Determination of Nilotinib in Spiked Plasma, Urine, and Capsules by High-Performance Liquid Chromatography with Fluorimetric Detection

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Introduction
Nilotinib (NTB) [4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[(4-pyridin-3-ylpyrimidin-2-yl) amino] benzamide monohydrochloride monohydrate] (Fig. 1) is a tyrosine kinase inhibitor which has been approved by the Food and Drug Administration (FDA) for the treatment-intolerant adult patients in the chronic phase and accelerated phase of Philadelphia chromosome positive chronic myelogenous leukemia [1].NTB is well absorbed, and maximum peak plasma concentrations were reached in 3 h after oral administration.Mean bioavailability of NTB is about 30%.Its bioavailability is increased with food.Plasma protein binding rate of NTB in humans was about 99% [2].Studies have shown that NTB is metabolized mainly by CYP3A4 with oxidation and hydroxylation, and that none of the metabolites contribute considerably to its pharmacological activity.After a single oral dose NTB, recovery was evaluated over 7 days and found 4% in urine and about 90% in feces (69% as unchanged) [3].In the literature, there are only few analytical methods for determination of NTB in its pharmaceutical formulation.There are three high-performance liquid chromatography-ultraviolet (HPLC-UV) methods for quantification or pharmacokinetic studies of drug in the plasma [4], in cell culture [5], and in plasma, urine, and cell culture [6].Parise et al. described an HPLC-mass spectrometry (MS) method for NTB in plasma and serum [7].Furthermore, simultaneous assay of NTB with other antichronic myeloid leukemia drugs using solid phase extraction and ultra-performance liquid chromatographytandem mass (UPLC-MS/MS) [8], HPLC-UV [9][10], HPLC-MS/MS [11][12][13][14][15], and HPLC-MS [16,17] have been reported.
Due to the presence of very few methods for the analysis of the NTB, new drug, and some serious side effects of the drug, there is a need to develop accurate, more sensitive, and validated methods which may be used in quality control and clinical analysis.Although published LC-MS techniques have high sensitivity and selectivity, these instruments are expensive, not available in many laboratories, and are complicated to operate.For the analysis of NTB, an HPLC method using fluorescence detector has not been reported in the literature.HPLC-fluorescence detection provides better selectivity and sensitivity, and reduces interferences compared to HPLC-UV detection.Therefore, the aim of this research is to develop a sensitive validated reversed phase high-performance liquid chromatography with fluorescence detection method (RP-HPLC) for the determination of NTB in spiked plasma, urine samples, and capsules after derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl -Fig.1), which is a common derivatizing reagent for primary and secondary aliphatic amines.
There is also no report on the forced degradation study which can provide identification of degradation profile and stability of drug compounds in the literature.In this study, forced chemical and physical stress studies were carried out by HPLC-UV to investigate the stability of drug according to International Conference on Harmonization (ICH) Q1A (R2) [18] guideline for the first time.

Experimental Apparatus
The assay was carried out on a Shimadzu HPLC (Kyoto, Japan) with LC 10AT pump, SCL-10A VP system controller, RF 10 AXL fluorescence detector, 20 μL sample loop, and Class-LC 10 version 1.61 software.Detector was set at an excitation wavelength of 447 nm and an emission wavelength of 530 nm.
For the stability studies, the drug was separated from degradation products on Thermo Separation system (San Jose, CA) with the following parts: controller SN 4000, pump P 4000 and auto sampler AS 3000, fitted with 20 μL sample loop, and photodiode array detector UV 6000 LP.Data acquisition was performed with ChromQuest 5.0 software.Spectrophotometric and spectrofluorimetric analyses were measured by Shimadzu UV-160A (Kyoto, Japan) UV-vis spectrophotometer and Shimadzu RF-1501 spectrofluorometer, respectively.
A WTW pH 526 digital pH meter (Mettler Toledo, Switzerland) was used in the pH measurements.An aqua MAXTM-ultra water purification system (Young-lin instrument, South Korea) was used for water purification.

