Novel high-performance liquid chromatography–tandem mass spectrometry method for simultaneous quantification of BCR-ABL and Bruton’s tyrosine kinase inhibitors and their three active metabolites in human plasma
Yuji Mukaia, Tatsunari Yoshidab, Takeshi Kondoc, Nobuo Inotsumea, Takaki Todaa,⁎
a Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 7-15-4-1, Teine-ku, Sapporo, Hokkaido 006-8585, Japan
b Biotage Japan Ltd., 1-14-4 Kameido, Koto-ku, Tokyo 136-0071, Japan
c Blood Disorders Center, Department of Hematology, Aiiku Hospital, Minami 4-jo Nishi 25-chome 2-1, Chuo-ku, Sapporo, Hokkaido 064-0804, Japan


Tyrosine kinase inhibitor Therapeutic drug monitoring Plasma concentration


Therapeutic drug monitoring is important in patients taking BCR-ABL and Bruton’s tyrosine kinase inhibitors (TKIs). Some TKI active metabolites with long elimination half-lives, such as dihydrodiol ibrutinib (DHI), N- desmethyl imatinib (N-DI), and N-desmethyl ponatinib (N-DP), have been characterized, indicating that these active metabolites should be monitored along with the parent compounds. However, there are currently no methods for the simultaneous quantification of BCR-ABL and Bruton’s TKIs and their three active metabolites. The present study aimed to develop and validate a method for the simultaneous quantification of nine phar- macologically active compounds (bosutinib, dasatinib, DHI, ibrutinib, imatinib, N-DI, N-DP, nilotinib, and po- natinib) using high-performance liquid chromatography–tandem mass spectrometry. A 150-μL sample of plasma was analyzed after purification with supported liquid extraction. The method has a run time of 7 min and was successfully validated over the following calibration ranges: 0.25–75 ng/mL for N-DP, 0.5–150 ng/mL for da- satinib and ponatinib, 10–3000 ng/mL for imatinib and nilotinib, and 1–300 ng/mL for the other analytes. Stability of the analytes after short- and long-term storage in the presence of plasma matriX was examined, and all analytes were found to be stable under all tested conditions. The recovery was ≥83%, and the relative standard deviation of internal-standard normalized matriX effects ranged from 3.9 to 13.9%. Dilution integrity up to 4-fold was ensured. The applicability of the method for all analytes was demonstrated using patient samples.

1. Introduction
BCR-ABL and Bruton’s tyrosine kinase inhibitors (TKIs) are essential agents in the treatment of hematologic malignancies [1]. Imatinib is a key drug in the treatment of chronic myeloid leukemia, and the re- lationship between its plasma/serum concentration and efficacy has been well studied [2–5]. In clinical practice, adjustment of the imatinib dose based on therapeutic drug monitoring (TDM) is required. Recent reports expanded the applicability of TDM to include other BCR-ABL TKIs, such as bosutinib, dasatinib, nilotinib, and ponatinib [6–10]. High inter-individual pharmacokinetic variability of these TKIs has been

reported, however, which could be explained in part by the effects of food-drug and drug-drug interactions [7,11–13]. It should be noted that dasatinib and imatinib are time-dependent inhibitors of CYP3A4, such that these TKIs have the capacity to inhibit their own metabolism upon chronic exposure [1]. These findings indicate the importance of TDM for determining the optimal TKI dose.
The benefit of TDM for treatment regimens involving ibrutinib is under exploration. Some studies have shown that the pharmacokinetics of ibrutinib are highly sensitive to drug-drug interactions via CYP3A4 and hepatic metabolism [14,15], suggesting that rational use of the drug necessitates monitoring the plasma concentration of ibrutinib in Abbreviations: Ctrough, trough plasma concentration; DHI, dihydrodiol ibrutinib; HPLC, high-performance liquid chromatography; IS, internal standard; IS-norm, internal standard normalized; LC–MS/MS, liquid chromatography–tandem mass spectrometry; LLOQ, lower limit of quantification; ME, matriX effect; MF, matriX factor; MRM, multiple reaction monitoring; N-DI, N-desmethyl imatinib; N-DP, N-desmethyl ponatinib; QC, quality control; %RE, percent relative error; %RSD, percent relative standard deviation; SPE, solid-phase extraction; MTBE, tert-butyl methyl ether; TDM, therapeutic drug monitoring; TKI, tyrosine kinase inhibitor
⁎ Corresponding author at: Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 7-15-4-1 Maeda, Teine-ku,
Sapporo, Hokkaido 006-8585, Japan.
E-mail address: [email protected] (T. Toda).


