Anal. Chem. 2001, 73, 2178-2182
Simultaneous Quantification of Etoposide and Etoposide Phosphate in Human Plasma by Capillary Electrophoresis Using Laser-Induced Native Fluorescence Detection Uta Birgitta Soetebeer,† Marc-Oliver Schierenberg,‡ Harald Schulz,‡ Georg Hempel,† Peter Andresen,‡ and Gottfried Blaschke*,†
Institute of Pharmaceutical Chemistry, University of Mu¨nster, Hittorfstrasse 58-62, D-48149 Mu¨nster, Germany, and Faculty of Physics, University of Bielefeld, Universita¨tsstrasse 25, D-33615 Bielefeld, Germany
A method using capillary electrophoresis with UV laserinduced native fluorescence detection was developed as a sensitive and selective assay for the simultaneous determination of etoposide and etoposide phosphate in human plasma. Laser-induced native fluorescence detection with a frequency-doubled argon ion laser at an excitation wavelength of 257 nm was used for the simultaneous assay of etoposide and etoposide phosphate in plasma to improve the sensitivity compared to that obtained with UV absorption. The detection system consists of an imaging spectrograph and an intensified CCD camera which views an illuminated 1.5-mm section of the capillary. This setup is able to record the whole emission spectra of the analytes to achieve additional wavelengthresolved electropherograms. In the concentration range of 200 µg/L-50 mg/L in plasma for etoposide and 100 µg/L-20 mg/L for etoposide phosphate, coefficients of correlation were better than 0.998. Within-day variation determined with three different concentrations showed accuracies ranging from 91.0 to 109.3% for etoposide and from 91.2 to 109.9% for etoposide phosphate (n ) 6) with a precision of about 8%. Day-to-day variation presented accuracies ranging from 91.8 to 107.9% for etoposide and from 94.4 to 109.3% for etoposide phosphate with a relative standard deviation less than 6% (n ) 5). To our knowledge, this is the first method for the simultaneous quantification of etoposide and etoposide phosphate in plasma samples. Etoposide, a topoisomerase II inhibitor, is a semisynthetic derivative of podophyllotoxin1-3 which has been used for the treatment of both hematologic and solid malignancies over the past 20 years. It is sparingly water soluble and must be solubilized * Corresponding author: Tel.: 49 251 8333311. Fax: 49 251 8332144. E-mail:
[email protected]. † University of Mu ¨ nster. ‡ University of Bielefeld. (1) Acherrath, W.; Niederle, N.; Raettig, R.; et al. Cancer Treat Rev. 1982, 9 (Suppl A), 3-13. (2) Chen, Gl.; Yung, L.; Rowe, T. C.; et al. J. Biol. Chem. 1984, 259, 1356013566. (3) Ross, W.; Rowe, T.; Glisson, B.; et al. Cancer Res. 1984, 44, 5857-5860.
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in a formulation which includes polysorbate 80, poly(ethylene glycol), citric acid, and ethanol.4 Administration of etoposide itself has been associated with serious side effects probably due to the solvents.5 Etoposide phosphate is the water-soluble phosphate ester of etoposide which behaves as a prodrug with minimal activity until hydrolyzed, presumably by endogenous phosphatases.6 Once administered, etoposide phosphate is rapidly converted in vivo to etoposide.7 Analytical methods used so far for the determination of etoposide phosphate or etoposide are high-performance liquid chromatography (HPLC) with fluorimetric, UV, or electrochemical detection8-14 and capillary electrophoresis (CE) with UV absorption detection with a highly sensitive cell (Z-cell).15 There is no method available for the simultaneous assay of etoposide and etoposide phosphate. This study describes a sensitive and highly selective CE method with laser-induced native fluorescence detection for the simultaneous quantification of etoposide and etoposide phosphate in plasma samples. A frequencydoubled argon ion laser (λem ) 257 nm) was used. Lasers in the (4) Dorr, R. T.; von Hoff, D. D.; et al. Cancer chemotherapy handbook; Appleton and Lang: Norwalk, CT, 1993; pp 460-464. (5) Toxicity of Chemotherapy; Antman, K. H., Mayer, K. J., Frei, E., Perry, M. C., Yarbro, J. W., Eds.; Grune and Stratton: Orlando, 1984; pp 525-526. (6) Senter, P. D.; Saulinier, M. G.; Schreiber, G. J. