Abstract
Background
We propose a simple, sensitive, and fast high‐performance liquid chromatography ultraviolet detection (HPLC‐UV) method for the quantitative determination of bosutinib in human plasma.
Methods
Plasma samples were processed using an Oasis hydrophilic‐lipophilic balance extraction cartridge (1 mL, 30 mg). Bosutinib and the internal standard imatinib were separated using a mobile phase of 0.5% Na2 PO 4H2O (pH 3.5)‐acetonitrile‐methanol (55:25:20, v/v/v) on a CAPCELL PAK C18 MG II reversed‐phase column 250 nm×4.6 nm i.d., at a flow rate of 1.0 mL/min, with ultraviolet detection at 250 nm.
Results
The calibration curve exhibited linearity over the bosutinib concentration range of 25‐1500 ng/mL at 250 nm, with coefficient of variation for intraday precision of 2.42%, 6.04%, and 1.11% for 100, 250, and 1500 ng/mL, respectively, of bosutinib. The lower limit of detection was 20 ng/mL. The extraction recovery rates for bosutinib ranged from 84.36% to 85.82%. The intra‐ and interday precision was below 8.7%, and the accuracy ranged from −5.95% to 5.85% over the linear range. No notable matrix effects or astaticism were observed.
Conclusion
The proposed HPLC‐UV method was successfully applied as an assay to detect bosutinib in human plasma.
Keywords: assay, bosutinib, chromic myeloid leukemia, high‐performance liquid chromatography
Bosutinib (bosutinib hydrate, 4‐[(2, 4‐Dichloro‐5‐methoxyphenyl) amino]‐6‐methoxy‐7‐[3‐(4‐methylpiperazin‐1‐yl) propyloxy] quinoline‐3‐carbonitrile monohydrate) is a dual inhibitor of Src and Abl tyrosine kinases, with minimal activity against platelet‐derived growth factor receptor or receptor kinase c‐KIT.1, 2 Many patients with Philadelphia‐chromosome‐positive (Ph+) leukemia who received bosutinib as second‐line therapy showed a complete hematologic response, in addition to a cytogenetic response, following the development of resistance/intolerance to imatinib.3 Similar results were found in third‐ and fourth‐line settings following the development of resistance/intolerance to imatinib and dasatinib and/or nilotinib.3 Treatment with tyrosine kinase inhibitors has been shown to result in adverse events, with large variations among individuals. Such adverse events lead to interruption of treatment and weight loss. The cytogenetic and molecular responses of patients with chronic myeloid leukemia (CML) to imatinib have been reported to be correlated with the trough plasma concentration of imatinib.4, 5 Similar to first‐generation imatinib, bosutinib requires therapeutic drug monitoring (TDM) for optimal efficacy.
Several LC‐MS/MS analytical methods have been reported for the determination of bosutinib in human plasma.6, 7 However, these methods are not always available in standard hospital laboratories. Several high‐performance liquid chromatography (HPLC)‐based methods, with ultraviolet (UV) detection have been reported for the quantitation of imatinib in plasma.8 However, no HPLC‐UV methods have been reported for the determination of bosutinib in human plasma. The simple, sensitive method proposed herein for determining the concentration of bosutinib in plasma consists of one‐step solid‐phase extraction, followed by HPLC‐UV.
Bosutinib (purity 99.6%) and imatinib were obtained from Funakoshi Co (Tokyo, Japan). An Oasis hydrophilic lipophilic balance (HLB) extraction cartridge (1 mL, 30 mg) was purchased from Waters (Milford, MA, USA). All other reagents used were of analytical grade, except methanol, which was HPLC grade.
Stock solutions for generating standard curves of bosutinib and imatinib were prepared by dissolving the dry reagents in methanol to yield concentrations of 1.0 mg/mL. Working standard solutions of bosutinib (25, 50, 100, 250, 500, and 1500 ng/mL) and imatinib (500 ng/mL) were prepared by serial dilution with methanol. All the solutions were stored at 4°C.
A Waters™515 HPLC pump, equipped with a Waters™486 tunable absorbance detector was used. The flow rate was 0.5 mL/min, and sample detection was carried out at 265 nm using a CAPCELL PAK C18 MG II (Shiseido, Tokyo, Japan) reversed‐phase column (250 nm×4.6 nm i.d.) at ambient temperature. The column contains packing material made of porous spherical silica, which is coated with a silicone polymer monolayer containing octdecyl (C18) groups. The mobile phase was 0.5% KH2PO4 (pH3.5; adjusted with 50% phosphoric acid), acetonitrile, and methanol (55:25:20, v/v/v), which was degassed in an ultrasonic bath prior to use.