Materials and Chemicals
NTB certified to contain 99.89% was kindly supplied by Novartis Pharmaceuticals (Istanbul, Turkey).All solvents and chemicals with HPLC grade were purchased from Merck.4-Chloro-7-nitrobenzofurazan (NBD-Cl) was obtained from Sigma-Aldrich (St. Louis, MO, USA).Drug-free human plasma was kindly supplied by Dr. Siyami Ersek Cardiovascular Surgery Training and Research Hospital (Istanbul, Turkey), kept in freezer (−20 °C) until analysis, and thawed in warm water when used.

Chromatographic Conditions
Separations were carried out on a Waters Symmetry C 18 -column (250 mm × 4.6 mm i.d. 5 μm).Ten millimolar of phosphoric acid and acetonitrile (40:60 v/v) was used as mobile phase in isocratic mode with 1 mL min −1 flow rate at ambient temperature.The mobile phase was filtered through a Millipore vacuum filter system, set with a 0.45-μm HV filter.

Solutions
Stock solution of NTB at 0.5 mg mL −1 concentration (expressed as base compound) in methanol was prepared.The preparation of working standard solution at 0.01 mg mL −1 was made by appropriate dilutions from stock solution in acetonitrile-water mixture 1:1, v/v).Under these conditions, the stock solution remained stable at least 2 weeks.
NBD-Cl solution at 4.0 mg mL −1 in methanol was prepared daily.To prepare 0.1 M borate buffer (pH between 7.0 and 10.5), 0.75 g of potassium chloride and 0.62 g of boric acid were dissolved in 50 mL; the pH of the solutions was adjusted with 0.1 M NaOH and then filled to 100 mL with distilled water.

Procedures Examination of Reaction Conditions for Derivatization of NTB
To obtain optimum derivatization reaction of NTB with NBD-Cl, various parameters including pH of buffer, temperature, reaction time, reagent amounts, and extraction solvent were tested individually by either spectrofluorimetric or HPLC-fluorimetric detection method.

The Effect of Buffer pH
To investigate the effect of buffer solution on the reaction efficiency, 500 mL of borate buffer at various pH (7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5) and 300 μL of NBD-Cl solution (4 mg mL −1 ) were added to 250 mL of NTB solution (0.01 mg mL −1 ) in a tube, separately and stirred by a vortex mixer.The samples were kept for 10 min at 80 °C in hot water bath.The tubes were cooled in an ice bath, and 200 mL 0.1 N HCl was added.Then, derivative mixtures were extracted with 5 mL of chloroform using the vortex and the chloroform layer was separated.The fluorescence intensity of the solutions was measured at 530 nm after excitation at 447 nm by spectrofluorometer.Blank experiment was performed in the same manner.

Effect of Temperature and Reaction Time
To examine the effect of temperature and reaction time on the reaction, 500 mL of borate buffer pH of 9 and 300 μL of NBD-Cl solution (4 mg mL −1 ) were added to a series of tube that contained 250 mL of NTB solution (0.01 mg mL −1 ) separately and stirred by the vortex mixer.Each sample was heated individually at temperatures of 50, 60, 70, and 80 °C for 10, 15, 20, 30, 40, 50, and 60 min.The samples were cooled in an ice bath, and the procedure and measurement were followed as described above.

Effect of Concentration of NBD-Cl
To determine adequate amount of reagent for the reaction, 500 μL of buffer at pH 9 was added into a series of tubes which contained 250 μL NTB (0.01 mg mL −1 ), after addition of NBD-Cl solution to be 5 to 1,200 mole times of drug, worked up as described above.In this assay, chloroform layer was evaporated under a stream of nitrogen gas at 40 °C, and the residue was dissolved in the mobile phase and analyzed by HPLC.

