Chromatographia (2011) 74:151–155 DOI 10.1007/s10337-011-2019-1
FULL SHORT COMMUNICATION
MEKC Determination of Nilutamide in Human Serum Using Sweeping in High Salt Sample Matrix Joanna Znaleziona • Vı´teˇzslav Maier • Jan Petr • Jan Chrastina • Juraj Sˇevcˇ´ık
Received: 8 November 2010 / Revised: 17 March 2011 / Accepted: 21 March 2011 / Published online: 21 April 2011 Ó Springer-Verlag 2011
Abstract The determination of the antiandrogen drug nilutamide in human serum by MEKC using flutamide as an internal standard is described. Several parameters influencing the separation, such as the running electrolyte composition concerning the micelle concentration and pH, are discussed. MEKC separation was achieved within 7 min using 50 mM sodium borate pH 9.0 with the addition of 50 mM sodium dodecylsulfate at ?20 kV. The proposed method was applied to determination of nilutamide in spiked human serum samples after protein precipitation with acetonitrile. The increasing of sensitivity for determination of nilutamide in serum was done by sweeping in a high salt concentration sample matrix when the injection of a larger volume of sample diluted in 150 mM NaCl was applied. The limit of detection after the preconcentration step for nilutamide was 26 lg L-1. Keywords Micellar electrokinetic chromatography High salt sample stacking Sweeping Serum Nilutamide
J. Znaleziona J. Sˇevcˇ´ık Department of Analytical Chemistry, Faculty of Science, Palacky´ University in Olomouc, 17. Listopadu 12, Olomouc 77146, Czech Republic V. Maier (&) J. Petr Regional Centre of Advanced Technologies and Materials, Department of Analytical Chemistry, Faculty of Science, Palacky´ University in Olomouc, 17. Listopadu 12, Olomouc 77146, Czech Republic e-mail: [email protected]
J. Chrastina Faculty of Health Sciences, Palacky´ University in Olomouc, Tr. Svobody 8, Olomouc 77146, Czech Republic
Introduction Nilutamide (5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl) phenyl]imidazolidine-2,4-dione), as a non-steroidal antiandrogen drug, is one of the compounds used to treat prostate cancer. This antiandrogen is applied in the monotherapy of prostate cancer to block the action of androgen hormones . It can also be used in hormone therapy . Determination of nilutamide is important for both drug quality control and drug monitoring in body fluids. To the best of our knowledge, no analytical methods have been published on the determination of nilutamide in tablets or in biological fluids so far. The unchanged nilutamide represents the major active compound . Creavena et al.  concluded that the decay of nilutamide in human plasma is very slow based on results obtained by 14Carbonnilutamide, radioimmunoassay, and HPLC. The mean steady-state plasma concentration of nilutamide in patients, after repeated dose treatment, was 6–7 mg L-1 for the 300 mg daily dose and 3–4 mg L-1 for the 150 mg daily dose. The structurally similar antiandrogen drug is flutamide. Flutamide and nilutamide are not administered together in medicinal therapy; thus, flutamide can be employed as the internal standard for precise determination of low concentration levels of nilutamide. The chemical structures of the structurally similar antiandrogens nilutamide and flutamide are shown in Fig. 1. MEKC is a well-established separation method for analysis of drugs in body fluids. Separation is based on different distributions of analytes between the pseudostationary phase of micelles and the bulk phase of the running electrolyte. MEKC is a useful technique particularly for the separation of neutral and charged small molecules with high efficiency, short analysis time, and minimum sample
J. Znaleziona et al.
Fig. 1 Chemical structures of the studied nilutamide and flutamide (internal standard)
and reagent consumption [5–8]. If using UV detection, one of the drawbacks of MEKC is the relatively poor concentration sensitivity. To improve the concentration sensitivity, several on-line sample preconcentration techniques such as sweeping have been developed [9, 10]. Sweeping is based on the interaction between the analyte and micellar phase and electrophoresis to induce the interaction. The preconcentration mechanism of sweeping when using micelles as a buffer additive relies on the electrokinetic focusing of large injection volumes of analytes that possess a strong affinity for the micellar phase. The velocity of analytes in the sample zone is modified upon interaction with the additive, resulting in analytes being ‘‘swept’’ or focused into a narrow band . The aims of this work were to develop a sensitive method for the determination of the antiandrogen drug nilutamide using flutamide as the internal standard and to apply the developed method for the determination of nilutamide in spiked human serum samples.