Received 17 October 2019; Received in revised form 2 December 2019; Accepted 4 December 2019

specific populations such as those with low CYP3A4 activity. Recent reports also showed that adherence to ibrutinib therapy is critically important for achieving a clinical response in patients with chronic lymphocytic leukemia, small lymphocytic lymphoma, and non-Hodg- kin’s lymphoma [16,17]. Adherence to therapy is also important for patients taking imatinib [18] and dasatinib [19].
Several active metabolites with longer elimination half-lives than the parent TKIs, including dihydrodiol ibrutinib (DHI), N-desmethyl imatinib (N-DI), and N-desmethyl ponatinib (N-DP), have been char- acterized [20–22]. These studies indicated that data regarding the plasma concentrations of DHI, N-DI, and N-DP would be useful if de- velopment of dose-dependent adverse events was suspected. As the elimination half-life of DHI is longer than that of ibrutinib [22], mon- itoring the plasma concentration of DHI would be useful for evaluating adherence to ibrutinib therapy. Thus, the concentrations of the active metabolites DHI, N-DI, and N-DP should be monitored along with that of the respective parent compounds.
Numerous methods for determining the plasma concentrations of TKIs and their active metabolites using high-performance liquid chro- matography (HPLC) with ultraviolet detection and liquid chromato- graphy (LC)–tandem mass spectrometry (MS/MS) have been reported [9,10,23–25]. In addition to LC conditions, the optimum sample clean- up procedures also vary based on the target analyte, which renders clinical application of specific analytical methods for routine TDM of the various TKIs impractical. However, several recent reports described the simultaneous quantification of more than 5 TKIs [26–28]. In addi- tion, Huynh et al. [29] developed a method for the simultaneous quantification of 14 compounds including all BCR-ABL and Bruton’s TKIs. As the authors employed protein precipitation with salting-out as the clean-up method for human plasma, the processed samples con- tained numerous plasma components, such as phospholipids. Repeated injection of such crude samples into MS/MS instruments is undesirable, because the instruments must be cleaned frequently after analysis.
To our knowledge, there are no methods currently available for the simultaneous determination of the plasma concentrations of bosutinib, dasatinib, DHI, ibrutinib, imatinib, N-DI, N-DP, nilotinib, and pona- tinib. The present study therefore aimed to develop and validate a method for the simultaneous quantification of the abovementioned pharmacologically active compounds using LC-MS/MS.
2. Materials and methods
2.1. Chemicals and reagents
Bosutinib (purity: 98%), dasatinib (98%), DHI (96%), ibrutinib
(98%), imatinib mesylate (98%), N-DI (98%), nilotinib (98%), pona-
tinib (96%), bosutinib-d8 (98%), dasatinib-d8 (98%), and imatinib-d8 (97%) were purchased from Toronto Research Chemicals (North York, ON, Canada). Ibrutinib-d5 (99%) and N-DP (98%) were obtained from TLC Pharmaceutical Standards (Aurora, ON, Canada) and Alsachim (Illkirch Graffenstaden, France), respectively.
Blank pooled human plasma (lot: BJ9363A) and siX different lots of drug-free human plasma collected from individuals (both containing EDTA-2 K as an anticoagulant) were purchased from Cosmo Bio (Tokyo, Japan) and Clinical Trials Laboratory Services (London, UK), respec- tively. HPLC-grade acetonitrile, methanol, and tert-butyl methyl ether (MTBE) were obtained from Kanto Chemical (Tokyo, Japan). LC- MS–grade formic acid and Suprapur®-grade ammonia solution (25%) were purchased from Merck (Darmstadt, Germany), and MS-grade ammonium formate was purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Analytical procedures
LC-MS/MS analysis was performed on an Agilent 1200 series LC system (Santa Clara, CA, USA) coupled to an API3200 Q Trap System

