capillary (by forced air), a fluorescence detector (Argos 250B, Flux Instruments, Switzerland) with a fluorescence flow-cell mounted on the modified cassette for the Agilent CE, and equipped with a Xe-Hg lamp, a 240–400 nm band-pass as excitation filter and a 418 nm cut-off emission filter, and a DAD (Agilent Technologies). Studies were carried out using a fused-silica capillary of 58.5 cm × 75 μm id × 375 μm od (BGB Analytik, Switzerland), with two detection windows (Fig. S2, Supporting Information). The software package ChemStation was used to control the instrument. Chem Elut cartridges (3 mL) from Agilent Technologies (Madrid, Spain) were used for the solid-supported liquid–liquid extraction (SS-LLE) of human urine samples. Calibration curves and analytical figures of merit were performed by means of the ACOC program [37], in MATLAB code. This article is protected by copyright. All rights reserved.

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2.3 CE method Before the first use, the capillary was conditioned by rising at 900 mbar with 1.0 M NaOH for 15 min at 50°C, followed by ultrapure water for 15 min, and BGE (phosphate buffer, pH 11.34; 20 mM) for 15 min, at 25°C. At the beginning of each day, the capillary was rinsed with ultrapure water, 0.1 M NaOH and BGE for 10 min in each case, at 900 mbar. For the separation, the capillary was previously flushed with BGE for 3 min (900 mbar) and, after the separation, in post-conditioning, was rinsed with 0.1 M NaOH for 3 min at 2 bar and ultrapure water for 2 min at 2 bar. Samples were hydrodynamically injected at 30 mbar for 9 s, and then followed by an injection of running buffer at 30 mbar during 1 s to minimize the sample loss. Separation was performed at 25 kV for 6 min at 25°C. Under these conditions the current were 112–115 µA. The buffer vials were filled with new filtered BGE solution each three injections.

2.4 Human urine analysis A 2.0 mL aliquot of a pool of human urine spiked with the analytes was transferred to a Chem Elut cartridge and, after 2 min, the analytes were eluted with 2.0 mL of ethyl acetate/methanol mixture (95:5, v/v), and the eluate was collected in a round-bottom flask. Elution was repeated with another 5.0 mL of the same mixture and, finally, with 5.0 mL of chloroform. The combined eluates were evaporated at 40°C until dryness under pressure, and reconstituted in 1.0 mL of MeOH followed by 1.0 mL of BGE. The separation of analytes was carried out under the conditions described in Section 2.3. SN-38 was fluorimetrically monitored, while the rest of compounds were photometrically detected at 265, 272, 288 and 360 nm, for 5-FU, TF, LV and CPT-11, respectively.


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3 Results and Discussion 3.1 Optimization of detection parameters and electrophoretic separation Among the analyzed compounds, only CPT-11 and its metabolite SN-38 are fluorescent while the rest of compounds only present spectrophotometric characteristics. To obtain the best conditions for the fluorimetric and UV detection of these compounds, previous studies in relation to its spectral behavior were carried out. First, the structures and pKa values were revised; LV presents the lowest pKa value (3.76), while CPT-11 and SN-38 show the highest values. Previous studies carried out in our research group show that the highest pKa value of CPT-11 is 8.93 [38], while SN-38 shows a pKa value of 8.65 [39]. Thus, solutions of each analyte in a concentration level of 0.50 mg L -1 were prepared in presence of borate buffer (pH 9.2; 100 mM), and the absorption spectra for each analyte were obtained. The wavelengths of maximum absorbance were 265, 272, 288, 360 and 410 nm, for 5-FU, TF, LV, CPT-11 and SN-38, respectively. In these conditions, the excitation and emission spectra of CPT-11 and SN-38 were also registered. CPT-11 presents an excitation maximum centered at 360 nm and an emission maximum centered at 440 nm. SN-38 shows two excitation maxima at 340 and 410 nm, and a single emission maximum at 548 nm. Regarding the electrophoretic separation of the target compounds, several buffers were evaluated as BGE (borate buffer (pH 9.2; 20 mM) and (pH 10.12; 20 mM), and phosphate buffer (pH 11.34; 20 mM) and (pH 11.93; 20 mM)). The results obtained shown that higher pH provided shorter migration time with complete resolution of the corresponding peaks. Phosphate buffer (pH 11.34; 20 mM) was selected as BGE optimum for providing a reasonable total analysis time (around 6 min), Fig. 1. In these conditions, the separation of all compounds, including CPT used as internal standard (IS), showed a good separation efficiency, as demonstrated by the number of theoretical plates values obtained for each analyte, which are in all cases higher than 30000. This article is protected by copyright. All rights reserved.

