Research article Received: 29 January 2014,

Revised: 27 March 2014,

Accepted: 4 April 2014

Published online in Wiley Online Library: 7 July 2014

(wileyonlinelibrary.com) DOI 10.1002/bio.2700

Capillary electrophoresis coupled with electrochemiluminescence for determination of atomoxetine hydrochloride and the study on its interactions with three proteins Hua-jin Zeng,a Ran Yang,b Ying Zhang,b Jian-jun Lib and Ling-bo Qub,c* ABSTRACT: A simple, rapid and sensitive method for the determination of atomoxetine hydrochloride (AH) by capillary electrophoresis with electrochemiluminescence detection (CE-ECL) using tris(2,2′-bipyridyl) ruthenium (II) was developed. Under optimized conditions, the determinations of AH in capsules and rat plasmas and the study on its interactions with three plasma proteins, including bovine serum albumin, cytochrome c and myoglobin were performed successfully. Relative to some previous studies, in this paper the methodologies for the determination of AH in aqueous solution and spiked plasma systems were established, respectively. By comparing the difference between the two work curves of two systems, the matrix effect in plasma samples on the determination of AH by the CE-ECL method was discussed in detail. The results indicated that the effect of the matrix in plasma samples should not be ignored even if no obvious interference was found in the electropherograms and the establishment of method validation in complex samples by the CE-ECL method was necessary. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: capillary electrophoresis; electrochemiluminescence; atomoxetine hydrochloride; interaction; protein

Introduction

124

Atomoxetine hydrochloride (AH), (–)-N-methyl-3-phenyl-3-(otolyloxy)-propylamine hydrochloride (Fig. 1), is the first nonstimulant orally administered, selective norepinephrine reuptake inhibitor. The US Food and Drug Administration (FDA) approved it in November 2002 for the treatment of attention-deficit/ hyperactivity disorder in patients 6 years or older (1,2). Now, it has been used in the clinic in many countries. However, based on published results (3,4) and data on file with the manufacture of AH, some adverse effects, including abdominal pain, decreased appetite, vomiting (5), would occur in about 5% of patients. Therefore, to utilize AH further and effectively and enhance clinical safety, a simple, rapid, efficient method is needed for the determination of AH in biological samples, mostly in clinical monitors. Several analytical methods, including high-performance liquid chromatography (HPLC) (6–8), gas chromatography (9), liquid chromatography-mass spectrometry (10,11) and electrochemistry (12), have been developed for the determination of AH. These methods are reliable but expensive, not environmental-friendly and time-consuming. In recent years, there are increasing interests in coupling capillary electrophoresis (CE) separation with high-sensitive tris (2,2′-bipyridyl) ruthenium (II) (Ru(bpy)32+)-based electrochemiluminescence (ECL) detection for nitrogen-containing compounds analysis (13–18) because of its simplicity, rapidity, inexpensive instrumentation, high sensitivity for amines, good selectivity and low consumption of solvent and sample (19). However, in most of the published papers, the detection of the target drugs in biological samples was always carried out under the work curve of the standard system

Luminescence 2015; 30: 124–130

in which no complex matrix was considered. Theoretically speaking, such results are not reliable because the ECL intensity was very easily interfered by many factors in the complex matrix. Therefore, it is necessary to discuss the problem whether the matrix could be ignored when the complex samples were analyzed by the CE-Ru(bpy)32+-ECL method. As with many other drugs, AH may undergo some degree of reversible binding to plasma proteins, a process that may significantly affect its distribution in the body and the elimination rate. Most of the papers have focused on the study of interactions between drugs and serum albumin, a very important protein for transporting and depositing the drugs (20–22). As we know, there are still many other proteins such as cytochrome c (Cyt-C) and myoglobin (Mb) in the plasma. Studies on the binding of AH with the three above-mentioned proteins might be helpful for us to learn about its internal behaviors. Taking these factors into account, in this paper, a novel CE method with Ru(bpy)32+-ECL detection for the determination of AH in biological samples and its capsules and the study of its interactions with three proteins, including bovine serum albumin

* Correspondence to: Ling-bo Qu, Department of Chemistry, Zhengzhou University, Zhengzhou 450001, China. Email: [email protected] a

School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China

b

Department of Chemistry, Zhengzhou University, Zhengzhou 450001, China

c

College of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, China

Copyright © 2014 John Wiley & Sons, Ltd.

Determination of AH and study on its interactions with three proteins series of standard solutions with concentration in the range of 5.0 × 10–9 to 7.0 × 10–7 g/mL was obtained by further dilution of the stock solution with distilled water. All the solutions were stored at 4°C and filtered through a 0.22 μm membrane before use.

Figure 1. Molecular structure of atomoxetine hydrochloride.