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4842-4846. (7) Rose, W. C.; Basler, G. A.; Trail, P. A.; et al. Invest New Drugs 1990, 8, 525-532. (8) Kaul, S.; Igwemezie, C. N.; Stewart, S. Z.; Fields, S. Z.; Kosty, M.; Levithan, N.; Bukowski, D.; Gandara, D.; Goss, G.; Dwyer, P.; Schacter, L. P.; Barbhaiya, R. H. J. Clin. Oncol. 1995, 13 (11), 2835-2841. (9) Soni, N.; Meropol, N. J.; Pendyala, L.; Noil, D.; Schacter, L. P.; Gunton, K.; Creaven, P. J. J. Clin. Oncol. 1997, 15 (2), 766-772. (10) Brooks, D. J.; Srinivas, N. R.; Alberts, D. S.; Thomas, T.; Igwemezie, C. N.; McKinney, L. M.; Randolph, J.; Schacter, L. P.; Kaul, S.; Barbhaiya, R. H. Anti-Cancer Drugs 1995, 6, 637-644. (11) Porter, D.; Boddy, A.; Thomas, H.; Lind, M.; Newell, D.; Calvert, A. H.; Robson, L.; Brampton, M.; Abrahamsen, K.; Winograd, B. Semin. Oncol. 1996, 23 (No. 6, Suppl. 13), 34-44. (12) Thompson, D. S.; Greco, F. A.; Miller, A. A.; Srinivas, N. R.; Igwemezie, L. N.; Hainsworth, J. D.; Schacter, L. P.; Barbhaiya, R. H.; Garrow, G. C.; Hande, K. R. Clin. Pharmacol. Ther. 1995, 57 (No. 5), 499-507. (13) Chabot, G. G.; Armand, J. P.; Terret, C.; deForni, M.; Abigerges, D.; Winograd, B.; Igwemezie, L. N.; Schacter, L. P.; Kaul, S.; Ropers, J.; Bonnay, M. J. Clin. Oncol. 1996, 14 (No. 7), 2020-2030. (14) Manouelov, K. K.; McGuere, T. R.; Gordon, B. G.; Gwilt, P. R. J. Chromatogr. 1998, B681, 317-322. (15) Mrestani, Y.; Neubert, R. Electrophoresis 1998, 19, 3022-3025. 10.1021/ac001467v CCC: $20.00
© 2001 American Chemical Society Published on Web 04/10/2001
deep UV range are already available, but these lasers have rarely been used for detection in CE routinely. MATERIALS AND METHODS 1. Chemicals. Etoposide and etoposide phosphate (as Etopophos) were purchased from Bristol Myers Squibb (Munich, Germany). Methylenedioxymethamphetamine (MDMA), which was used as an internal standard (IS), was a gift from the Department of Forensic Medicine of the University of Mu¨nster. All chemicals were of analytical grade. Sodium tetraborate (Borax) and sodium hydroxide were purchased from Merck (Darmstadt, Germany). The water was deionized and bidistilled. 2. Apparatus. A modified SpectraPHORESIS 100 CE system (Thermo Separation Products, ThermoQuest Analytische Systeme, Egelsbach, Germany) and fused silica capillaries with 363 µm o.d., 50 µm i.d., an effective length of 55 cm, and a total length of 75 cm were used. The UV-LIF detector consisted of a frequency-doubled argon ion laser (Lexel 95 SHG, Lexel Laser, Polytec, Waldbronn, Germany) operating at 257 nm with a power of 200 mW to provide the excitation wavelength. An on-column detection window was created by removing a 4-mm section of the polyimide coating on the fused silica tubing. This is different from many others LIFCZE systems, where the laser beam is focused to a spot of less than 100 µm, thus illuminating the analyte for only several milliseconds.16-19,22 In the system described here, the capillary is illuminated with the laser profile at a length along the detection window of 1.5 mm and a height according to the inside diameter of the capillary (50 µm) using a 40-mm focal length cylindrical quartz lens for focusing. The resulting fluorescence from this section is imaged onto a spectrograph with an attached intensified CCD camera (Imager, La Vision Bio Tec, Bielefeld, Germany). This setup is used to achieve wavelength resolution of the emitted light, allowing the additional registration of the emission spectra which are read out by the CCD camera.18,20,21,23 The fluorescence is collected during the entire residence time of the analyte band in the capillary section of about 1.5 mm, thus enhancing significantly the sensitivity of the LIF system. The laser-induced natural fluorescence (LINF) signal was collected at an angle of 90° to the excitation light with a spherical aluminum mirror (diameter 5 cm, f/number 1.1) to suppress chromatic aberration. An imaging spectrograph (Multispec 77417, L.O.T.