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3.2 Analysis of real samples: extraction procedure optimization Since some of the tested analytes absorb at low wavelengths (265–288 nm), the direct injection of the sample was not possible because the urine is a complex matrix with several compounds absorbing at these wavelengths, thus it presents a background that interferes in the analysis of the compounds of interest. For this reason, a clean-up step of the sample, previous to the analysis, was required to eliminate interferences. Recently, a review of the extraction techniques most employed in biomedical analysis has been published [40]. LLE, and SPE with C18 cartridges, are the most widely applied methods to remove possible contaminants in the analysis of chemotherapeutic compounds in biological samples. Firstly, extraction procedures with C18 cartridges were assayed with urine samples spiked with the compounds of interest. Different solvents, such as MeOH, ACN, n-butanol and tetrahydrofuran, were assayed as extracting solvent, but 5-FU was not extracted from the cartridge in any case. Bearing this in mind, LLE was employed as urine samples cleaning procedure. Several solvents (ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, n-butanol and acetonitrile) were individually evaluated by extracting spiked urine samples with a volume ratio urine sample/solvent 1:1 and agitation time of 90 s. Of all solvents assayed, none provides a simultaneous extraction of all compounds. Chloroform provided the best results for the extraction of CPT-11, SN-38 and TF, with recoveries near to 100%. 5-FU was only extracted with n-butanol and LV with ethyl acetate. With the object to get an adequate extraction of all analytes, SS-LLE has been assayed. This technique offers advantages against the traditional extraction techniques: high recoveries, removal of the matrix effect, minimum sample handling, not requires solvent preconditioning, prevents the formation of emulsions, and reduced sample volumes [41]. It is conceptually analogous to traditional LLE, however, it uses an inert support material such as modified diatomaceous earth, with a high surface area for extraction interface and a modified flow-through technology. This article is protected by copyright. All rights reserved.

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A SS-LLE procedure has been previously proposed to analyze 5-FU in plasma and urine samples [42], using an ethyl acetate/methanol mixture (95:5, v/v) as extracting solvent. Now, this procedure has been modified and extended to the analysis of the five analytes in human urine samples. Chem Elut cartridges, packed with inert diatomaceous earth sorbent, have been employed. Parameters such as extraction pH, solvent of elution and volume, among others, were carefully studied and optimized. For that, an aliquot of 2.0 mL of a human urine sample was spiked with 0.50 mg L-1 of SN-38 and with 10.0 mg L-1 of the rest of compounds. Firstly, the capability of ethyl acetate/methanol mixture (95:5, v/v) to extract all target compounds was tested. Under these conditions CPT-11 was not eluted and, for this reason, a second extracting solvent was employed: chloroform. Several combinations and volumes of these solvents were tested to achieve the maximum recovery as possible. Finally, the following combination of solvents was chosen: 2.0 mL of ethyl acetate/methanol (95:5, v/v), followed by 5.0 mL of the same mixture, and 5.0 mL of chloroform. The influence of pH, was studied in a range comprised between 1 and 8. For that, 0.5 mL of trichloroacetic (pH 1), 0.5 mL of chloroacetic/chloroacetate (pH 2 and 3), acetic/acetate (pH 4, 4.4, 5, 5.6 and 6) or TRIS (pH 7 and 8) buffers were added to the urine sample before the extraction. The obtained results showed that at low values of pH (1–3), urine interferences, which overlap with TF signal, are also eluted. Between pH 3 and 6, the maximum extraction recoveries were achieved for 5-FU, and for LV the best results were obtained at pH 4.4 and 6. Finally, as a compromise, pH 5, fixed with acetic/acetate buffer was selected as optimum.