Atomoxetine hydrochloride capsule analysis

(BSA), Cyt-C and Mb, was developed. Furthermore, considering the effect of the matrix in samples on the detection of the analyte, the two methodologies for the determination of AH in aqueous solution and spiked plasma systems were established, respectively. By comparing the difference between the two work curves of the two systems, the matrix effect in plasma samples on determination of AH by CE-ECL method was assessed in detail.

Ten pills of AH capsule (removed the capsule) were weighed and grounded to a fine powder by use of a pestle and mortar. The powder was dissolved in double-distilled water and the resulting solution was diluted to 50 mL in a calibrated flask. Suitable aliquots from this solution were taken and diluted with distilled water and filtered through 0.22 μm membrane for the determination of AH. The obtained solutions were injected and analyzed by the work curve of the standard solutions.

Experimental

Rat plasma analysis

Chemicals and materials

Rat plasma sampling. The plasma sampling procedure of AH used here was a modification of the technique reported by Hao et al. (23). Fifty μL 0.1 M NaOH and 500 μL ethyl acetate were added to 100 μL of the plasma. The resulting solution was thoroughly vortex-mixed for 3 min. After centrifugation at 1065 g for 20 min, the supernatant was transferred to a test tube and evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 100 μL 1.0 × 10–4 mol/L HCl solution and stored at 4°C until use.

AHtomoxet and its capsule were obtained from Henan Topfound Pharmaceutical Co. (Zhumadian, China). BSA, Cyt-C and Mb were purchased from Sigma Chenical Co. (USA). Tris (2,2′-bipyridyl) ruthenium(II) chloride hexahydrate was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). The phosphate buffer used in the detection cell as well as the electrophoresis running buffer was prepared by mixing different volumes of disodium hydrogen phosphate (Na2HPO4, 0.1 M) and sodium dihydrogen phosphate (NaH2PO4, 0.1 M). The pH was monitored with a PHS-3C pH meter (Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China). Before CE analysis, the required sample solutions and phosphate buffer were filtered through 0.22 μm membrane (Shanghai Xinya Purification Material Factory, Shanghai, China). All the reagents used were commercially available and of analytical grade. Double-distilled water was used throughout. Apparatus A CE-ECL detection system (Xi’an Remex Electronics Co. Ltd., Xi’an, China), equipped with a high-voltage power supply for electrokinetic injection and separation, potential control system, chemiluminescence detector and data processor, was used. Data acquisition and recording of electropherograms were accomplished with MPI-A software. The construction of the ECL detection cell was composed of a 500 μm diameter Pt disc working electrode, Pt wire counter electrode and Ag/AgCl reference electrode (KCl saturated). A high-voltage power supply was used to supply high voltage for the injection and separation of samples. Separations were performed in a 40 cm long fusedsilica capillary (Yongnian Photoconductive Fiber Factory, Hebei, China) with a 25 μm i.d. and 375 μm o.d. When first used, the new capillary was flushed with 0.1 M NaOH for 10 h and distilled water for 30 min. Before each run, the capillary was flushed with 0.1 M NaOH for 5 min and then with the corresponding running buffer until the baseline of ECL is flat. The running electrolyte (about 300 μL) was refreshed about 30 runs. Preparation of standard solutions

Luminescence 2015; 30: 124–130

Male and female Sprague– Dawley rats weighing 220–250 g were obtained from the Henan Laboratory Animal Center (Zhengzhou, China) and kept in an environmentally controlled breeding room for 2 days before starting the experiments. They were fasted for 12 h with free access to water before the experiments and fed with a standard laboratory food and water during experiments. All procedures involving animals were in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of People’s Republic of China. Three rats were administered an oral dose of 4 mg/kg AH. Thirty minutes before administration of AH, 0.5 mL of blank blood sample was withdrawn. At 3.0, 3.5, 4.0, 4.5 and 5.0 h after

Animals and drug administration.

Copyright © 2014 John Wiley & Sons, Ltd.

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The stock solution of AH was prepared by dissolving AH in doubledistilled water to give a final concentration of 1.0 × 10–4 g/mL. A

The plasma samples of standard calibration in the range of 8.0 × 10–9 to 2.0 × 10–6 g/mL were prepared by spiking the appropriate amount of stock solution into 100 μL of drug-free rat plasma. The calibration curves were constructed by plotting the ECL intensity to the corresponding plasma concentrations. The precision and accuracy of the method were evaluated by analyzing five replicate plasma samples at three concentration levels (4.0 × 10–8, 1.0 × 10–7, 1.0 × 10–6 g/mL). The concentration of each examined sample was calculated using calibration curves of plasma samples. Accuracy was defined as the rate of the calculated value by the standard curve to that of its true value, expressed as recovery rate (%). Precision was evaluated as the relative standard deviation (RSD) of five replicates detection. The extract recovery of AH was assessed by comparing the mean peak height of the regularly prepared plasma samples (4.0 × 10–8, 1.0 × 10–7 and 1.0 × 10–6 g/mL) with the mean peak height of the spike-after-extraction plasma samples, which represented 100% recovery. To prepare the spiked-after-extraction samples, blank plasma sampling was processed according to the procedure for the plasma sample preparation as described above. Method validation.