-Oriel, Darmstadt, Germany) with a 1200 lines/mm grating, holographic blazed at 250 nm is used. With the present CCD camera, the covered spectral range is 160 nm wide, adjustable between 180 and 400 nm.24 A CCD camera was used with a readout rate of 9.9 Hz and a binning factor of 10 × 80.20 (16) Lee, T. E.; Yeung, E. S. Anal. Chem. 1992, 64, 3045. (17) Lee, T. E.; Yeung, E. S. J. Chromatogr. 1992, 595, 319. (18) Timpermann, A. T.; Sweedler, J. V. Analyst 1996, 121, 45R-52R. (19) Lillard, J.; Yeung, E. S. Anal. Chem. 1996, 68, 2897. (20) Sweedler, J. V.; Shear, J. B.; Fishman, H. A.; Zare, R. N.; Scheller, R. H. Anal. Chem. 1991, 63, 496. (21) Timperman, A. T.; Olderburg, K. E.; Sweedler, J. V. Anal. Chem. 1995, 67, 3421. (22) Schierenberg, M. O. UV Laser induzierte wellenla¨ngenaufgelo¨ste Detektion der nativen Fluoreszenz von biologischen Proben in der Kapillarelektrophorese, Dissertation, December 2000. (23) Nouadje, G.; Simeon, N.; Couderc, F. Analusis 1996, 24, 360. (24) Fuller, R. R.; Moroz, L. L.; Gilette, R.; Sweedler, J. V. Neuron 1998, 20, 173.
Figure 1. Schematic description of the experimental setup.
The wavelength-resolved CE-LINF data were processed with custom algorithms using Mathcad 7 (Mathsoft Inc.). Figure 1 shows a schematic description of the experimental setup. 3. Electrophoretic Conditions. A 150 mM tetraborate buffer adjusted to pH 10.6 with 150 mM NaOH was used as the running buffer. The applied voltage was +20 kV, and the temperature was maintained at 19.0 °C. The injection was performed hydrodynamically. Detection was started 6 min after the electrophoretic run. 4. Internal Standard Solution. As the internal standard (IS), a stock solution of 3,4-methylenedioxymethamphetamine (MDMA) (500 µg/L) in water was prepared weekly and stored at 4 °C. 5. Standard Solutions. Stock solutions of 1 g/L etoposide were prepared in methanol and stored at 4 °C. Etopophoslyophilized substance was dissolved in 113.6 mL of deionized and bidistilled water/methanol (1:1) to obtain a concentration of 1 g/L etoposide phosphate. Standard solutions from 2 to 500 mg/L for etoposide and from 1 to 200 mg/L for etoposide phosphate were prepared daily by dilution with deionized and bidistilled water. 6. Plasma Samples. Pooled blank plasma collected from a male healthy volunteer was used to prepare spiked plasma samples. A 10-µL aliquot of the respective standard solution of etoposide and etoposide phosphate was added to 80 µL of blank plasma in order to obtain calibration series of 0.2, 0.5, 1, 5, 10, 20, and 50 mg/L for etoposide and 0.1, 0.2, 0.5, 1, 5, 10, and 20 mg/L for etoposide phosphate. Each sample was precipitated with 150 µL of acetonitrile and centrifuged for 10 min. The supernatant was transferred into a clean tube and evaporated in a gentle stream of nitrogen. The sample was reconstituted in 100 µL of deionized and bidistilled water containing 500 µg/L internal standard. 7. Validation. 7.1. Calibration. The calibration curves were obtained daily by analyzing plasma samples with known concentrations of the analytes. Linear regression analysis according to the IS method was performed by plotting the peak area ratio of etoposide or etoposide phosphate/internal standard against the known concentrations of the samples. 7.2. Precision and Accuracy. Intraday variation was assessed from six replicated determinations of three concentrations in the tested range (0.2, 1, and 20 mg/L for etoposide and 0.1, 0.5, and 10 mg/L for etoposide phosphate). Intraday accuracy was expressed as the mean of the results relative to the theoretical values (%). The intraday precision of the method was expressed Analytical Chemistry, Vol. 73, No. 10, May 15, 2001
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Figure 2. Structure of etoposide (1) and etoposide phosphate (2) and their emission spectra.