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In SS-LLE, the aqueous sample is adsorbed and distributed as a thin film over the solid support. In consequence, it is necessary to wait a few minutes before elution of the target compounds. Different waiting times between the addition of the sample to the cartridge and the elution step (0, 2, 5, 10 and 15 min) were tested, and it was found that the extraction efficiency was kept constant from 2 min, and this time was selected as optimum. This behavior shows that the equilibrium state is reached very quickly decreasing the total duration of the extraction procedure. Under these conditions, the final recoveries of the compounds were approximately 100% for CPT-11, SN-38 and TF, and 65% and 47% for 5-FU and LV, respectively. Fig. 2 shows the electropherograms obtained in presence and in absence of urine registered at the optima UV wavelength of each analyte, and using fluorescence detection for CPT-11 and SN-38. Also, electropherograms corresponding to blank urine sample unspiked with the analytes at each wavelength have been depicted in the same Fig. 2.

3.3 Evaluation of matrix effects Matrix effects were tested by comparing the slopes from standard addition and external standard calibrations. This comparison was carried out with ACOC program [37], at 95% of confidence level. Results obtained showed that, except for TF, matrix suppression effect was observed for all compounds. Matrix-matched calibration method can be used to compensate the matrix effect when matrix suppression phenomena cannot be eliminated, if blank samples are available. To apply this type of calibration, it is necessary to check if the matrix effect found for each analyte is independent of the sample. For that, slopes of external calibration curves in water were compared with slopes of external calibration curves from three urine samples. All samples were taken from healthy people, to assure the complete absence of the target compounds in


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them, of different ages (woman of 26 years old and two men of 27 and 50 years old, respectively). Then, slope ratios matrix/solvent (urine samples/external calibration) were obtained for each compound, considering a tolerable signal enhancement or suppression effect if the slope ratio ranged from 0.9 to 1.1, whereas values higher than 1.1 or lower than 0.9 imply a strong matrix effect. From the values of the slope ratios matrix/solvent (Fig. S3, Supporting Information) is possible to deduce that the matrix effect is irrelevant for TF and 5FU, and is important for CPT-11, SN-38 and LV (Fig. S4, Supporting Information). For this reason, the analysis of the urine samples has been carried out using matrix-matched calibration.

3.4 Method validation The method was validated in terms of linearity, precision, accuracy, LOD, and LOQ. Matrix-matched calibration curves were used, and for this, aliquots of a pool of blank human urine samples, collected from four healthy volunteers, were spiked with the analytes at four concentration levels in the ranges 1–30 mg L-1 for CPT-11 and LV, 2–20 mg L-1 for TF and 5-FU and 0.05–3.5 mg L-1 for SN-38, and submitted to the SS-LLE procedure. Each standard was prepared in triplicate. Peak areas at the optimum UV wavelengths for each analyte were selected as analytical signal. For CPT-11 and SN-38 peak areas from the signal obtained with the fluorescence detector was also employed. Calibration curves with good linearity and with determination coefficients higher than 0.99 for all compounds were obtained. The LOD and LOQ were calculated, as the concentrations corresponding to 3 and 10 times the SD of the signal from baseline from the electropherograms. Results are summarized in Table 1. To evaluate the repeatability (intraday precision, n=5) of the method, one spiked blank urine sample with 16.0 mg L-1 CPT-11 and LV, 10.0 mg L-1 TF and 5-FU, and 0.30 mg L-1 SN-38 was assayed. Interday precision (reproducibility) was analyzed over five consecutive days at the same concentration levels. As can be seen, both intraday and interday precision values are lower than 7%, which indicates the good repeatability of the proposed method. Also, the obtained results in presence and in absence of IS were similar, therefore the use of the CPT was not necessary. This article is protected by copyright. All rights reserved.

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3.5 Application of the method to the analysis of chemotherapeutic compounds in human urine samples Due to the difficulty to obtain real urine samples, a fter the optimization of the conditions

for the extraction and separation of the chemotherapeutic compounds, the method was checked by analyzing a human urine pool from four healthy people, spiked at three concentrations levels with the five chemotherapeutics, in triplicate, and using the optimized SS-LLE procedure. To know the concentration levels in which these compounds are presents in human urine, the doses administered in the main first-line treatments applied to patients with colorectal cancer [9, 10, 43–46] were analyzed using a colorectal cancer nomogram [47], and considering a patient with a weight of 80 kg and a height of 1.76 m. In accordance with previous research about the excretion of these compounds, in the first 24 h, all compounds are excreted in a range of 4–22% [48–51], with the exception of SN-38, which is excreted in a range between 0.18 and 0.43% [48]. These percentages allow us to conclude that, in urine, all compounds are present in the range of mg L -1, with the exception of SN-38, which is present in levels of µg L -1. For this reason, analysis of CPT-11, TF, 5-FU and LV in urine was carried out with the DAD detector, while SN-38 was analyzed employing the fluorescence signal. The results obtained when urine samples were analyzed, are summarized in Table 2. It can be seen that satisfactory recoveries, comprised between 92.4 and 107.7%, were obtained in all cases.