General Procedures and Calibration Curve
50.0-300.0μL of standard drug solutions (0.01 mg mL −1 ) were transferred into a series of 10 mL capped tubes by an automatic pipette.To each tube, 500 μL of borate buffer of pH 9.0 and 500 μL of NBD-Cl solution were added and stirred briefly for 10 s with a vortex.The reaction solutions were heated in a water bath at 80 °C for 10 min.After chilling in an ice bath, 200 μL 0.1 N HCl was added and mixture was extracted with 5 mL of chloroform using a vortex.One milliliter of the chloroform layer was taken and evaporated under nitrogen gas at 40 °C, and the residue was dissolved in 1 mL of mobile phase and then was given to HPLC-fluorescence detection system.Blank analysis was made in the same way but without the drug under study.
The values of peak area have been corrected by subtracting the values of the peak area of blank samples from the values of the peak area of NTB samples.To determine the calibration curves, the corrected values of peak area were plotted against the concentrations of drug and the corresponding regression equation was obtained.

Procedure for Spiked Human Plasma
Aliquots of 0.1 mL of drug free plasma samples were transferred into tubes and added NTB solutions at six different concentration levels to obtain a final concentration range of 100-600 ng mL −1 .For precipitation of plasma proteins, 0.5 mL HCl was added to samples, mixed with a vortex for 3 min, and then extracted by adding 3 mL chloroform.The mixtures were centrifuged at 4,500 rpm for 15 min.The supernatant of these samples was evaporated to dryness under nitrogen.The residue was dissolved with 500 μL water-acetonitrile (50:50; v/v), and then assay was performed as described in section "General procedures and calibration curve."Blank analysis was made in the same way.

Procedure for Spiked Human Urine
The drug-free urine samples were obtained from a healthy volunteer and were stored in color capped glass bottles at −20 °C until analysis.Urine samples were diluted 100 times with water before analysis.Aliquots of 0.1 mL of urine were spiked with standard analyte solution in six different concentrations to obtain concentration of range of 100-600 ng mL −1 .Derivatization and analysis were carried out as described in section "General procedures and calibration curve."

Determination of Drug in Capsules
Capsules containing 200 mg NTB were prepared at Pharmaceutical Technology Department at the Faculty of Pharmacy, Istanbul University since it was not possible to obtain the commercially available capsules.The capsule contains the following ingredients: microcrystalline cellulose, lactose monohydrate, magnesium stearate, crospovidone, poloxemers, aerosol colloidal silicon dioxide, and PoliplasdoneXL-10.Five capsules were weighed and powdered separately.An amount of the powder equivalent to 200 mg NTB was transferred to a 100-mL volumetric flask with 50 mL of methanol and then shaken in an ultrasonic bath at room temperature for 1 h.The volume was completed to 100 mL with methanol and filtered.The solution of capsules was analyzed after appropriate dilutions as in section "General procedures and calibration curve."Content of the capsules was found using related regression equation of the calibration curve.

Liquid-Liquid Extraction
Plasma proteins were precipitated, and NTB was extracted from the plasma in one simple step; 100 μL of plasma was transferred into a series of tube and mixed with 250 µL NTB at 0.01 mg mL −1 on a vortex mixer.Studies were conducted as follows: (a) To four separate tubes containing plasma samples spiked with NTB, 500 µL methanol, 500 µL of acetonitrile, 1 mL of hexane-amyl alcohol mixture (20:80, v/v) and 50 µL perchloric acid-170 µL ACN-KH 2 PO 4 solution (40:60, v/v) were added and stirred with a vortex mixer.The samples were centrifuged at 4500 rpm for 15 min.The supernatants were evaporated under a stream of nitrogen gas.The residues were derivatized and analyzed after dissolving with 500 µL mixture of acetonitrile-water (50:50; v/v) as described in section "General procedures and calibration curve."(b) 0.5 mL 0.1 N HCl was added into two plasma samples spiked with NTB separately and mixed thoroughly.Then, to one sample, 3 mL of ethyl acetate, and the other, 3 mL of chloroform was added as the extraction solvent, mixed well, and centrifuged at 4,500 rpm.The supernatants were evaporated under stream of nitrogen gas, and the assay was carried out as described above.