(Sciex, Framingham, MA, USA). Separation was achieved on an L- column3 C18 (3 μm, 2.1 × 50 mm; Chemicals Evaluation and Research Institute, Tokyo, Japan) equipped with a matching guard column (5 μm, 2.0 × 5 mm). The column temperature was maintained at 55 °C, and the sample compartment in the autosampler was maintained at 4 °C. The aqueous and organic mobile phases were 10 mmol/L of am- monium formate (A) and acetonitrile (B), respectively, each containing 0.1% formic acid. A miXture of acetonitrile/methanol/water (25/25/ 50, v/v/v) containing 0.1% formic acid was used as the needle wash solvent. The initial flow rate and percentage of mobile phase B were set at 0.35 mL/min and 45%, respectively, with isocratic delivery to the column until 2.2 min. A divert valve allowed delivery of the eluate to the MS/MS detector from 0.3 to 2.2 min. After elution of all analytes, the column was washed with the mobile phase consisting of 95% B at a flow rate of 0.50 mL/min and subsequently re-equilibrated to the initial mobile phase conditions. The total run time was 7.0 min.
Samples were analyzed by electrospray ionization (ESI), with the instrument operated in positive ion mode and nebulizing, turbo spray, and curtain gas optimal values set at 30, 40, and 15 psi, respectively. Ion source temperature was set at 600 °C, and the ESI needle voltage was set at +5.5 kV. Multiple reaction monitoring (MRM) was employed using nitrogen as the collision gas. Quantifier and qualifier MRM were set for each analyte, and the ratios of qualifier MRM to quantifier MRM were used for the identification of analytes. Data were acquired using the scheduled MRM algorithm of Analyst® software, ver. 1.6.3. (Sciex).

2.3. Stock solutions, calibration standards, and quality control (QC) samples
Stock solutions of DHI, all TKIs, and all internal standards (ISs) were prepared in methanol at 0.1, 0.5, and 1.0 mg/mL, respectively, except for ibrutinib-d5, which was prepared in acetonitrile. Stock solutions of N-DI and N-DP were prepared in a miXture of 0.1% formic acid/me- thanol (50/50, v/v) or dimethyl sulfoXide at 0.2 mg/mL, respectively. Working solutions for all TKIs, metabolites, and ISs were prepared by diluting with methanol, and those for calibration standards and QC samples were separately prepared using different stock solutions. All stock and working solutions were stored at −20 °C until use.
Spiked plasma was prepared by adding 10 μL of methanol-based working solution for analytes to 140 μL of blank pooled plasma. Calibration standards were prepared at the following concentrations: 0.25, 0.5, 1.25, 5, 12.5, 25, and 75 ng/mL for N-DP; 0.5, 1, 2.5, 10, 25,
50, and 150 ng/mL for dasatinib and ponatinib; 10, 20, 50, 200, 500,
1000, and 3000 ng/mL for imatinib and nilotinib; and 1, 2, 5, 20, 50, 100, and 300 ng/mL for the other analytes. QC samples were prepared at four concentrations (lower limit of quantification [LLOQ], low QC, medium QC, and high QC): 0.25, 0.75, 30, and 60 ng/mL for N-DP; 0.5,
1.5, 60, and 120 ng/mL for dasatinib and ponatinib; 10, 30, 1200, and
2400 ng/mL for imatinib and nilotinib; and 1, 3, 120, and 240 ng/mL for the other analytes.

2.4. Sample clean-up method
A 150-μL sample of spiked plasma was diluted with 150 μL of 1% ammonia aqueous solution containing ISs (20 ng/mL for bosutinib-d8;
10 ng/mL for dasatinib-d8 and imatinib-d8; and 5 ng/mL for ibrutinib- d5). The diluted plasma was loaded onto an ISOLUTE® SLE+ 400-μL capacity column (Biotage, Uppsala, Sweden) and held for 5 min. The analytes were eluted three times with 800-μL aliquots of MTBE, and then the extract was evaporated to dryness. The residue was recon- stituted in 80 μL of acetonitrile/methanol (50/50, v/v), followed by 120 μL of water. After centrifugation for 5 min at 18,000g, 20 μL of the supernatant was injected into the LC-MS/MS system.