Fifty microliters of imatinib (500 ng) were added as an internal standard to a 100 μL plasma sample, and the plasma sample was then diluted with 900 μL of water and mixed in a vortexer for 30 seconds. This mixture was applied to the Oasis HLB extraction cartridge, which had been activated previously with methanol and water (1.0 mL each). The cartridge was then washed with 1.0 mL of water and 1.0 mL of 60% methanol in water and eluted with 100% methanol. The eluates were evaporated to dryness with nitrogen at 40°C using an evaporator (Iwaki, Tokyo, Japan). The residues were dissolved in 50 μL of methanol (vortex mixing for 30 seconds). Then, 50 μL of the mobile phase were added to each sample, followed by vortex mixing for another 30 seconds. An aliquot of 50 μL of each sample was then processed on the HPLC apparatus.
Calibration curves samples for bosutinib were prepared from pooled human blank plasma obtained from healthy subjects. The calibration curves were constructed for six concentrations of bosutinib (25, 50, 100, 250, 500, and 1500 ng/mL). Calibration curves at these concentrations were selected because the blood concentration of bosutinib in patients with CML is 100‐250 ng/mL in the stationary state.9 Calibration graphs were constructed from the peak height ratio of bosutinib to the imatinib internal standard from the HPLC chromatograms and the nominal concentration of bosutinib. The calibration curves were calculated by the least squares method.
The extraction efficiencies of bosutinib at the said concentrations, as well as those of imatinib at the applied concentrations, were calculated in replicates (n=5) by comparing the respective peak height obtained by extraction of the control samples from blank plasma with those obtained from the same amounts of unextracted control samples in methanol. The control samples were prepared by mixing solutions containing the same amount of compound that was added to the plasma blank.
Both the intra‐ and interday precision of the assay was determined. The intraday precision and accuracy were determined by analyzing spiked controls that were run in random order five times over the course of 1 day. The interday precision and accuracy were assessed by comparing the assay on different days for 5 days. The precision of the method at each concentration was determined by comparing the coefficient of variation (CV), which was obtained by calculating the standard deviation (SD) as a percentage of the calculated mean concentration. The estimated accuracy of each spiked control was obtained by comparing the nominal concentration with the assayed concentration. The limits of quantitation (LOQ) were determined as the lowest nonzero concentration measured with an intraday CV within 20% and an accuracy of less than ±20% [8], and the limit of detection (LOD) was defined as the (3.3×SD)/(slope of analytical calibration).
Bosutinib was stable in plasma for at least 3 hours at room temperature.10 A study of the long‐term stability of bosutinib showed that it was stable in plasma for at least 28 days when stored at −20°C.11
The present study was carried out in accordance with the guidelines for care in human studies adopted by the Ethics Committee of Oita University of Medicine and sanctioned by the Japanese government.
The peaks of bosutinib and imatinib were clearly separated using a mobile phase of 0.5% KH2PO4 (pH 3.5), acetonitrile, and methanol (55:25:20, v/v/v) on a CAPCELL PAK C18 MG II column at a flow rate of 1.0 mL/min. Imatinib was used as an internal standard for the method. In clinical settings, imatinib is not used in combination with bosutinib, which is reserved for use in third‐ and fourth‐line settings following the development of resistance or intolerance to dasatinib and/or nilotinib after the development of resistance or intolerance to imatinib. The total analytical time of the chromatogram was 20 minutes. A bosutinib peak was detected at a retention time of 15 minutes. The chromatograms are those from the 100 μL plasma samples spiked with bosutinib (25, 50, 100, 250, 500, and 1500 ng), with imatinib (500 ng) used as an internal standard. There was no interference by plasma components or bosutinib metabolites.
A calibration curve for bosutinib was constructed by the peak height method. A good linear relationship and wide dynamic range were observed at concentrations of bosutinib over 25‐1500 ng/mL. A typical calibration curve (obtained using the least‐squares method) was expressed as y=0.0024x−0.0144 (r 2=.9996), where y was the peak height ratio and x was the concentration in ng/mL. The correlation coefficient was 0.9996.
The proposed method was used for the determination of the recovery of bosutinib in human plasma, as shown in Table 1. The recovery of bosutinib was determined by adding three known bosutinib concentrations (100, 250, and 1500 mg/mL) to drug‐free plasma. The extraction recovery rates for bosutinib ranged from 84.36% to 85.82% (Table 1).
Table 1.