Method Validation
The validation of the developed method was analyzed in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, recovery, stability, and specificity in accordance with ICH guidelines [18,19].

Linearity and Sensitivity
Calibration curves of NTB-NBD derivatization in standard solution, spiked plasma, and urine were constructed by linear least squares regression using plotting peak area of the drug versus concentration.The values of peak area for the drug have been corrected by subtracting the values of the peak area of blank samples.
LOD and LOQ of drug for proposed methods were calculated with the following equation: LOD = 3S a /b and LOQ = 10S a /b, where S a is the standard deviation of the intercept and b is the slope of calibration curve [19].

Accuracy and Precision
Intra-day and inter-day accuracy and precision were validated by NTB-NBD derivative at three different concentrations (100, 300, and 500 ng mL −1 ) in standard solution, spiked plasma, and urine.Determinations were carried out in five replicates within same day for intra-day and on five separate days for inter-day precision.The precision and the accuracy were expressed as percent relative standard deviation (RSD %) and percent recovery (%), respectively.

Recovery
Recovery in spiked human plasma and urine samples was calculated from the ratio of the slope of the regression equation of calibration curve for standard solution with obtained slopes regression equations for plasma and urine.

Stability and Specificity
The stability of derivatization of NTB at 300 ng mL −1 concentration for the standard solution, plasma, and urine was investigated in terms of storage conditions in mobile phase in darkness at room temperature for 24 h and periodically analyzed by HPLC with fluorescence detection.
The stability of NTB in plasma and urine samples was tested by keeping the spiked samples at room temperature and +4 °C for 10 days and then analyzed by the developed method.The analytical results obtained during the storage conditions were compared with the results of the freshly prepared derivative.
The specificity and stability were tested by forced degradation of NTB in different conditions according to ICH guidelines Q1A (R2) [18].For each stress condition, 5 mL of 0.01 mg mL −1 NTB solution was transferred to 10 mL volumetric flask and 0.5 mL of 0.1 N NaOH and 0.1 N HCl were added separately.Then, NTB solutions were subjected to forced degradation under acidic and basic conditions at 70 °C for 15 min-24 h.Oxidative stress was carried out using 30% H 2 O 2 at room temperature up to 140 min.After subjection to chemical stress, the test samples were completed to 10 mL with mobile phase to obtain concentration of 5 μg mL −1 .The drug was placed in a thermally controlled oven at 70 °C up to 3 days for thermal stress in solid as well as in methanol solution at 5 μg mL −1 .For photolytic stress, the drug was exposed to UV light at 254 nm for 3 days.After the specified time intervals, samples were stored in a refrigerator at 4 °C until analysis.
The separation of drug and its degradation products was performed on a C 18 -column (Phenomenex, 250 mm × 4.6 mm i.d., 5 μm) using acetonitrilewater (70:30, v/v) as mobile phase with 1 mL min −1 flow rate in an isocratic elution mode by HPLC-diode array detection (DAD).The detector was set at 266 nm.

Optimization of Derivatization of NTB Conditions The effect of pH
The effect of pH on the reaction was investigated using borate buffer at pH 7.0-10.5 with a spectrofluorometer.Highest fluorescence intensity was obtained when the pH was 9.0 (Fig. 2).In addition, the amount of buffer solu-

Effect of temperature and reaction time
For optimum formation of derivatives, different temperatures (50-80°C) and reaction times (10-60 min) were tested.The result of analysis showed that the reaction was completed in 10 min at 80 °C (Fig. 3).

Effect of the concentration of NBD-Cl
To examine the effect of the concentration of NBD-Cl, reagent solution was added to be 5 to 1,200 mole times.For optimum derivatization, 500 mole times of NBD-Cl was found to be adequate.
In order to prevent interference with high fluorescence of NBD-Cl, the derivatives of reaction solution were extracted with various immiscible organic solvent.For this purpose, chloroform, ethyl acetate, and dichloromethane as extracting solvents were used, and chloroform was found most suitable.The derivative formed was found to be stable in chloroform for at least 6 h.