2.5. Assay validation
The method was validated in accordance with published guidelines [30,31].
2.5.1. Linearity and carry-over
Calibration curves were generated for each analyte by plotting the peak area ratio versus the theoretical concentration of 7 non-zero ca- libration standards over 3 different days. A linear equation was ob- tained using the least squares model with a weighting factor of 1/X2. The LLOQ was defined as the lowest concentration exhibiting a signal/ noise ratio > 10 that also met the acceptance criteria in relation to accuracy and precision, as described below. Carry-over was evaluated by injecting total blank samples, containing neither analytes nor ISs, following injection of the highest calibration standard. The peak area observed in the carry-over sample for each analyte was compared to that obtained in the LLOQ analysis. Carry-over was considered negli- gible when the peak area ratio (blank/LLOQ) was < 20%.
2.5.2. Accuracy and precision
Accuracy and precision were evaluated using QC samples at four concentrations as described above. The concentrations of QC samples were determined using calibration curves prepared on the same day. Accuracy was expressed as percent relative error (%RE), and intra- and inter-assay accuracy were determined using siX replicates for all con- centrations in each analytical batch. Precision was expressed as percent relative standard deviation (%RSD), and intra- and inter-assay precision were determined using siX replicates for all concentrations in each analytical batch. The intra- and inter-assay %RE as well as %RSD were considered acceptable when the values were within the range of ± 15% ( ± 20% for the LLOQ).
2.5.3. Stability
The stability of each analyte in the presence of plasma matriX was assessed at low and high QC levels in five replicates, under the fol- lowing conditions: bench-top stability (held at room temperature for 5 h), processed sample stability (kept in the autosampler maintained at 4 °C for 24 h), freeze–thaw stability (after three freeze–thaw cycles), and long-term stability (at −20 °C for up to 3 months). Stability was expressed as the mean ratio of the peak area after storage to that of a freshly prepared sample; samples were considered stable when the ratio was ≥85%.
2.5.4. Recovery and matrix effects (MEs)
Recovery and MEs were investigated at low and high QC levels using five replicates, according to the method reported by Matuszewski et al. [32]. MEs were examined using siX lots of drug-free plasma, and the matriX factor (MF) was determined based on the ratio of the peak area obtained in the presence of plasma matriX to that in the absence of ma- triX. The IS-normalized MF (IS-norm MF) was then calculated by dividing the MF of each analyte by the MF of the corresponding IS, and a %RSD of the IS-norm MF for each analyte of ≤15% was considered acceptable.
2.5.5. Selectivity
The selectivity of the method was assessed by analyzing drug-free plasma obtained from siX individuals. The signal intensity of each analyte was compared to that obtained with spiked plasma at the LLOQ and considered acceptable when the peak ratio (blank/LLOQ) was < 20% for each analyte or < 5% for each IS. Cross-talk and effects as- sociated with impurities were also examined prior to the validation process using IS solutions, and the IS concentrations were then adjusted accordingly.
2.5.6. Dilution integrity
Dilution integrity was examined using spiked plasma samples at concentrations 2-fold higher than that of the highest calibration

standard. The samples were processed as described above, and the re- constituted solutions were diluted 4-fold using processed blank plasma as the control matriX. Based on the calculated concentration of each sample, %RE and %RSD were determined and considered acceptable when the values were within the range of ± 15% and ≤15%, respec- tively.

2.6. Clinical application
The present method was applied to determine the plasma con- centrations of TKIs and their metabolites in patients. Outpatients over 16 years old taking any BCR-ABL or Bruton’s TKI for at least 7 days were eligible to participate in the study. Venous blood was collected in a tube containing EDTA-2 K and then centrifuged for 5 min at 1900g. The resulting plasma samples were stored at −30 °C until analysis. The clinical portion of the study was approved by the ethics committees of Aiiku Hospital and Hokkaido University of Science and conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent prior to participation in the study.

3. Results and discussion
3.1. Method development
3.1.1. LC-MS/MS assay
Parameters of the MS/MS detector were optimized for each analyte using 100 ng/mL methanol-based solutions. Various parameters were manually tuned for analysis of imatinib, nilotinib, and ponatinib to avoid saturation of the detector response (Supplementary Table S1). To eliminate the effect of cross-talk between analytes that have similar product ions, a time scheduled MRM algorithm was programed and the MRM scan order was optimized prior to analysis. Typical chromato- grams and the ratio of ions are shown in Fig. 1.