H.-j. Zeng et al. administration, the same volume of blood sample was withdrawn via orbit vein into heparinized centrifugation tubes and then centrifuged at 1065 g for 10 min at 4°C. The separated plasma was frozen at – 20°C before assay. When analyzed, 100 μL of the plasma was taken and pretreated as plasma sampling.

Interaction investigations The interactions between AH and three proteins were carried out by a microdialysis sampling technique, based on the method in Huang et al. (24). First, a dialysis bag with 100 μL 10 μM protein and 100 μL of 10 mM AH in it was incubated in 5 mL phosphate buffer solution (pH 6.6) at 37°C and the ECL intensity of AH outside the bag was detected every half an hour until no remarkable change in ECL intensity was found, which indicated that the binding had already reached its equilibrium. Then, a series of different volumes of 10 mM AH and 100 μL 10 μM protein were mixed in the dialysis bag and then incubated in 5 mL phosphate buffer solution (pH 6.6) at 37°C, while keeping their total volume at 1 mL. After the equilibrium, the ECL intensity outside the dialysis bag was measured and then the concentration of free AH could be estimated. The binding curve was plotted to estimate the binding site number and binding constant.

2+

1600 1400 1200 1000 800 600 400 200 0

a

1

1.05

1.1

1.15

Electrochemiluminescence behavior of Ru(bpy)32+ and atomoxetine hydrochloride The ECL behaviors of Ru(bpy)32+ and AH were studied at the Pt electrode. As shown in Fig. 2, after 0.5 μg/mL AH was added to the Ru(bpy)32+ solution (curve a), the ECL intensity increased significantly and reached a maximum at 1.15 V (curve b). This accorded with the oxidation potential of Ru(bpy)32+, which indicated that the oxidation of Ru(bpy)32+ played an important role in the AH-ECL reaction. Optimization of capillary electrophoresis– electrochemiluminescence conditions Effects of detection voltage. In the phenomenon of ECL, the intensity of light depends on the rate of light-emitting reaction, and the reaction rate is decided by the detection voltage (25). In this study, the influence of detection voltage on ECL intensity was studied. The ECL intensity enhanced with the increasing of voltage from 1.0 to 1.15 V and then decreased after 1.15 V (shown in Fig. 3a). Therefore, 1.15 V was selected as the detection voltage for the following experiments. Effect of Ru(bpy)32+ concentration. One of the most important detection parameters is the optimal concentration of Ru(bpy)32+ added to the detection cell. The effect of Ru(bpy)32+ concentrations (from 2 to 10 mM) on the ECL intensity was investigated. A low concentration of Ru(bpy)32+ leads to low background noise, while a high concentration leads to better sensitivity as well as the increasing S/N. However, when the concentration is too high, more consumption of the expensive reagent (Ru(bpy)3CI2·6H2O) will be required and the working curve will not be linear any more (15). When the concentration of Ru(bpy)32+ was 5 mM, high ECL intensity and high sensitivity were obtained (shown in Fig. 3b). Beyond 5 mM of Ru(bpy)32+, the ECL intensity was not increased any more, but the background noise increased. To get a higher S/N value, high ECL efficiency and moderate reagent consumption, 5 mM Ru(bpy)32+ was chosen in the experiment. After operating for 2 h or 30 runs, the Ru(bpy)32+ solution needed replenishing to ECL intensity (counts)

ECL intensity (counts)

Figure 2. The ECL intensity–potential curve: (a) 5 mM Ru(bpy)3 in 70 mM phosphate buffer of pH 8.0; (b) 0.5 μg/mL atomoxetine hydrochloride in (a) at a Pt electrode. ECL, electrochemiluminescence.

Results and discussion

1.2

1.25

1.3

1.35

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2+

7

8

9

10

80

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300 250

d

200 150 100 50 0 5

5.5

Detection buffer concentration (mM) Figure 3. Effect of detection voltage (a), Ru(bpy)3 ECL intensity. ECL, electrochemiluminescence.

6

C (mM) ECL intensity (counts)

ECL intensity (counts)

Detection voltage (V)

6

6.5

7

7.5

8

8.5

9

pH

concentration (b), detection buffer concentration (c) and pH of the phosphate buffer (d) in ECL detection cell on the

Copyright © 2014 John Wiley & Sons, Ltd.