Liquid-Liquid Extraction
Plasma proteins were precipitated, and NTB was extracted from plasma using different solvents including methanol, acetonitrile, hexane-amyl alcohol mixture, perchloric acid-ACN-KH 2 PO 4 , HCl-chloroform, and HClethyl acetate were tested as described in experimental section.HClchloroform solvent system was selected for the high rate of recovery of NTB from the spiked plasma.

Optimization of Separation Conditions
In order to obtain proper analysis conditions for the NTB-NBD derivative, methanol-water and acetonitrile-water in different portions were tested as mobile phase.In addition, phosphoric acid in different portions was tested instead of water for mobile phase.The best result in terms of analysis time and sharp peak was obtained by using acetonitrile-10 mM phosphoric acid (40:60 v/v) with 1 mL min −1 flow rate.The average retention time of the derivatization product was about 5.12 min in pure and in biological fluids (RSD % was 2.3% for 15 independent analysis).A typical chromatogram of NTB-NBD derivative in selected conditions is shown in Fig. 4.

Linearity and Sensitivity
For the determination of the linear range of NTB in standard solution, spiked plasma and urine, concentrations between 100 and 600 ng mL −1 of drug at six different concentrations were analysed under the optimized conditions (Figs. 5 and 6).Five independent analyses were performed for each concentration.Correlation coefficients (r 2 ) were greater than 0.9997, indicating good linearity (Table I).
The LOD and LOQ values were 0.007 and 0.023 ng mL −1 for standard drug solution and 15.59 and 51.98 ng mL −1 for plasma samples, respectively.For urine samples, LOD and LOQ were 29.28 ng mL −1 and 97.60 ng mL −1 , respectively (Table I).

Accuracy and Precision
For standard solution, plasma, and urine analysis, percent relative standard deviation (RSD %) values of intra-day and inter-day precision were ranged from 0.73 to 1.94 and 0.56 to 2.90%, respectively.The accuracy, expressed as percent recovery (%) values for intra-day and inter-day ranged from 97.75 to 100.74% and 97.75 to 102.39%, respectively, over the three concentration levels evaluated in studied samples (Table II).tions.In this study, liquid-liquid extraction method used in the preparation of plasma protein sample was simple, rapid, and provided high recovery rate compared to solid-phase extraction methods [9,10].Quite small amounts of biological samples were adequate for assays.The retention time in the proposed method was considerably shorter than the published HPLC-UV methods [4,6,9,10].Considering the values for the linearity and LOD values, the proposed method is more sensitive than published methods for assay of NTB alone or in combination with different drugs [4,8,12].The developed method proved adequate for analysis of NTB in the biological fluids and in its pharmaceutical preparation.

Fig. 2 .
Fig. 2. The effect of pH on derivatization of NTB with NBD-Cl

Fig. 3 .
Fig. 3.The effect of temperature and time on the derivatives of NTB-NBD-Cl

Fig. 5 .
Fig. 5. Chromatogram of (a) blank human plasma with NBD-Cl and (b) human plasma spiked with 400 ng mL −1 NTB for derivatization with NBD-Cl under optimized conditions

Fig. 6 .
Fig. 6.Chromatogram of (a) blank human urine with NBD-Cl and (b) human urine spiked with 400 ng mL −1 NTB for derivatization with NBD-Cl under optimized conditions

Fig. 7 .
Fig. 7. Chromatogram of NTB solutions after being subjected to hydrogen peroxide conditions by RP-HPLC-DAD method.The inset shows UV spectrum of (a) degradation product and spectrum of (b) NTB Unauthenticated | Downloaded 12/27/23 07:16 PM UTC

Table I .
Statistical parameters for analysis of derivatives of NTB with NBD-Cl (C is the concentration of drug in ng mL −1 , A is the peak area, a is slope, and b is intercept).
a Average of five determinations.b A = aC + b

Table II .
Intra-day and inter-day precision and accuracy of NTB in standard solution, plasma, and urine by proposed method (n = 5)