Experimental Apparatus and Conditions All experiments were performed on a CE instrument (HP3D CE, Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector. The detection wavelength was 200 nm. Uncoated fused-silica capillaries (MicroSolv Technology Corp., Eatontown, NJ) of 50 lm i.d., 40.0 cm effective length, and 48.5 cm total length were used. The capillary was rinsed 15 min with 1 M sodium hydroxide, 15 min with deionized water, and then 5 min with the running electrolyte at the beginning of each working day. The capillary was rinsed with the running electrolyte for 5 min between individual runs. Injection was performed at 50 mbar pressure for 5 s. For sample enrichment, the 150 s injection at 50 mbar was applied on extracted sera samples. A voltage of ?20 kV was applied for all experiments. The capillary thermostat was set at 25 °C. All of the measurements were performed five times unless stated otherwise. The analytes were identified by spiking (standard addition method).
Standards (flutamide, nilutamide) were supplied by SigmaAldrich (St. Louis, MO). Methanol, acetonitrile, ethylacetate, sodium hydroxide, boric acid, phosphoric acid, SDS, and sodium chloride were also purchased from Sigma. All reagents were of analytical grade purity. Running electrolytes were prepared by dissolving an appropriate amount of boric acid or phosphoric acid in deionized water (18 MX cm, Millipore, MA) and titrating it to the desired pH using 50% (w/w) sodium hydroxide solution. An adequate amount of sodium dodecylsulfate was added at the end, and the solution was sonicated for 15 min at ambient temperature. The stock standard solutions of flutamide and nilutamide were prepared separately in 10 mg mL-1 concentration in methanol and then diluted to the appropriate lower concentration by deionized water. Stock standard solutions and diluted solutions were stored in a refrigerator at -20 °C. Real Sample Preparation Human blood was obtained from healthy volunteers. Centrifugation at 5,0009g for 15 min was applied to obtain blood sera as the supernatant for the next experiments. Blood serum (200 lL), spiked with nilutamide and internal standard (flutamide), was mixed with acetonitrile (200 lL) as the deproteination and extraction solvent. After centrifugation for 10 min at 3,0009g, the acetonitrile phase (supernatant) was transferred to a plastic conical tube and dried in a gentle nitrogen stream. The residues were reconstituted with 200 lL of 150 mM NaCl in deionized water and directly injected into the capillary. The blank serum samples were prepared by the same deproteination procedure.
Results and Discussion Capillary Zone Electrophoresis—Brief Study of Acidobasic Properties Acid-base properties of the studied antiandrogen nilutamide have not been published yet. Nilutamide is a hydantoin derivative of flutamide. Reddy et al.  published a pK0 a value of 4.58 for flutamide in the mixture of ethanol:Britton-Robinson buffer (20:80, v/v). From this point of view, we studied the migration behavior of both analytes in a pH range from 2.5 to 13.0 in electrolytes based on sodium phosphate, sodium borate, and sodium hydroxide as a first approach. Both antiandrogens comigrated with the electroosmotic flow in the pH range 2.5–12.5, and their separation according to differences of
MEKC Determination of Nilutamide in Human Serum
free solution mobilities (by capillary zone electrophoresis) was not possible. Only the slight migration of nilutamide as a very weak anion was observed at pH 13.0, but flutamide still migrated with the electroosmotic flow. The obtained results showed that the separation of both drugs by capillary zone electrophoresis is not possible. Moreover, the UV spectra of both drugs in the studied pH range did not show any changes significant for dissociation equilibrium, which verified that our results concerning the acid-base properties of both compounds were correct. Unfortunately, these results were not in agreement with the published results for flutamide . In the next step, the separation of the studied antiandrogens by MEKC was performed to obtain good resolution for the target analyte and internal standard. MEKC Separation Firstly, the influence of the SDS concentration on migration times and resolution of the drugs was investigated in the range