Luminescence 2015; 30: 124–130

Determination of AH and study on its interactions with three proteins

Optimization of separation conditions In the CE-ECL system, CE conditions not only decide the separation efficiency but also influence ECL intensity. To obtain a high ECL signal and good separation efficiency for AH detection, the injection voltage and injection time of sample solutions, separation voltage, and concentration and pH value of the separation buffer were examined. Figure 4(a) shows the effect of injection voltage on ECL intensity and theoretical plate number (N) as injection time set at 8 s, and Fig. 4(b) shows that ECL intensity and N are influenced by the injection time ranging from 4 to 20 s. The number of theoretical plates (N) was calculated according to the following equation (24,28): N ¼ 5:54 tm =W 1=2

2

7000

550

6500

500

6000

450

5500

400

5000

350 300

4500

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Theoretical plates (N)

ECL intensity (counts)

600

3500 4

6

8

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12

14

16

18

20

1,200

b

1,100 1,000 900 800 700 600 500 400 300

4

6

8

10

12

14

16

18

20

4900 4700 4500 4300 4100 3900 3700 3500 3300 3100 2900

Theoretical plates (N)

Injection voltage (kV)

Injection time (s) Figure 4. Effect of injection voltage (a, n = 3, RSD 3.7%) and injection time (b, n = 4, RSD 6.1%) on the ECL intensity (triangle) and on the calculated number of theoretical 2+ plates, N (square). 10.0 μg/mL atomoxetine hydrochloride; 5 mM Ru(bpy)3 and 70 mM phosphate buffer in the detection reservoir; detection potential, 1.15 V, electrokinetic injection, 8 s at 12 kV; separation voltage, 15 kV. ECL, electrochemiluminescence.

1100

600

1000

500

900

400

800

300

700

200

600

100

500

0 5

7.5

10

12.5

15

17.5

20

Separation voltage (kV) Figure 5. Effect of separation voltage on the ECL intensity (square, n = 4, RSD 6.6%) and on inject time (triangle, n = 4 RSD 3.4%). 10.0 μg/mL atomoxetine hydro2+ chloride; 5 mM Ru(bpy)3 and 70 mM phosphate buffer in the detection reservoir; detection potential, 1.15 V, electrokinetic injection, 10 s at 12 kV; separation voltage, 15 kV. ECL, electrochemiluminescence.

(1) decreasing from 500 to 120 s. The separation voltage higher than 12.5 kV resulted in much higher noise of the baseline, which is attributed to the increase of Joule heating in capillary. Furthermore, the strong flow of effluent from the capillary might reduce the concentration of Ru(bpy)3+ 3 near the Pt working electrode surface, resulting in reducing the efficiency of light emission. To obtain a short migration time and high ECL intensity for the detection of AH, the 12.5 kV was selected as the optimum separation voltage. The pH of the separation buffer affected the ECL intensity directly because the light-emitting reaction is pH dependent and pH value strongly influences the electro-osmosis flow (13). Solutions of different pH values (from 5 to 9.5) were prepared to study the relationship between pH value and ECL intensity. At low pH value, ECL intensity increased with the elevation of pH value, but at values higher than 7.5, the ECL intensity decreased. Therefore, the separation buffer of pH 7.5 was selected for the experiments.

Copyright © 2014 John Wiley & Sons, Ltd.

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where tm is the migration time and W1/2 is the width at half height of the analyte peak during electrophoresis. As illustrated in Fig. 4, long injection time and high voltage led to strong ECL signal and low-separation efficiency due to the introduction of more analyte in the detection cell. However, when the shorter injection time and lower injection voltage were used, it was difficult to obtain favorable ECL intensity though high column efficiency could be achieved. Taking two opposite effects into account, injection voltage of 12 kV and injection time of 10 s were chosen in the next experiments to obtain higher ECL intensity and a larger N for the detection of AH. The separation voltage affected greatly ECL intensity and migration time of analytes. When the separation voltage changed from 5 to 20 kV, the ECL intensity kept increasing and reached the maximum value at 12.5 kV, and then slowly decreased (shown in Fig. 5). However, the migration time kept

Luminescence 2015; 30: 124–130

7500

a

Migration time (s)

Effects of detection buffer pH. As the ECL reaction of Ru (bpy)32+ with alkylamine depends on the buffer pH value to a great extent (26), the ECL intensity as a function of the buffer pH value over the pH range from 5.0 to 9.0 (0.5 as a unit) was investigated (shown in Fig. 3d). At a lower pH value, lower ECL response for AH is observed, attributed to the protonation of AH. However, when the pH increased up to 8.0, the ECL intensity increased with the protonation on weakened AH. OH– ion can compete with the secondary amine group to react with Ru (bpy)33+ and lead to considerable consumption of Ru(bpy)33+ at higher pH (27). The phenomenon that the ECL intensity decreased above pH 8.0 can be explained with the reduced availability of Ru(bpy)33+, which can react with AH. Thus, the optimized pH value was set at 8.0 in this study.

650

ECL intensity (counts)

Effect of concentration of detection buffer. The effect of the buffer concentration on the detection cell on the ECL intensity was investigated. The concentration changed from 10 to 90 mM and the highest ECL intensity of AH was obtained at 70 mM (shown in Fig. 3c). The ionic strength of background electrolyte is very low to transfer electrons, which were produced in the electrochemical steps that resulted in the decreased ECL efficiency. While the concentration of background electrolyte is very high, the quantity of Ru(bpy)32+ ions near the working electrode will be reduced because other ions may replace Ru(bpy)32+ near the electrode. The two factors reach a compromise at a concentration of 70 mM.