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4 Concluding remarks An electrophoresis capillary method, with both fluorimetric and UV detection, to analyze five compounds used in chemotherapy for colorectal cancer has been developed. The sample was hydrodynamically injected in the system during 9 s and the separation has been performed by employing a phosphate buffer (pH 11.34; 20 mM) as BGE and using a voltage of 25 kV and 25°C of temperature. In these conditions, a good peak resolution was achieved in less than 6 min. Once the method was optimized, it was applied to the analysis of the target compounds in human urine samples. For that, due to the background, a SS-LLE was optimized to eliminate interferences from urine. A very good extraction recovery was obtained for CPT-11, SN-38 and TF, approximately of 100%, but only around 50% of recovery was achieved for 5-FU and LV. Finally, this method was validated by the analysis of the compounds in spiked urine samples. Matrix effects were found for all compounds except for TF and 5-FU, for that reason, matrix-matched calibration was employed. The detection limits of the developed CE–FD–DAD procedure are adequate to determine the normal levels of the target analytes in urine samples.

Acknowledgements The authors gratefully acknowledge to Junta de Extremadura and European FEDER Funds (Consolidation Project of Research Group FQM003, Project GR10033). M.C. Hurtado-Sánchez thanks to Consejería de Economía, Comercio e Innovación of Junta de Extremadura and European Social Fund for a fellowship (DOE 04/01/2011).

The

authors

have

declared


no

conflicts

of

interest.

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Fig. 1. Electropherograms of a 10 mg L-1 stock standard solution of each chemotherapeutic. Conditions: capillary, 58.5 cm x 75 μm id; capillary temperature, 25°C; 9 s hydrodynamic injection; applied voltage, 25 kV; current generated, 112–115 µA; phosphate buffer (pH 11.34; 20 mM) as BGE; UV detection at 265 nm; Fluorescence detection conditions see Section 2.2


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Fig. 2. Electropherograms corresponding to a stock standard solution ( _____), an unspiked blank urine sample (--·--), and a spiked human urine sample, after optimized SS-LLE procedure (_ _ _ _ ). [CPT-11] = [TF] = [5-FU] = [LV] = 10 mg L-1 ; [SN-38] = 0.5 mg L-1. Separation and fluorescence detection conditions as indicated in Figure 2. UV detection: 5FU at 265 nm; TF at 272 nm; LV at 288 nm, and CPT-11 at 360 nm. In enlarged figures, each electropherogram has been shifted along the y axis for better visualization.


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Table 1. Validation parameters of the proposed method. Precisiona

Detector

Linear range (mg L-1)

Intercept ±SD

Slope ±SD (mg L-1)

R2

DAD (360 nm)

1.0–30.0

(–0.4) ±0.3

5.56 ±0.02

0.9934

0.19

0.63

1.3

3.4

FD

0.05–3.5

0.9 ±0.3

16.4 ±0.2

0.9990

0.01

0.03

1.9

1.7

DAD (418 nm)

1.0–30.0

(–1.1) ±0.6

1.75 ±0.03

0.9986

0.22

0.73

1.6

3.8

FD

0.05–3.5

(–0.8) ±0.7

31.7 ±0.6

0.9974

0.01

0.03

2.4

2.9

TF

DAD (272 nm)

2.0–20.0

1.7 ±0.2

1.12 ±0.02

0.9984

0.23

0.77

4.5

6.1

5-FU

DAD (265 nm)

2.0–20.0

(–0.6) ±0.2

0.84 ±0.02

0.9967

0.25

0.83

5.4

3.3

LV

DAD (218 nm)

2.0–30.0

0.3 ±0.1

0.67 ±0.01

0.9994

0.30

1.00

3.1

4.7

Compound

LOD (mg LOQ (mg Intraday L-1) L-1) (n=5)

Interday (n=5)

CPT-11

SN-38

a

Expressed as RSD (% RSD). Standard solutions employed containing 16.0 mg L-1 CPT-11; 16 mg L-1 SN-38; 10.0 mg L-1 TF; 10.0 mg L-1 5-FU and 16.0 mg L-1 LV in DAD detection and 1.0 mg L-1 CPT-11 and 1.0 mg L-1 SN-38 in FD.