as the relative standard deviation (RSD) of the assays prepared for intraday accuracy. Interday variation was determined by analyzing replicates of spiked plasma samples with the same concentrations on five separate days. Interday accuracy was expressed as the mean of the assays relative to the theoretical values (%). The interday precision was expressed as the RSD of the assays made for the interday accuracy. 7.3. Detectability. The limit of detection (LOD) was defined as the analyte concentration resulting in a signal-to-noise (S/N) ratio of 3:1. The limit of quantification (LOQ) was defined as the analyte concentration that could be analyzed with acceptable precision and accuracy according to the ICH guidelines.25 7.4. Selectivity. Selectivity was determined by comparing a blank sample with a plasma sample spiked with etoposide, etoposide phosphate, and internal standard. 8. Application. Plasma samples from a 12-year-old patient with a weight of 37.8 kg were collected during a 24 h i.v. administration of 380 mg/m2 etoposide phosphate after 23 h and after 21 h on the next cycle, respectively. The concentrations of etoposide and etoposide phosphate were calculated from the calibration equations obtained in section 7.1 (above). RESULTS AND DISCUSSION The combination of the characteristic fluorescence emission spectra and the retention time of the analytes in CE allows a more complete indentification compared to that obtained with the migration time alone, which would not be possible with filterbased, single-channel fluorescence detectors. When excited at 257 (25) ICH Harmonised Tripartite Guideline, Q2A, Text on Validation of Analytical Procedures, Recommended for Adoption at Step 4 of the ICH Process on 27 October 1994 by the ICH Steering Committee.
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Figure 3. Blank plasma compared to a spiked plasma sample. Experimental conditions: 150 mM borax buffer, pH 10.6, applied voltage +20 kV. Concentration of the sample: 10 mg/L etoposide and 5 mg/L etoposide phosphate.
nm, both etoposide and etoposide phosphate exhibited almost identical emission sprectra, as shown in Figure 2. To obtain the optimum S/N ratio for these analytes, the emitted light was collected in the range from 295 to 330 nm. The main advantage of the method is the capability for simultaneous quantification of etoposide and etoposide phosphate in plasma samples. Several other methods have been described in the literature for determination of etoposide and etoposide phosphate in different biological fluids,7-14 but there is no technique available for simultaneous quantification of them. Figure 3 shows an electropherogram of a plasma sample compared to the signal for blank plasma. The approximate migration times of etoposide, etoposide phosphate,
Table 1. Results of the Validation (Interday): Simultaneous Determination of Etoposide and Etoposide Phosphate in Human Plasma (n ) 5) etoposide theor concn (mg/L) 20
day 1 2 3 4 (av) 5
average (mg/L) interday precision 1
etoposide phosphate
found concn (mg/L)
theor concn (mg/L)
19.2 21.2 19.9 19.7 19.9
96.4 105.9 99.6 98.6 99.5
20.0 3.5
100.0
average (mg/L) interday precision
0.930 1.072 1.017 0.981 1.000
93.0 107.9 101.8 98.1 100.0
0.5
1.000 5.2
100
1 2 3 4 (av) 5
average (mg/L) interday precision 0.2
accuracy (%)
1 2 3 4 5
average (mg/L) interday precision
10
day
found concn (mg/L)
1 2 3 4 (av) 5
1 2 3 4 (av) 5
average (mg/L) interday precision
0.183 0.197 0.195 0.195 0.194
91.8 98.6 97.5 97.5 97