3.1.2. Sample clean-up method
Huynh et al. reported an ultra HPLC-MS/MS method for the si- multaneous quantification of 14 TKIs; their method involved protein precipitation with salting-out to extract the analytes from plasma [29]. However, samples processed using protein precipitation still contain many plasma components, such as phospholipids. In addition to en- dogenous substances, the samples prepared by Huynh et al. contained zinc sulfate at a final concentration of 14.3 mmol/L [29]. As our ana- lytical method requires a large injection volume (20 μL) to achieve sufficient sensitivity for all analytes of interest, repeated analysis of such crude samples rendered the curtain plate and orifice of the MS/MS instrument dirty, resulting in a loss of sensitivity during analysis of the batch (data not shown).
Merienne et al. reported a sample clean-up method for the si-
multaneous quantification of 17 TKIs using solid-phase extraction (SPE) with OASIS® MCX cartridges [28]. The authors reported absolute re- coveries of bosutinib, dasatinib, and imatinib of over 80%, whereas the recoveries of nilotinib and ponatinib were approXimately 25 and 65%, respectively. Thus, another clean-up method is required to improve the sensitivity for determining plasma concentrations of nilotinib and po- natinib.
The current method employed a supported liquid extraction method using an ISOLUTE SLE+ column. According to the protocol provided by the manufacturer, we examined several dilution and extraction solvents and found that a 2-fold dilution of plasma with 1% ammonia aqueous solution and elution with MTBE (800 μL × 3) provided the best re- covery for all analytes (as described in Section 3.2.4.). Supported liquid extraction is not required for the conditioning and wash processes, whereas those processes are generally required for SPE. The sample purification method developed in the present study is thus more con- venient compared to SPE-based methods [28].

Fig. 1. Typical chromatograms for analytes examined at the lower limit of quantification and internal standards in spiked plasma. The signal intensities for all multiple reaction monitoring (MRM) windows were collected and integrated using Analyst® software. Data, including retention time and signal intensity, were converted into comma-separated values format. The chromatograms were depicted using Microsoft EXcel® for Office 365 (Redmond, WA, USA). Blue and orange lines represent signals obtained from spiked plasma for quantifier and qualifier MRM windows, respectively, and purple lines represent signals obtained from blank plasma for quantifier MRM windows of each analyte. For ISs, the blue, orange, green, and yellow lines denote imatinib-d8, dasatinib-d8, bosutinib-d8, and ibrutinib-d5, respectively. Ion ratio: the mean ratio (SD) of qualifier MRM to quantifier MRM over the calibration range.

3.2. Method validation
3.2.1. Linearity and carry-over
Each analyte exhibited a linear calibration curve, with a mean coefficient of determination (R2) ≥ 0.99 for each analyte over the ca- libration range (Table 1). Analyte concentrations back-calculated from the corresponding linear equations were accurate within the range of ± 15% ( ± 20% for the LLOQ) for at least 75% of calibration stan- dard samples in each analytical run.
Significant carry-over was observed for bosutinib, N-DI, N-DP, and ponatinib, with ratios of the carry-over blank peak area to that of the LLOQ sample of 32, 61, 42, and 22%, respectively. However, no carry-over into the second blank samples was detected. The highest concentration of the calibration range for bosutinib, N-DP, and ponatinib was at least 3-fold higher than the reported corresponding trough plasma concentration

(Ctrough) [2,9,10,21,23,28,33–40] (Table 2), indicating that predicted carry-over after the analysis of clinical samples to determine the Ctrough of these analytes would be one-third or less of the current result. In contrast, the reported Ctrough of N-DI after administration of 400 mg of imatinib mesylate is approXimately 200 ng/mL [23]. As this Ctrough is close to the highest concentration of the calibration range for this compound, sig- nificant carry-over could be observed after analysis of clinical samples to determine the Ctrough of N-DI. However, the effect of such carry-over on the accuracy of N-DI quantification results should be negligible, as the calculated concentration based on the carry-over peak would be less than the LLOQ (i.e., < 1 ng/mL). Although the effect of carry-over on the ac- curacy of quantification results for the analytes described here should be negligible, a blank sample should be injected following the analysis of clinical samples in determining the Ctrough of bosutinib, N-DI, N-DP, and ponatinib in order to eliminate any potential carry-over effect.