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Table 2. Recoveries of the compounds analyzed in spiked human urine samples.

Compound

Concentration (mg L-1) Added

CPT-11

7.00

SN-38

0.35

TF

6.00

5-FU

6.00

LV

7.00

Found 6.92 6.87 6.74 0.37 0.38 0.36 5.48 5.56 5.59 5.62 5.89 5.98 7.67 7.33 7.52


%Recovery ±SD


97.8 ±1.3

12.0

105.1 ±1.8

0.55

92.4 ±1.0

10.00

97.2 ±3.1

10.00

107.2 ±2.4

12.00

Found 11.72 11.04 11.15 0.55 0.56 0.55 10.57 10.24 10.33 10.27 10.08 10.47 12.52 12.22 12.02

% Recovery ±SD


94.2 ±3.0

25.00

101.0 ±0.9

1.20

103.8 ±1.7

17.00

107.7 ±2.0

17.00

102.10 ±2.1

26.00

Found 23.78 23.94 23.61 1.28 1.27 1.24 16.23 16.37 16.50 17.02 17.12 17.31 26.40 23.34 27.24

% Recovery ±SD

95.1 ±0.7

105.3 ±1.4

96.3 ±0.8

100.9 ±0.9

103.8 ±2.0

Simultaneous determination of polyamines and acetylpolyamines in human urine by capillary electrophoresis with fluorescence detection.

Determination of Gabapentin in Human Plasma and Urine by Capillary Electrophoresis with Laser-Induced Fluorescence Detection.

vis detection.

Large volume sample stacking for rapid and sensitive determination of antidiabetic drug metformin in human urine and serum by capillary electrophoresis with contactless conductivity detection.

Determination of aminoglycoside antibiotics in pharmaceuticals by capillary zone electrophoresis with indirect UV detection coupled with micellar electrokinetic capillary chromatography.

Simultaneous determination of aromatic amino acids in human blood plasma by capillary electrophoresis with UV-absorption detection.

Development of a New Microextraction Fiber Combined to On-Line Sample Stacking Capillary Electrophoresis UV Detection for Acidic Drugs Determination in Real Water Samples.

Determination of melamine impurities by capillary zone electrophoresis with UV- and quadrupole time-of-flight mass spectrometric detection.

Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection.

Rapid differentiation of commercial juices and blends by using sugar profiles obtained by capillary zone electrophoresis with indirect UV detection.

Simultaneous determination of caffeine, paracetamol, and ibuprofen in pharmaceutical formulations by high-performance liquid chromatography with UV detection and by capillary electrophoresis with conductivity detection.

Determination of gibberellins in soybean using tertiary amine labeling and capillary electrophoresis coupled with electrochemiluminescence detection.

Capillary electrophoresis coupled with electrochemiluminescence detection for the separation and determination of thyreostatic drugs in animal feed.

Simultaneous determination of omeprazole and their main metabolites in human urine samples by capillary electrophoresis using electrospray ionization-mass spectrometry detection.

Analysis of underivatized cellodextrin oligosaccharides by capillary electrophoresis with direct photochemically induced UV-detection.

Characterization of sulphoalkyl ether derivatives of beta-cyclodextrin by capillary electrophoresis with indirect UV detection.

Comparison of different sorbent materials for solid-phase extraction of selected drugs in human urine analyzed by UHPLC-UV.

Electromembrane extraction of salivary polyamines followed by capillary zone electrophoresis with capacitively coupled contactless conductivity detection.

Simultaneous determination of citrulline and arginine in human blood plasma by capillary electrophoresis with ultraviolet absorption detection.

Simultaneous determination of phenylurea herbicides in yam by capillary electrophoresis with electrochemiluminescence detection.

Determination of tamoxifen and its metabolites in human plasma by nonaqueous capillary electrophoresis with contactless conductivity detection.

Sensitive determination of four β2-agonists in pig feed by capillary electrophoresis using on-line sample preconcentration with contactless conductivity detection.

Quantitative determination of native proteins in individual human erythrocytes by capillary zone electrophoresis with laser-induced fluorescence detection.

Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis.