Novel spectrofluorimetric determination of brigatinib in bulk powder and human urine samples via ion-pair complex formation using eosin Y

Halah S. Almutairi a, Mona M. AlShehri a, Mohammed M. Alanazi a, Ibrahim A. Darwish a, Hany W. Darwish a,b

Abstract

The developed spectrofluorimetric method was successfully applied to the analysis of brigatinib (BRG) in its bulk powder form, and human urine sample. It is based on the investigation of the fluorescence spectrum behavior of the BRG-eosin Y complex. The relative fluorescence intensity (RFI) was recorded at 560 nm after excitation at 480 nm. The principle of the proposed method was thoroughly explained. All experimental parameters affecting method development were optimized. Moreover, the obtained results were fully discussed and statistically analyzed. The molar ratio method was applied to study the stoichiometric relationship between BRG and eosin Y complex. The method revealed a ratio of 1:3 for BRG-eosin Y afforded the highest RFI. The developed method was validated over the concentration range of 62.5–4000 ng mL1. The results were compared positively with the reported method.

Keywords:
Brigatinib
Spectrofluorimetry
Ion-pair complex
Eosin Y

1. Introduction

Brigatinib (BRG) is a second-generation ALK inhibitor. In treating patients with lung cancer and patients that are resistant to other inhibitors,BRG causesaneffectivedualinhibitionofALK and a transmembrane protein epidermal growth factor receptor in humans [1]. BRG was rapidly approved by the U.S. food and drug administration (FDA) on April 28, 2017 [2]. The chemical structure of BRG is shown inFig. 1.The IUPACis 5-chloro-4-N-(2-dimethylphosphorylphenyl)2-N-(2-methoxy-4-(4-(4-methylpiperazin-1-yl) piperidin-1-yl] phenyl] pyrimidine-2,4-diamine [3].
Six LC-MS quantification methods for BRG in rat and human plasma were reported [4–9]. Although LC-MS exhibits excellent selectivity and sensitivity, as well as rapid analysis time, it is expensive and requires highly skilled technical expertise, which may not be readily available and/or affordable for most laboratories in resource-limited countries. Spectrofluorimetry, on the other hand, is a specific, highly selective, sensitive, and simple technique for the analysis of drug molecules in biological fluids [10–12].
Eosin Y, tetrabromofluorescein disodium salt, is a yellowish-red dye with green fluorescence (Fig. 2). It is an acidic dye containing one carboxyl group. Eosin Y, with a molecular formula of C20H6Br4Na2O5 and a molar mass of 691.85, is chemically known as disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen9-yl) benzoate [13–15].
Mono- or di-anionic forms of eosin Y can interact with a cationic form of the drug molecule via electrostatic interactions and hydrophobic forces. Hence, eosin Y is applied for the determination of several basic drugs via the formation of binary [13–14] or ternary complexes with drug molecules [13–18] and measured either directly without extraction or indirectly by extraction in an organic solvent [19].
In this study, the ion-pair complex formed between BRG and eosin Y was utilized for the development of a selective, sensitive spectrofluorimetric method for the BRG analysis of bulk materials and human urine. All factors were optimized, and the method was validated as per for the International Council for Harmonization (ICH) guidelines [20].

2. Experimental

2.1. Chemicals and reagents

BRG standard was purchased from Med Chem. Express (Princeton, NJ, USA). Eosin Y was obtained from Sigma (BioChemika, China), and was prepared as 0.2% aqueous w/v. Anhydrous potassium dihydrogen orthophosphate was purchased from WINLAB (Leicestershire, UK). Phosphate buffer (0.01 M), covering a pH range of 3–7, was freshly prepared. Analytical-grade sodium sulphate anhydrous was purchased from Macclesfield (Cheshire, UK). Sodium hydroxide pellets and orthophosphoric acid (85%) were purchased from BDH Chemicals Ltd. (Poole, UK). Chloroform and HPLC-grade methanol were purchased from Sigma (St. Louis, USA). Dimethyl sulfoxide (DMSO) was supplied by Loba Chemie Pvt. Ltd. (Mumbai, India). Acetone was purchased from Fisher Scientific UK Ltd. (Leicestershire, UK). Ultrapure water was produced by a Milli-Q water purification system, Millipore Ltd. (Bedford, USA). Human urine obtained from healthy adult male volunteers and was kept frozen until analysis.

2.2. Instruments

- Fluorescence spectra were recorded on a Spectramax M5 from Molecular Devices (California, USA), with a standard quartz cell having a path length of 1 cm and a bandwidth of 5 nm for excitation and emission monochromators. A quinine sulfate solution (0.01 mg mL1) was frequently utilized for the calibration of the fluorescence spectrometer. Data acquisition was performed via Softmax ProGXP software.
- The pH measurements were performed on a microprocessor laboratory pH meter obtained from Mettler-Toledo International Inc. (Zurich, Switzerland).
- A magnetic stirrer was purchased from IKA (Wilmington, USA).
- The ultrasonic sonicator cleaning system was supplied by XTRA150H (Elma, England).
- The electric digital balance was provided by Mettler-Toledo International Inc. (Zurich, Switzerland).
- Ultrapure water was produced by a Milli-Q water purification system, Millipore Ltd. (Bedford, USA)
- The biomedical freezer was supplied by Sanyo (Onoda, Japan).
- Medicool fridge was provided by Sanyo (Onoda, Japan).
- Vortex was obtained from Clifton Cyclon (Weston, England).

2.3. Preparation of standard solutions and calibration curve

A BRG stock solution was prepared by dissolving 0.005 g of BRG in 0.5 mL DMSO in a 10-mL volumetric flask. The volume was completed to the mark by the same solvent to obtain a final concentration of 0.5 mg mL1. The solution was stable for 1 month if kept in the refrigerator. A suitable aliquot of the standard stock solution was transferred into a 25-mL volumetric flask, and the final volume was diluted to the mark with methanol to yield a working standard solution with a final concentration of 25 mg mL1.

2.4. General analytical procedure

Suitable aliquots of the working standard solution (25 mg mL1) were quantitatively transferred into a series of separating funnels containing 1 mL of buffer and 6 mL of eosin Y dye. The mixtures were mixed well and diluted with deionized water to ~ 10 mL. The resulting solutions were extracted by 3 8 portions of chloroform for ~ 2 min each time. The resulting extracts were combined into dry 25-mL volumetric flasks, and the final volume was completed with chloroform to yield serial dilutions in the concentration range of 62.5–4000 ng mL1. Then, the contents of the flasks were treated with a small amount of chloroform, washed with anhydrous sodium sulfate, and shaken well for ~ 1 min. After the solid sodium sulfate settled down, the emissions of the clear final diluted solutions were measured within 10 min against a reagent blank treated similarly at a wavelength of kex 480 nm and kem 560 nm.

2.5. Stoichiometric relationship

The molar ratio method [21] was applied for stoichiometric investigation. The stock solution (2 mg mL1) was prepared by dissolving 0.02 g of the standard material in 10 mL of DMSO. First, the working solution (2.89 104 M) was prepared by taking a suitable aliquot of the stock solution and diluting to 10 mL with methanol. Second, 1 mL of the working solution (2.89 104 M) was separately treated with 1, 1.5, 2, 3, 4, 5, and 6 mL of the eosin Y dye solution (2.89 104 M), followed by the addition of 1 mL of the buffer and subsequent dilution with deionized water to ~ 10 mL. The formed complex was extracted and analyzed as explained under the general analytical procedure section.

2.6. Preparation of the urine sample

The stock solution (2 mg mL1) was prepared by dissolving 0.02 g of the standard material in 10 mL of DMSO. A suitable aliquot of the standard stock solution was transferred and diluted to 2 mL with methanol to yield a working standard solution of 0.4 mg mL1. Next, 600 mL of the working solution was spiked in 10 mL of urine sample to obtain a final concentration of 24 mg mL1. Different volumes (i.e., 0.5, 1, 1.5 mL) were taken from the spiked urine sample and separately treated with 6 mL of the 0.2% eosin Y dye solution, followed by the addition of 1 mL of potassium dihydrogen orthophosphate buffer pH 4.35. The formed complex was extracted with 3 8 portions of chloroform for ~2 min. The resulting extracts were combined into a dry 25-mL volumetric flask and diluted with chloroform. The formed complex was analyzed as explained under the general analytical procedure section.

3. Results and discussion

BRG comprises piperazine and piperidine moieties, containing three tertiary amines. In acidic media (pH 4.35), these tertiary amines are readily protonated, subsequently affording three positive centers in the drug molecule. Eosin Y ionizes in an aqueous solution, affording a negative charge on its carboxylic group. Thus, one mole of protonated BRG reacts with three mole of eosin Y to afford an ion-pair complex. The formed complex is appropriately extracted by chloroform. Therefore, the formation of the ion-pair complex is crucial for the spectrofluorimetric determination of BRG.
According to Scheme 1, BRG (I) combines with the anion of eosin Y (II) and is transferred from the aqueous phase into the organic phase as an ion pair (III).

3.1. Preliminary investigation

Several preliminary investigations were conducted for the spectrofluorimetric determination of BRG. Initially, derivatization was attempted using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBDCl) as the derivatizing agent; however, no reaction occurred possibly due to the steric hindrance, which prevented the reactivity of the secondary amine of BRG. Second, a fluorescein dye was used by the direct quenching and ion-pair complex method; however, these methods were limited due to the absence of a linear relationship between the decrease in RFI values and BRG concentration in the former method and the high fluorescence background in the later one. Third, the micellar-enhanced spectrofluorimetric method by using different surfactants was attempted, but no response was observed due to the absence of the native fluorescence of BRG.
Finally, a successful ion-pair complex method was developed by using eosin Y. Preliminary experiments were performed to determine the accurate strength of methanol for use as a solvent of BRG. Unsatisfactory results were observed using absolute methanol rather than methanol–water mixture in different proportions. However, best results were obtained by the use of 5% v/v methanol: water rather than higher concentrations of methanol in water or even absolute methanol as the solvent, related to the poor solubility of BRG in different proportions of the methanol: water mixture although the fluorescence intensities of the analyte and reagent blank increased with increased percentages of methanol in the aqueous phase before extraction; however the difference between them diminished. Hence, better results can be obtained by maintaining the total volume of the aqueous layer to ~10 mL; thus, the maximum percentage of methanol: water is ~5% m/m for BRG.

3.2. Excitation and fluorescence spectra

Eosin Y and its ion pair complex exhibit almost identical excitation and emission wavelengths. Fig. 3 shows the excitation and emission spectra of the extracted complex, with the maximum emission at 560 nm after excitation at 480 nm.

3.3. Optimization of reaction parameters

To establish the optimum pH as well as volumes of buffer and dye recommended for the maximum RFI, a series of experiments were conducted using changing one factor at a time approach. Then, the general suggested procedure was followed. All studies were performed at room temperature (~25 C).

3.3.1. Effect of pH

The effect of the aqueous phase pH for ion-pair extraction was examined using a potassium dihydrogen orthophosphate buffer over a pH range of 3–7 (Fig. 4). The maximum RFI was obtained at pH 4.35 for BRG, which was utilized for subsequent investigation.

3.3.2. Effect of buffer volume

In addition, the effect of the buffer volume at the selected pH for ion-pair extraction was examined. The maximum RFI was obtained using 1 mL of phosphate buffer for BRG, which was utilized for subsequent investigation (Fig. 5).

3.3.3. Effect of dye volume

Under the established experimental conditions regarding the pH and buffer volume, the effect of the dye volume on the extraction of the ion-pair complex were also examined. The maximum RFI was obtained using 6 mL of eosin Y for BRG, which was utilized for subsequent investigation (Fig. 6).

3.3.4. Effect of diluting solvent

Different solvents were tested as a diluting solvent, such as methanol, chloroform, ACN, and water. Compared to other solvents, water afforded superior RFI values, possibly related to the preference of water molecules to interact with themselves via hydrogen bonding without interfering with the formed complex [21].