8000

700

ECL intensity (counts)

eliminate the change in Ru(bpy)32+ concentration and maintain good reproducibility.

H.-j. Zeng et al.

Detection limit, linearity and reproducibility The optimized CE-ECL conditions were as follows: separation capillary, 40 cm length (25 μm i.d.); sample injection, 10 s at 12 kV; separation voltage, 12.5 kV; running buffer, 30 mM sodium phosphate of pH 7.5; detection potential, 1.15 V; 70 mM of sodium phosphate buffer (pH 8.0) containing 5 mM of Ru(bpy)32+ in the ECL detection cell. The calibration curve for AH is linear over the concentration range from 5.0 × 10–9 to 7.0 × 10–7 g/mL with a regression curve of y = 39.004c + 11.169 (r = 0.9997, n = 5), where y is the ECL intensity and c is concentration of AH (1 × 10–7 g/mL). The limit of the detection calculated as S/N = 3 by dilution of the standard sample was 2.1 × 10–9 g/mL. The RSD of the ECL intensity and the migration time for eight consecutive injections of 5.0 × 10–7 g/mL AH were 2.83% and 1.10%, respectively. Applications To examine the application for practical analysis, the proposed method was applied to the determination of AH in its pharmaceutical preparation (AH capsule) and rat plasma. Determination of atomoxetine hydrochloride in capsule. Table 1 shows the results for the AH capsule by using the proposed method. Satisfactory analytical recovery rates at three different AH levels between 8.0 × 10–8 g/mL and 12.0 × 10–8 g/mL were obtained ranging from 97.7% to 101.0% and the coefficient of variation at these AH levels was 0.8–4.5% (n = 3). The content of AH in capsule was 9.23 mg/capsule (n = 3), which is 92.3% of commercial specification (10.0 mg/capsule). Determination of atomoxetine hydrochloride in rat plasma. According to the plasma sampling procedure, the blank sample and spiked sample of rat plasma were pretreated. The electropherograms showed that no significant interference has occurred. The calibration curve between ECL intensity and

the concentration of AH spiked in rat plasma ranged from 8.0 × 10–9 to 2.0 × 10–6 g/mL with a regression curve of y = 53.273c + 20.594 (r = 0.9946, n = 5), where y is the ECL intensity and c is concentration of AH (1 × 10–7 g/mL). The detection limit is 2.8 × 10–9 g/mL (S/N = 3). The relative recovery and RSD for rat plasma at concentration of 4.0 × 10–8, 1.0 × 10–7 and 1.0 × 10–6 g/mL were 93.4%, 101.3%, 104.5% and 3.2%, 4.1%, 4.5%, respectively (n = 5). The extract recoveries at concentrations of 4.0 × 10–8, 1.0 × 10–7 and 1.0 × 10–6 g/mL were 79.3%, 83.7% and 85.6%, respectively. The electropherograms of the blank plasma sample, plasma sample after oral administration of AH are shown in Fig. 6. The results for the mean content in plasma samples and the recoveries are summarized in Table 2, along with the RSD. The mean AH concentration in plasma at 3.0, 3.5, 4.0, 4.5 and 5.0 h after intravenous administration of 4 mg/kg AH was 3.70 × 10–7, 4.48 × 10–7, 6.68 × 10–7, 4.61 × 10–7 and 3.86 × 10–7 g/mL (n = 5) by the standard addition method, respectively. The recovery and RSD were 98.4–104.9% and 2.7–4.9%, respectively. In comparing the standard curve in rat plasmas to that in standard solutions, the slope and y-intercept of the standard curves are different (shown in Fig. 7). This suggested there must be some factors in the matrix of the plasma samples that influenced the ECL intensity. Therefore, the effect of the matrix of the plasma samples should not be ignored even if no clear interference was found in the electropherograms. This suggested that the establishment of the systemic method validation of the complex samples by CE-ECL method was necessary. In addition, compared with the published papers for the determination of AH (shown in Table 3), the proposed method was also a valuable and suitable tool for use in pharmacokinetic studies. 1800

ECL intensity (counts)

The effect of the buffer concentration on the AH detection was also investigated. Fixed pH value at 7.5, the buffer concentration was changed from 5 to 50 mM. Results showed that the variation in the ECL intensity was small when the buffer concentration changed. The highest ECL intensity was obtained when the buffer concentration was 30 mM. It was also found that with the increase of the buffer concentration, the migration time of AH increased and the baseline became unstable. This was due to the effect of the increase of the ionic strength, which resulted in the increase of Joule heating. Simultaneously, electro-osmosis flow decreased with the increase of the buffer concentration and the migration time became longer. Therefore, 30 mM of the buffer concentration was chosen as the optimum condition.

b

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Time (s) Figure 6. Typical electropherograms of capillary electrophoresis–ECL. (a) Blank rat plasma sample; (b) extract of plasma sample from a rat after 10 min of oral administration of 4 mg/kg atomoxetine hydrochloride. Capillary electrophoresis–ECL conditions: separation capillary, 40 cm length (25 μm i.d.); sample injection, 10 s at 12 kV; separation voltage, 12.5 kV; running buffer, 30 mM sodium phosphate of pH 7.5; detection potential, 1.15 V; 70 mM of sodium phosphate buffer (pH 8.0) containing 2+ 5 mM of Ru(bpy)3 in ECL detection cell. ECL, electrochemiluminescence.

Table 1. Determination of atomoxetine hydrochloride in its capsule (n = 3) Labeled (mg/capsule) 10

Found by this method (mg/capsule)

Original quantity (g/mL)

9.23 (n = 3)

8.5 × 10–8

Added (g/mL) – 8.0 × 10–8 10.0 × 10–8 12.0 × 10–8

Found (g/mL) 8.5 × 10–8 16.12 × 10–8 18.47 × 10–8 20.71 × 10–8

Recovery (%)

RSD (%)

– 97.7 99.8 101.0

4.5 4.4 4.3 0.8

128

RSD, relative standard deviation.

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Luminescence 2015; 30: 124–130

Determination of AH and study on its interactions with three proteins Table 2. Determination of atomoxetine hydrochloride in rat plasma after oral administration of 4 mg/kg atomoxetine hydrochloride (n = 5) Time (h)

Plasma content (g/mL)

Added (g/mL)

–7

3 3.5 4 4.5 5

Found (g/mL)

–7

3.70 × 10 4.48 × 10–7 6.68 × 10–7 4.61 × 10–7 3.86 × 10–7

–7

1 × 10 1 × 10–7 1 × 10–7 2 × 10–7 2 × 10–7

4.87 × 10 5.75 × 10–7 7.56 × 10–7 6.65 × 10–7 5.81 × 10–7

Recovery (%)

RSD (%)

103.6 104.9 98.4 100.6 99.2

2.7 4.8 3.6 4.9 2.7

RSD, relative standard deviation.

constant. The data analysis of drug protein often assumes one type of binding site on the protein and equation (2) can therefore be simplified to:

1200

ECL intensity (counts)

b 1000 800

r ¼ ½C bound =½Ptotal  ¼ n·K·½C free =ð1 þ K·½C free Þ

600 400

a 200 0

0

2

4

6

8

10

12

14

16

18

20

c (1×10-7 g/mL-1) Figure 7. The standard curve of atomoxetine hydrochloride in standard solutions (a) and rat plasmas (b). Capillary electrophoresis–ECL conditions: separation capillary, 40 cm length (25 μm i.d.); sample injection, 10 s at 12 kV; separation voltage, 12.5 kV; running buffer, 30 mM sodium phosphate of pH 7.5; detection potential, 2+ 1.15 V; 70 mM of sodium phosphate buffer (pH 8.0) containing 5 mM of Ru(bpy)3 in ECL detection cell. ECL, electrochemiluminescence.

Study on the interaction between atomoxetine hydrochloride and three proteins In most cases, the drug is bound to m types of independent binding sites on the protein. The fraction, r, of bound drug molecules per protein molecule is given by Busch as follows (29): r¼

m C bound K i ½C free  ¼ ∑ ni 1 þ K i ½C free  Pfree i¼1

(2)

Where [Cbound], [Ptotal] and [Cfree] are the concentrations of bound drug, total protein and free drug, respectively; ni represents the number of sites of class i and Ki is the binding

(3)

where n, K correspond to the number of binding sites and the binding constant, respectively (30). According the equation (3) and the [Cfree] obtained, the binding curves of AH with BSA, Cyt-C and Mb were established as shown in Fig. 8. The non-linear relationship between the fraction of bound drug molecules per protein molecule and the concentration of free AH can be fitted to the following equation: r ¼ 15:29:5103 ½C free = 1 þ 9:5103 ½C free  r ¼ 10:21:2104 ½C free = 1 þ 1:2104 ½C free 



(4)



(5)

 and r ¼ 17:17:4103 ½C free = 1 þ 7:4103 ½C free 

(6)

for BSA, equation (4); Cyt-C, equation (5); and Mb, equation (6). Therefore, the binding site numbers are 15.2, 10.2 and 17.1 and the binding constants are 9.5 × 103, 1.2 × 104 and 7.4 × 103 M–1 for BSA, Cyt-C and Mb, respectively. The value of the binding constants is of the order of 103 M–1, indicating that a strong interaction exists between AH and protein. These results were consistent with the data on specifications of AH, which indicates that AH has a high protein binding rate and a rapid absorption after oral administration.

Table 3. Comparison of various methods for determination of atomoxetine hydrochloride Methods

Linear range

Limit of detection

HPLC-UV RP-LC HPLC-FL HPLC-UV LC-MS

3.12–200 ng/mL 1–10 μg/mL 1–1000 ng/mL 0.05–3.0 μg/mL 0.25–25 ng/mL (plasma) 1–200 ng/mL (urine) 8.0 × 10–9–2.0 × 10–6 g/mL 5.0 × 10–9–7.0 × 10–7 g/mL

2.5 ng/mL – 0.3 ng/mL 0.05 μg/mL 0.25 ng/mL 1 ng/mL 2.8 ng/mL 2.1 ng/mL

CE-ECL

Recovery (%)

Analytical time

92.4–103.0 5.2 min 98.13–101.50 3.34 min 95–105 13.6 min 95.67–108.80 13.9 min 88.0–100.7 (human plasma) 2.3 min 96.0–110.0 (urine) 98.4–104.9 (rat plasma) 3.7 min 97.7–101.0 (capsule)

Analytical samples

References

Human plasma Dosage form Human plasma Human plasma Human plasma and urine Rat plasma and capsule

1 4 7 8 10 This work

Luminescence 2015; 30: 124–130

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CE-ECL, capillary electrophoresis with electrochemiluminescence; FL, fluorescence; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS,-mass spectrometry; RP, reversed phase; UV, ultraviolet.

H.-j. Zeng et al. 18

c

16

a

14

Value of r

12

b

10 8 6 4 2 0

0

1

2

3

4

5

6

7

8

9

Figure 8. Binding curve of atomoxetine hydrochloride with bovine serum albumin (a), cytochrome c (b) and myoglobin (c) in phosphate buffer solution (pH 6.6) at 37°C; [Cfree] is the concentration of unbound atomoxetine hydrochloride.

Conclusion In this work, the CE-ECL method, as a sensitive, simple, rapid and repeatable method, was first developed for the analysis of AH in rat plasma and AH capsule. Parameters that affect separation and detection were optimized and all the solutions were dissolved in the water, avoiding the use of the organic reagents. Considering the effect of the matrix in the plasma samples on the detection of the target drugs, in the work described in this paper, the methodology of determination of AH in rat plasma was systemically investigated and the results obtained may be significant for further application of this method. In addition, using the proposed method, the interactions between AH and three proteins (BSA, Cyt-C and Mb) were investigated and the number of binding sites and binding constant were estimated.

References

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1. Mendez L, Singh P, Harrison G, Huang YS, Jin X, Cho SC. Academic outcomes in Asian children aged 8-11 years with attention-deficit/ hyperactivity disorder treated with atomoxetine hydrochloride. Int J Psychiatry Clin Pract 2011;15:145–56. 2. Holzer B, Lopes V, Lehman R. Combination use of atomoxetine hydrochloride and olanzapine in the treatment of attention-deficit/ hyperactivity disorder with comorbid disruptive behavior disorder in children and adolescents 10-18 years of age. J Child Adolesc Psychopharmacol 2013;23:415–18. 3. Spencer T, Heiligenstein JH, Biederman J. Results from 2 proof-ofconcept, placebo-controlled studies of atomoxetine in children with attention-deficit/hyperactivity disorder. J Clin Psychiatry 2002;63:1140–7. 4. Michelson D, Faries D, Wernicke J. Atomoxetine in the treatment of children and adolescents with attention-deficit/hyperactivity disorder: A randomized, placebo-controlled, dose-response study. Pediatrics 2001;108:1–9. 5. Caballero C. Nahata MC. Atomoxetine hydrochloride for the treatment of attention-deficit/hyperactivity disorder. Clin Ther 2003;25:3065–83. 6. Patel C, Patel M, Rani S, Nivsarkar M, Padh H. A new high performance liquid chromatographic method for quantification of atomoxetine in human plasma and its application for pharmacokinetic study. J Chromatogr B 2007;850:356–60. 7. Patel SK, Patel NJ. Development and validation of a stabilityindicating RP-HPLC method for determination of atomoxetine hydrochloride in tablets. J AOAC Int 2010;93:1207–14. 8. Prajapati HR, Raveshiya PN, Prajapati JM. RP-HPLC Determination of atomoxetine hydrochloride in bulk and pharmaceutical formulations. E-J Chem 2011;8:1958–64.