0.1
1 2 3 4 (av) 5
0.193 2.8
96.5
average (mg/L) interday precision
accuracy (%)
9.7 10.2 10.0 10.1 10.7
97.4 102.6 100.3 101.1 106.8
10.2 3.4
101.6
0.505 0.516 0.497 0.478 0.471
101.1 103.3 99.4 95.6 94.4
0.493 3.8
98.8
0.106 0.107 0.105 0.105 0.109
106.8 107.0 105.2 102.7 109.3
0.106 2.3
106.2
Table 2. Results of the Validation (Intraday): Simultaneous Determination of Etoposide and Etoposide Phosphate in Human Plasma (n ) 6) etoposide theor concn (mg/L) 20
average (mg/L) intraday precision
etoposide phosphate found concn (mg/L)
accuracy (%)
theor concn (mg/L)
found concn (mg/L)
accuracy (%)
18.9 19.4 20.2 18.8 20.7 20.1
94.6 97.1 101.2 94.2 103.7 100.6
10
9.8 9.7 9.6 9.7 10.9 10.7
98.2 97 96.4 97.4 109.9 107.5
19.7 3.9
98.6
average (mg/L) intraday precision
10.1 5.9
101.1
1
0.989 0.981 0.975 0.955 0.949 1.036
98.9 98.1 97.5 95.5 95.0 103.6
average (mg/L) intraday precision
0.981 3.2
98.1
0.2
0.189 0.189 0.200 0.218 0.189 0.182
94.8 94.6 100.4 109.3 95.0 91.0
average (mg/L) intraday precision
0.195 6.7
97.5
and the internal standard were 6, 11, and 16 min, respectively. The total analysis time was 17 min. No interference of endogenous substances with the studied compounds was observed because the emission spectra were recorded and appropriate spectral ranges were individually selected for etoposide and etoposide phosphate in the data processing. The calibration curves of
0.5
average (mg/L) intraday precision 0.1
average (mg/L) intraday precision
0.469 0.456 0.511 0.503 0.489 0.439
93.9 91.2 102.3 100.8 97.8 107.9
0.478 5.9
95.6
0.108 0.109 0.092 0.104 0.092 0.108
108 109.1 92.5 104.9 92.8 108.7
0.102 7.7
102.7
etoposide and etoposide phosphate were linear over the whole concentration range. Correlation coefficients were higher than 0.998. Tables 1 and 2 list the results obtained from the inter- and intraday precision and accuracy studies. For all samples inter- and intraday precision was better than 10%. The inter- and intraday Analytical Chemistry, Vol. 73, No. 10, May 15, 2001
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The LOD given by the S/N ratio of 3:1 was determined to be 100 µg/L for etoposide and 30 µg/L for etoposide phosphate; thus, the sensitivity is improved about 30 times compared to that of CE methods using UV detectors. The limit of quantification was determined to be 0.2 mg/L for etoposide and 0.1 mg/L for etoposide phosphate. The electropherograms of plasma samples from a cancer patient are shown in Figure 4. After administration, etoposide phosphate is rapidly hydrolyzed to etoposide. The application of this method for pharmacokinetic studies is in process.
CONCLUSION CE-LINF detection proved to be a rapid, specific, sensitive, and accurate method for the simultaneous quantification of etoposide and etoposide phosphate in human plasma samples.
ACKNOWLEDGMENT This work was supported by the Forschungsvereinigung der Arzneimittelhersteller e.V. (FAH), the Arbeitsgemeinschaft industrieller Forschungseinrichtungen (AiF, project no. 11782N/ 1), the Bundesministerium fu¨r Wirtschaft und Technik (BMWI), and the Fonds der Chemie. Figure 4. Plasma sample of a cancer patient during a 24 h i.v. administration of 380 mg/m2 etoposide phosphate: (a) after 23 h; (b) after 21 h on the next cycle.
Received for review December 12, 2000. Accepted January 27, 2001.
accuracy ranged between 90 and 110%. These results meet the requirements for the validation of biological samples.24
AC001467V
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