of 10 to 150 mM in 50 mM sodium borate running electrolyte pH 9.0 (Fig. 2a). Sodium borate running electrolyte was chosen because sodium borate electrolyte exhibits low generation of the electric current (low Joule heating) in the capillary in comparison with phosphate electrolytes with SDS. The resolution decreased and retention times increased with increasing SDS micelle concentration in the running electrolyte, but efficiency values exhibit the maximum for both studied drugs at a concentration of 50 mM SDS. As a compromise between good resolution and high separation efficiencies, 50 mM SDS concentration in the running electrolyte was chosen for the next experiments. The influence of sodium borate concentration on the separation of studied antiandrogens in the running electrolyte with 50 mM SDS was studied in the range of 10 to 100 mM, too. The highest peak efficiency and sufficient
Fig. 2 a The influence of the SDS concentration of 50 mM sodium borate pH 9.0 on resolution of nilutamide and flutamide. Conditions: 50 mM sodium borate pH 9.0, U = 20 kV, sample injection 50 mbar/ 5 s, k = 200 nm, concentration of each 50 mg L-1 of antiandrogen.
resolution ([5.7) were achieved with 50 mM sodium borate (data not shown). Finally, the study of the influence of the pH value of the running electrolyte was carried out in the range of 8.0 to 10.5 using 50 mM sodium borate with 50 mM SDS (Fig. 2b). In this case, the pH value mainly influenced the electroosmotic mobility because the effect of pH on the dissociation equilibrium of studied analytes is negligible; see ‘‘Capillary Zone Electrophoresis—Brief Study of Acidobasic Properties.’’ The pH 9.0 was chosen as the best value according to the highest efficiency of both peaks, acceptable resolution ([6.0), and the best repeatability of analyses. The final conditions for separation of nilutamide and flutamide were 50 mM sodium borate pH 9.0 with 50 mM SDS at 20 kV. An example of the separation of both drugs at final conditions is shown in Fig. 3a. On-Line Sample Enrichment Under the best conditions where both nilutamide and the internal standard flutamide were baseline separated, the limit of detection for nilutamide (LOD, based on 3 9 S/N ratio) was 1,030 lg L-1, and the limit of quantification (LOQ, based on 10 9 S/N ratio) was 4,130 ± 4 lg L-1. The LOQ value did not satisfy the demands for the determination of lower concentrations of the antiandrogen drug in serum samples (a therapeutic serum concentration level of nilutamide in case of a lower dosage of 150 mg per day is around 3 mg L-1). An on-line sample enrichment methodology that allows quantification of lower concentrations of nilutamide in serum after single or repeated doses is needed. On-line sample enrichment was done by sweeping in high salt matrices as observed by Palmer et al. . The mentioned sample enrichment is induced by addition of the
b The influence of the pH of 50 mM sodium borate with addition of 50 mM SDS on resolution (other conditions are the same as in Fig. 2a)
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Fig. 3 a Electropherogram of MEKC separation of flutamide and nilutamide standards. Conditions: 50 mM sodium borate pH 9.0 with 50 mM SDS, U = 20 kV, sample injection 50 mbar/5 s, k = 200 nm, concentration of each antiandrogen is 50 mg L-1. b The MEKC electropherogram of spiked human blood serum after
sample enrichment step. Conditions: sample injection 50 mbar/150 s, k = 200 nm. Concentration of flutamide (IS) was 150 lg L-1 and spiked concentration of nilutamide was 100 lg L-1. Other conditions are the same as in a)
salt to a sample matrix to create conditions that lead to the preconcentration of analytes. The high salt concentration in the sample matrix induced the higher conductivity in the sample zone above that in the separation buffer. Field amplification occurred within the applied voltage in the buffer zone and the charged SDS micelle stack at the detector side of the sample matrix (not the neutral analytes). Consequently, the stacked SDS micelles strongly interacted with neutral analytes because of their high zonal concentration. The mechanism of the SDS micelle preconcentration has been explained as the field amplification based stacking , sweeping , and recently the transient isotachophoresis . In our case, sodium chloride was chosen as the the salt to create the higher conductivity sample. The concentration range from 100–200 mM of NaCl varied in the sample, and a long plug of the sample was injected into the capillary (50 mbar 9 100 s). The influence of the concentration of NaCl in the sample matrix