wileyonlinelibrary.com/journal/luminescence

9. Farid NA, Bergstrom RF, Ziege EA, Parli CJ, Lemberger L. Single-dose and steady-state pharmacokinetics of tomoxetine in normal subjects. J Clin Pharmacol 1985;25:296–301. 10. Choi CI, Bae JW, Lee HI, Jang CG, Sohn UD, Lee SY. Determination of atomoxetine metabolites in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci 2012;885–886:103–8. 11. Choi CL, Jang CG, Bae JW, Lee SY. Validation of an analytical LC-MS/ MS method in human plasma for the pharmacokinetic study of atomoxetine. J Anal Chem 2013;68:986–91. 12. Pérez-Ortiz M, Muñoz C, Zapata-Urzúa C, Álvarez-Lueje A. Electrochemical behavior of atomoxetine and its voltammetric determination in capsules. Talanta 2010;82:398–403. 13. Yin JY, Xu YH, Li J, Wang EK. Analysis of quinolizidine alkaloids in Sophora flavescens Ait. by capillary electrophoresis with tris (2,2′bipyridyl) ruthenium (II)-based electrochemiluminescence detection. Talanta 2008;75:38–42. 14. Min Z, Ma YJ, Ren XN, Zhou XY, Li L, Chen H. Determination of sinomenine in Sinomenium acutum by capillary electrophoresis with electrochemiluminescence detection. Anal Chim Acta 2007;587:104–9. 15. Deng BY, Xu QX, Lu H, Ye L, Wang YZ. Pharmacokinetics and residues of tetracycline in crucian carp muscle using capillary electrophoresis on-line coupled with electrochemiluminescence detection. Food Chem 2012;134:2350–4. 16. Tao YW, Zhang XJ, Wang JW, Wang XX, Yang NJ. Simultaneous determination of cysteine, ascorbic acid and uric acid by capillary electrophoresis with electrochemiluminescence. J Electroanal Chem 2012;674:65–70. 17. Deng BY, Kang YH, Li XF, Xu QM. Determination of erythromycin in rat plasma with capillary electrophoresis-electrochemiluminescence detection of tris (2,2′-bipyridyl) ruthemium (II). J Chromatogr B 2007;857:136–41. 18. Chen FM, Zhang YD, Nakagawa Y, Zeng HL, Chen L, Nakajima H, Uchiyama K, Lin JM. A piezoelectric drop-on-demand generator for accurate samples in capillary electrophoresis. Talanta 2013;107:111–17. 19. Yin XB, Wang EK. Capillary electrophoresis coupling with electrochemiluminescence detection: a review. Anal Chim Acta 2005;533:113–20. 20. Xiao JB, Shi J, Cao H, Wu SD, Ren FL, Xu M. Analysis of binding interaction between puerarin and bovine serum albumin by multispectroscopic method. J Pharm Biomed Anal 2007;45:609–15. 21. Wang YQ, Zhang HW, Zhang GC. Studies of the interaction between palmatine hydrochloride and human serum albumin by fluorescence quenching method. J Pharm Biomed Anal 2006;41:1041–6. 22. Kandagal PB, Ashoka S, Seetharamappa J, Shaikh SMT, Jadegoud Y, Ijare QB. Study of the interaction of an anticancer drug with human and bovine serum albumin: Spectroscopic approach. J Pharm Biomed Anal 2006;41:393–9. 23. Hao J, Feng F, Sun Q, Wang H. Study on pharmacokinetic interaction of atomoxetine and fluoxetin in rats. Pharm Clin Res 2008;16:111–13. 24. Huang Y, Pan W, Guo ML, Yao SZ. Capillary electrophoresis with endcolumn electrochemiluminescence for the analysis of chloroquine phosphate and the study on its interaction with human serum albumin. J Chromatogr A 2007;1154:373–8. 25. Zhou XM, Xing D, Zhu DB, Tang YB, Jia L. Development and application of a capillary electrophoresis-electrochemiluminescent method for the analysis of enrofloxacin and its metabolite ciprofloxacin in milk. Talanta 2008;75:1300–6. 26. Brune SN, Bobbitt DR. Effect of pH on the reaction of tris (2,2′bipyridyl) ruthenium (III) with amino-acids: Implications for their detection. Talanta 1991;38:419–24. 27. Zhang ZL, Li JJ, Qu LB, Yang R. Determination of pazufloxacin mesylas by capillary electrophoresis with electrochemiluminescence detection. Chin J Anal Chem 2008;36:941–6. 28. Wang JW, Yang ZM, Wang XX, Yang NJ. Capillary electrophoresis with gold nanoparticles enhanced electrochemiluminescence for the detection of roxithromycin. Talanta 2008;76:85-90. 29. Busch MHA, Carels LB, Boelens HFM, Kraak JC, Poppe H. Comparison of five methods for the study of drug-protein binding in affinity capillary electrophoresis. J Chromatogr A 1997;777:311–28. 30. Rudnev AV, Aleksenko SS, Semenova O, Hartinger CG, Timerbaev AR, Keppler BK. Determination of binding constants and stoichiometries for platinum anticancer drugs and serum transport proteins by capillary electrophoresis using the Hummel-Dreyer method. J Sep Sci 2005;28:121–7.

Copyright © 2014 John Wiley & Sons, Ltd.

Luminescence 2015; 30: 124–130

Capillary electrophoresis coupled with electrochemiluminescence for determination of atomoxetine hydrochloride and the study on its interactions with three proteins.

A simple, rapid and sensitive method for the determination of atomoxetine hydrochloride (AH) by capillary electrophoresis with electrochemiluminescenc...
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