Table 1
Calibration curve linearity.
Analyte Internal standard Linear range Slope Intercept R2

(ng/mL) Mean (n = 3) SD Mean (n = 3) SD Mean (n = 3) Range
Bosutinib Bosutinib-d8 1–300 0.090 0.0015 −0.026 0.011 0.9967 0.9958–0.9976
Dasatinib Dasatinib-d8 0.5–150 0.71 0.011 0.006 0.021 0.9931 0.9902–0.9952
DHI Ibrutinib-d5 1–300 0.12 0.0076 −0.005 0.005 0.9933 0.9908–0.9958
Ibrutinib Ibrutinib-d5 1–300 0.40 0.021 0.041 0.006 0.9957 0.9950–0.9964
Imatinib Imatinib-d8 10–3000 0.033 0.00040 −0.070 0.002 0.9946 0.9926–0.9962
N-DI Imatinib-d8 1–300 0.042 0.00057 −0.027 0.001 0.9951 0.9926–0.9976
N-DP Bosutinib-d8 0.25–75 0.32 0.021 −0.022 0.007 0.9943 0.9928–0.9958
Nilotinib Imatinib-d8 10–3000 0.017 0.00052 −0.002 0.010 0.9947 0.9902–0.9980
Ponatinib Bosutinib-d8 0.5–150 0.59 0.0090 0.020 0.005 0.9940 0.9936–0.9948
DHI, dihydrodiol ibrutinib; N-DI, N-desmethyl imatinib; N-DP, N-desmethyl ponatinib.
R2, coefficient of determination.
the first to provide data regarding the long-term stability of ponatinib
and N-DP stored for 3 months in human plasma.
For all analytes, the mean extraction recovery ranged from 83 to 119% (Table 4). A comparison with the SPE method using OASIS MCX

cartridges [28] indicated that the current method provided approXi-
mately 4-fold greater recovery of nilotinib. The reported extraction recovery of ibrutinib using SPE with an OASIS HLB cartridge is ap-
The %RSD values for IS-norm MFs fell within the range of ± 15% at both studied concentrations for all analytes, indicating that the addition of ISs compensated for any effects on ionization of the analytes asso- ciated with the presence of endogenous components.

DHI, dihydrodiol ibrutinib; N-DI, N-desmethyl imatinib; N-DP, N-desmethyl ponatinib.
QD, once daily; BID, twice daily; ND, not detected.
a Median Ctrough.
b Simulated mean Ctrough.
c Estimated Ctrough by multiplying the accumulation ratio (2.8) and Ctrough obtained after a single oral dose of ibrutinib (15 mg).
d Geometric mean Ctrough.

3.2.2. Accuracy and precision
The accuracy (%RE) and precision (%RSD) of the proposed method are summarized in Table 3. The intra- and inter-assay %RE and %RSD at all studied concentrations for all analytes fell within the pre-defined acceptance criteria. These results thus demonstrate the reproducibility and reliability of the analytical method.

Table 3

3.2.5. Selectivity
Selectivity was examined by analyzing siX lots of drug-free plasma samples. No significant interfering peaks were observed within the re- spective retention times for any of the MRM transitions of the target analytes or ISs. Endogenous substances in the plasma matriX exhibited negligible effects.

3.2.6. Dilution integrity
In terms of the accuracy and precision of sample dilution integrity,
%RE and %RSD ranged from −9.9 to 12% and 2.0–13%, respectively (Table 5). Assuming a clinical case in which the concentration was above the calibration range in the first analysis, such as the case of subject ID 1 described in the clinical application section, the remaining sample could be used to determine the accurate concentration after adequate dilution. Thus, we examined the dilution integrity of the post-

Accuracy and precision for quality control (QC) analyte samples at four concentrations over the calibration range.

Analyte LLOQ Low QC Middle QC High QC
Intra-assay Inter-assay Intra-assay Inter-assay Intra-assay Inter-assay Intra-assay Inter-assay
Bosutinib −2.1 7.4 2.4 8.3 −3.2 10.0 −7.3 7.8 3.5 7.4 −2.8 7.1 4.1 5.0 −3.3 6.9
Dasatinib −2.4 8.7 −5.0 10.4 −7.7 10.3 −8.5 10.0 −0.5 6.4 −3.3 6.9 −6.8 5.8 −4.3 7.9
DHI 1.5 14.4 6.0 13.5 −5.7 6.9 −4.1 8.5 −7.8 8.4 −7.9 6.0 −7.6 5.2 −6.3 6.1
Ibrutinib −9.1 5.2 −10.3 4.1 −5.7 2.0 −3.4 3.3 0.6 2.9 −3.8 4.2 −0.9 2.8 −3.1 2.9
Imatinib −6.1 7.8 −6.7 7.5 −11.7 7.3 −10.6 8.7 4.4 6.4 0.5 7.4 −2.4 5.8 −1.0 6.6
N-DI −3.2 4.8 −0.2 4.6 −7.2 5.7 −3.1 7.4 3.5 3.2 0.8 6.5 −0.4 4.0 2.8 6.1
N-DP −5.9 7.3 −2.8 10.3 −5.7 6.9 −3.1 6.3 −4.2 4.2 −5.0 5.8 −3.1 6.1 −7.0 6.8
Nilotinib −5.9 7.6 −2.4 7.5 −8.8 7.1 −0.5 8.9 −1.5 5.2 −2.7 8.8 2.6 3.3 −2.9 6.6
Ponatinib −11.1 8.8 −9.2 8.1 3.2 8.8 1.0 7.9 1.5 3.6 −0.2 3.8 −0.4 3.6 −4.3 4.7
DHI, dihydrodiol ibrutinib; N-DI, N-desmethyl imatinib; N-DP, N-desmethyl ponatinib. LLOQ, lower limit of quantification; QC, quality control.
Intra-assay, n = 6; Inter-assay, n = 18.
%RE, relative error = (measured − nominal)/nominal × 100; %RSD, relative standard deviation = (SD/mean) × 100.

Table 4
Recovery and matriX effects after supported liquid extraction of quality control (QC) samples at two concentrations (n = 5).

Table 6
Plasma concentrations of BCR-ABL and Bruton’s TKIs and their three active metabolites.
Mean %RSD Mean %RSD Mean %RSD Mean %RSD Bosutinib 102.6 4.4 109.5 9.8 96.4 6.2 103.5 7.1
Dasatinib 113.0 12.7 110.7 4.3 105.0 10.2 99.4 10.1
DHI 105.0 3.4 114.4 6.1 108.5 7.7 110.5 5.5
Ibrutinib 85.7 5.6 89.7 5.4 97.3 3.9 101.4 6.5
Imatinib 103.6 7.9 107.5 9.7 97.8 9.3 102.6 5.8
N-DI 83.4 13.9 102.2 3.4 85.3 11.4 86.7 6.5
N-DP 116.1 9.6 119.3 9.3 93.4 13.9 111.3 9.9
Nilotinib 101.8 7.9 111.8 6.0 118.5 10.9 94.7 7.0
Ponatinib 103.1 12.0 117.0 7.9 98.7 8.5 107.9 10.3

DHI, dihydrodiol ibrutinib; IS-norm MF, internal standard normalized matriX factor; N-DI, N-desmethyl imatinib; N-DP, N-desmethyl ponatinib.
QC, quality control; %RSD, relative standard deviation.

Table 5
Accuracy and precision for 4-fold diluted samples (n = 5).
Analyte %RE %RSD

1 Imatinib 400 mg QD 2.5 Imatinib: 1325 N-DI: 306
2 Nilotinib 300 mg BID 16 1285
3 Bosutinib 600 mg QD 3 172
4 Bosutinib 300 mg QD 15 86.4
5 Bosutinib 200 mg QD 4 112
6 Dasatinib 100 mg QD 7 31.1
7 Dasatinib 60 mg QD 1 8.48
8 Nilotinib 300 mg BID 0.6 1585
9 Dasatinib 20 mg QDa 24 0.75
10 Nilotinib 600 mg BIDb 2.5 1770
11 Bosutinib 300 mg QD 4.5 250
12 Ibrutinib 560 mg QD 1 Ibrutinib: 29.1 DHI: 59.7
13 Nilotinib 300 mg BID 5 821
14 Dasatinib 50 mg QD 4.5 69.0
15 Ponatinib 15 mg QD 3 Ponatinib: 28.9 N-DP:0.94
16 Dasatinib 100 mg QD 5 5.89
17 Imatinib 300 mg QD 17.5 Imatinib: 1550 N-DI: 226
18 Bosutinib 300 mg QD 5 221
19 Bosutinib 400 mg QD 17.5 109
20 Ponatinib 15 mg QD 1 Ponatinib: 41.6
21 Dasatinib 50 mg QD 4
22 Nilotinib 200 mg BID NA
23 Bosutinib 300 mg QD 7
24 Ponatinib 30 mg QD 22
Ponatinib: 141N-DP: 9.15
DHI, dihydrodiol ibrutinib; N-DI, N-desmethyl imatinib; N-DP, N-desmethyl