3.3.5. Extraction rate, extraction efficiency, and stability of extracts

A shaking time in the range of 0.5 to 3 min did not produce any change in fluorescence intensities; thus, a shaking time of 2 min is selected. Reproducible fluorescence readings were obtained after three successive extraction steps, each using ~ 8 mL of chloroform. The fluorescence of chloroform extracts was constant for ~ 15 min.

3.4. Stoichiometric relationship

The molar ratio method was utilized to examine the molar ratio of the formed complex. The method revealed a 1:3 ratio for BRGeosin Y; it afforded the highest RFI, followed by constant RFI values (Fig. 7). The stability constant of the formed complex was calculated according to the following equation by the molar ratio method as in Table 1 [22]. where, b0 is the instability constant of the formed complex, and the stability constant (bÞ= b10 . C is the initial total concentration of eosin Y, n is the eosin Y: BRG concentration ratio, anda ¼ RFImRFImRFIs, where RFIm is the relative fluorescence intensity when all of the BRG is complexed with eosin Y. RFIs is the relative fluorescence intensity of the stoichiometric molar ratio of BRG to eosin Y in the complex.
The value of calculated stability constant (log b) of the formed complex was equal to 10.21, which revealed good stability of the formed complex. according to the general suggested procedure described previously in the experimental section. The regression equation for the results was derived by using the least-squares method. RFI was linear with a good correlation coefficient in the concentration range of 62.5– 4000 ng mL1 (Table 2).
The limit of detection (LOD) was theoretically determined using the following formula: where, k = 3.3. SDa and b are the standard deviations of the intercept and slope, respectively. From triplicate measurements, the LOD was 40.2 ng mL1, while the limit of quantification (LOQ) was practically determined as the first point in the linearity range (62.5 ng mL1).
BRG afforded high sensitivity due to the presence of three tertiary amines in its structure. These tertiary amines were expected to form a unique stable complex with eosin Y under the selected experimental conditions, which was confirmed by the high sensitivity (LOD = 40.2 ng mL1) and good stability (log b = 10.21).

4.2. Accuracy and precision

The accuracy and precision of the proposed method was verified by the analysis of three concentration levels of BRG in triplicate over a period of three days. Precision and accuracy were reported as % relative standard deviation (%RSD) and % recovery, respectively. Low RSD values (<2%) and good recovery revealed a high degree of accuracy and precision of the proposed method (Table 3).

4.3. Robustness

To evaluate the robustness of the proposed method, the effect of small variation in some of the operational conditions, such as pH, dye and/or buffer volume, on the recoveries and standard deviation of the bulk drug was tested. The obtained results were not significantly affected within the examined ranges of variations under assay conditions thus, the proposed procedure can be robust (Table 4).

5. Application

5.1. Application to spiked human urine

After the oral administration of 180 mg of BRG, the apparent oral clearance in the steady-state is 12.7 L/h. The elimination of BRG is divided into 65% in feces and 25% in urine. From the elimination in both compartments, the unchanged form of BRG represented 41% of the total in feces and 86% in urine [23]. Hence, ~40 mg mL1 of the drug level is excreted unchanged in the urine, which is equal to the working range of the proposed method. The results summarized in Table 5 revealed a mean percentage recovery ± SD in the range of 91.86 ± 4.09–97.43 ± 2.66. The proposed method was successfully applied for the determination of the BRG-eosin Y complex in the spiked urine sample.

5.2. Comparison between the proposed and reported LC-MS method for the determination of BRG in biological fluids.

The statistical comparisons of the proposed and reported LC-MS method [4] via the t-test and variance ratio F-test were summarized in Table 6. Experimental values revealed no significant difference between both methods in terms of accuracy (t-test) and precision (F-test). Thus, both methods are suitable for the quantitation of BRG in biological samples.

6. Conclusion

Compared to chromatographic methods, which require a long operation time and are limited by tedious operation procedures, the spectrofluorimetric method is less tedious and less cumbersome. Moreover, the high sensitivity and specificity offered by this method permit the determination of drugs up to nano levels. This method was adopted for the quantitation of BRG in bulk powder and in spiked human urine due to its simplicity, costeffectiveness, high sensitivity, and wide concentration range. The main disadvantage of this method includes laborious multiple extraction steps.

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