Precision and accuracy of HPLC assay for the determination of bosutinib in human plasma (n=5)
| Added (μg/mL) | Intra‐day | Inter‐day | Recovery (%) | ||||
|---|---|---|---|---|---|---|---|
| Found Mean±SD | CV (%) | Accuracy (%) | Found Mean±SD | CV (%) | Accuracy (%) | ||
| Bosutinib | |||||||
| 25 | 27.08±0.91 | 3.36 | 8.31 | 27.55±1.56 | 5.65 | 10.18 | 84.49 |
| 100 | 105.85±2.56 | 2.42 | 5.85 | 94.05±8.18 | 8.70 | −5.95 | 85.42 |
| 250 | 235.34±14.22 | 6.04 | −5.86 | 261.34±9.94 | 3.81 | 4.54 | 84.36 |
| 1500 | 1467.50±16.24 | 1.11 | −2.17 | 1490.15±37.57 | 2.25 | −0.66 | 85.82 |
The CV and accuracy of the intraday and interday assays were determined at bosutinib concentrations of 25‐1500 ng/mL. The CV for intraday precision was 2.42%, 6.04%, and 1.11% at concentrations of 100, 250, and 1500 ng/mL, respectively (Table 1). The intraday accuracy was <10.18% (Table 1).
The LOQ for bosutinib was 25 ng/mL, and the LOD was 20 ng/mL. This sensitivity was achieved using a limited sample volume of 100 μL of plasma. Assays that require small sample volumes are useful for routine drug monitoring of patients.
We described a simple, robust, rapid, and inexpensive HPLC‐UV method for the determination of bosutinib, which had high sensitivity and a large dynamic range. This study demonstrated the reliability of this method for TDM of bosutinib in patients with CML. The results can be expected to extend the availability of TDM of bosutinib, as our HPLC‐UV method requires only standard equipment available in most hospital laboratories. We are conducting a prospective clinical trial to assess the usefulness of TDM of bosutinib performed using our assay method.
Sumimoto T, Nakahara R, Sato Y, Itoh H. A quantitative method for the determination of bosutinib in human plasma using high‐performance liquid chromatography and ultraviolet detection. J Clin Lab Anal. 2018;32:e22201 10.1002/jcla.22201
References
- 1. Puttini M, Coluccia AM, Boschelli F, et al. In vitro and in vivo activity of SKI‐606, a novel Src‐Abl inhibitor, against imatinib‐resistant Bcr‐Abl+ neoplastic cells. Cancer Res. 2006;66:11314. [DOI] [PubMed] [Google Scholar]
- 2. Golas JM, Arndt K, Etienne C, et al. SKI‐606, a 4‐anilino‐3‐quinolinecarbonitrile dual inhibitor of Src and Abl kinases, is a potent antiproliferative agent against chronic myelogenous leukemia cells in culture and causes regression of K562xenografts in nude mice. Cancer Res. 2003;63:375. [PubMed] [Google Scholar]
- 3. Khoury HJ, Cortes JE, Kantarjian HM, et al. Bosutinib is active in chronic phase chronic myeloid leukemia after imatinib and dasatinib and/or nilotinib therapy failure. Blood. 2012;119:3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Larson RA, Druker BJ, Guilhot F, et al. Imatinib pharmacokinetics and its correlation with response and safety in chronic‐phase chronic myeloid leukemia: a subanalysis of the IRIS study. Blood. 2008;111:4022‐4028. [DOI] [PubMed] [Google Scholar]
- 5. Picard S, Titier K, Etienne G, et al. Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard‐dose imatinib in chronic myeloid leukemia. Blood. 2007;109:3496‐3499. [DOI] [PubMed] [Google Scholar]
- 6. Hsyu PH, Mould DR, Abbas R, Amantea M. Population pharmacokinetic and pharmacodynamic analysis of bosutinib. Drug Metab Pharmacokinet. 2014;29:441. [DOI] [PubMed] [Google Scholar]
- 7. Abbas R, Hug BA, Leister C, Gaaloul ME, Chalon S, Sonnichsen D. A phase I ascending single‐dose study of the safety, tolerability, and pharmacokinetics of bosutinib (SKI‐606) in healthy adult subjects. Cancer Chemother Pharmacol. 2012;69:221. [DOI] [PubMed] [Google Scholar]
- 8. Miura M, Takahashi N, Sawada K. Quantitative determination of imatinib in human plasma with high‐performance liquid chromatography and ultraviolet detection. J Chromatogr Sci. 2011;49:412. [DOI] [PubMed] [Google Scholar]
- 9. Nakaseko C, Takahashi N, Ishizawa K, et al. A phase 1/2 study of bosutinib in Japanese adults with Philadelphia chromosome‐positive myeloid leukemia. Int J Hematol. 2015;101:154‐164. [DOI] [PubMed] [Google Scholar]
- 10. Shah VP, Midha KK, Findlay JW, et al. Bioanalytical method validation‐a revisit with a decade of progress. Pharm Res. 2000;17:1551‐1557. [DOI] [PubMed] [Google Scholar]
- 11. Xu Y, Huang X, Dai S, Xiao Y, Zhou M. A simple method for the determination of bosutinib in rat plasma by UPLC‐MS/MS. J Chromatogr B. 1004;2015:93‐97. [DOI] [PubMed] [Google Scholar]