DHI, dihydrodiol ibrutinib; N-DI, N-desmethyl imatinib; N-DP, N-desmethyl ponatinib.
QD, once daily; BID, twice daily; NA, not available.
a 3 days on and 1 day off.b 400 mg-200 mg.

%RE, relative error = (measured − nominal)/nominal × 100; %RSD, re-
the analytes examined were sufficient for TDM based on the Ctrough. The lative standard deviation = (SD/mean) × 100.
extraction samples with the blank samples containing control matriX. Dilution integrity was ensured up to 4-fold for all analytes.

3.3. Clinical application
The present method was applied to the determination of plasma concentrations of all nine pharmacologically active compounds. A total of 24 clinical samples (n = 7 for bosutinib, n = 6 for dasatinib, n = 1 for ibrutinib, n = 2 for imatinib, n = 5 for nilotinib, and n = 3 for ponatinib) were analyzed using the proposed method, and the results are shown in Table 6. The concentration of N-DI in plasma collected from a patient taking 400 mg/day of imatinib (subject ID 1) was cal- culated as > 300 ng/mL in the first analysis without sample dilution. The sample was reanalyzed after 4-fold dilution with the processed blank sample, resulting in a calculated concentration of 306 ng/mL. All other samples were quantified without dilution. The concentrations of ponatinib and N-DP in plasma collected from a patient taking 30 mg/ day of ponatinib (subject ID 24) were calculated as 141 and 9.15 ng/ mL, respectively. Although the blood sample was collected 22 h after the last drug intake, the concentration was approXimately 3-fold higher than that of the reported Ctrough (Table 2). As the patient was ad- ministered itraconazole along with ponatinib, this result could be ex- plained as being due to a drug-drug interaction via CYP3A4.
Overall, the proposed method was shown to be suitable for the analysis of clinical samples, regardless of the sampling time or potential drug-drug interactions, indicating that the calibration ranges for all of

proposed analytical method is therefore also suitable for pharmacoki- netic analyses of the TKIs examined in this study. Numerous analytical methods have been reported for monitoring the plasma concentrations of specific BCR-ABL and Bruton’s TKIs [9,10,23–29]. The current method is the first to enable the simultaneous determination of these TKIs and their active metabolites. This method could facilitate TDM and pharmacokinetic studies of BCR-ABL and Bruton’s TKIs to ensure ra- tional application of therapies for hematologic malignancies.
4. Conclusions
We developed the first method suitable for the simultaneous quantification of nine pharmacologically active analytes in relation to BCR-ABL and Bruton’s TKIs. The method requires only a simple clean- up process and has a run time of 7 min. The method was successfully validated and applied to the analysis of 24 clinical samples.
This work was supported by JSPS KAKENHI Grant Number JP18K14988.
CRediT authorship contribution statement
Yuji Mukai: Conceptualization, Methodology, Validation, Investigation, Resources, Writing – original draft, Funding acquisition. Tatsunari Yoshida: Conceptualization, Methodology, Resources, Writing – review & editing. Takeshi Kondo: Conceptualization, Investigation, Resources, Writing – review & editing. Nobuo Inotsume:

Conceptualization, Methodology, Writing – review & editing. Takaki Toda: Conceptualization, Methodology, Writing – review & editing.
Declaration of Competing Interest
Tatsunari Yoshida is an employee of Biotage Japan Ltd.
The authors would like to thank Mr. Rui Yoshida, Mr. Hayato Kobayashi, Ms. Miu Kawahara, Ms. Konomi Saito, Mr. Hiroyuki Watanabe and Ms. Haruna Muraki for their clinical assistance.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jchromb.2019.121928.

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