on peak areas of nilutamide and flutamide is shown in Fig. 4a. Addition of 150 mM NaCl gave the best results for on-line preconcentration of nilutamide. As the second parameter, the injection time (sample plug length) varied from 60 to 200 s by injection pressure of 50 mbar at a constant concentration of 150 mM NaCl in the sample matrix (Fig. 4b). The corrected area of nilutamide increased with increasing injection time up to 150 s (the corresponding sample plug volume was 753 nL). Therefore, an injection time of 150 s was chosen for the analyses of nilutamide. In all cases, the short plug of running electrolyte (50 mbar 9 2 s) was introduced after sample injection to avoid transferring analytes to the inlet vial. The electropherograms of blank blood serum and spiked serum after protein precipitation and preconcentration are shown in Fig. 3b. The described sample enrichment method allowed the determination of lower concentrations of nilutamide.
Fig. 4 a The influence of NaCl concentration in the sample matrix on peak area during the sample enrichment step (concentration of nilutamide and flutamide is 100 lg L-1). Other conditions are the same as in Fig. 3a. b The influence of injection time on peak area
during the sample enrichment step. Conditions: sample injection 50 mbar, sample matrix contains 150 mM NaCl in deionized water (concentration of nilutamide and flutamide is 100 lg L-1). Other conditions are the same as in Fig. 3a
MEKC Determination of Nilutamide in Human Serum
Intraday and interday reproducibility values of corrected migration times were 0.92 and 4.70%, respectively, and intraday and interday reproducibility values of corrected peak areas were 2.61 and 10.82%, respectively. The intraday precision was determined from the calibration curves between 90 and 500 lg L-1 of nilutamide (six points, each point measured ten times). The interday precision was determined by taking all the calibrations made in 3 days with flutamide as internal standard (150 lg L-1). The calibration equation for the method with a preconcentration step was y = (1.84 ± 0.05) x ? (0.25 ± 0.01), r = 0.992 (n = 5). Recoveries were tested for different precipitation agents (methanol, acetonitril, ethylacetate) in the spiked blood serum at a concentration level of 100 lg L-1. The found recoveries of nilutamide from blood serum were: 85 ± 2% for methanol, 95 ± 2% for acetonitrile, and 61 ± 3% for ethylacetate. Acetonitrile was finally chosen as the precipitation and extraction agent because it had the highest obtained recovery value. The LOD (based on 3 9 S/N ratio) was 26 lg L-1 (82 nmol L-1) for nilutamide, and the LOQ (based on 10 9 S/N ratio) was 87 ± 2 lg L-1, which represented the enrichment factor of 47.5. The on-line sample enrichment method increased the detection sensitivity of nilutamide; moreover, the method can be used conversely for the analysis of low concentrations of flutamide in human serum.
Conclusion A new MEKC method for the determination of nilutamide in human sera employing flutamide as the internal standard was described. The method allowed relatively fast separation of the studied analyte with small sample consumption after protein precipitation with acetonitrile and with an
on-line preconcentration step based on the injection of a large volume of the sample diluted in 150 mM NaCl-water solution. The method has the potential to monitor the actual serum concentration in tens of nanomolar levels of nilutamide in human sera or to be used in other pharmacokinetic studies. Acknowledgments This work was supported by the Research Project of the Ministry of Education of the Czech Republic (grant no. MSM6198959216), by the grant from Palacky´ University in Olomouc, Czech Republic, FZV_2010_001, and from the Operational Program Research and Development for Innovations—European Social Fund (project CZ.1.05/2.1.00/03.0058 of the Ministry of Education, Youth and Sports of the Czech Republic).
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