June 2022

Medical Mass Spectrometry Vol. 6 No. 1

Research Paper

Shifting the linear range of the calibration curves for the quantiﬁcation oforal molecular-targeted anticancer drugs and the active metabolite inhuman plasma using LC-ESI-MS/MS
1
1,2
2
2
2
Tensei Hirasawa , Masafumi Kikuchi *, Shinya Takasaki , Masaki Kumondai , Yu Sato ,
2

3

3

1,2

1,2

Toshihiro Sato , Eishi Imoto , Yoshihiro Hayakawa , Masamitsu Maekawa , Nariyasu Mano

Faculty of Pharmaceutical Sciences, Tohoku University, 6‒3 Aoba, Aramaki, Aoba-ku, Sendai 980‒8578, Japan
Department of Pharmaceutical Sciences, Tohoku University Hospital, Seiryo-machi, Aoba-ku, Sendai 980‒8574, Japan
3
Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604‒8511, Japan
1

2

Abstract

Many types of oral kinase inhibitors, including imatinib and geﬁtinib, have become clinically available andhave signiﬁcantly contributed to improving the survival outcomes in cancer patients. Therapeutic drug monitoring of someoral kinase inhibitors is important for ensuring the eﬃcacy and safety of these drugs. Liquid chromatography-electrosprayionization tandem mass spectrometry (LC-ESI-MS/MS) has been used for the simultaneous quantiﬁcation of these drugs
in the human plasma. However, the development of a simultaneous LC-MS/MS analytical method is diﬃcult owing to thediﬀerences in mass spectrometry (MS) sensitivity and the therapeutic range of each drug. In this study, we investigated thelinear range shifts of calibration curves by adjusting the collision energy defect, in-source collision-induced dissociation,
secondary product ion selected reaction monitoring, and isotopologue selected reaction monitoring to develop a simultaneous quantiﬁcation method for 20 oral kinase inhibitors and active metabolite of sunitinib. Although these four techniques
could easily adjust the number of ions introduced to the MS when used individually, it was diﬃcult to adapt 21 analytes
using only one technique. Therefore, it is important to utilize multiple techniques, considering the therapeutic concentration
range of each drug, in order to develop a method for the simultaneous quantiﬁcation of oral kinase inhibitors and active metabolites in molecular-targeted therapy.
Key words: oral kinase inhibitor, LC-ESI-MS/MS, collision energy, in-source collision-induced dissociation, simultaneousquantiﬁcation

Introduction

improving the survival outcomes in cancer patientscancer, which targets a speciﬁc molecule and controls its

Geﬁtinib, erlotinib, osimertinib, dacomitinib, lapatinib, andafatinib have been used in patients with non-small cell lung

1,2）

Molecular-targeted therapy is an essential treatment forfunction. Many types of oral kinase inhibitors have become.

cancer or breast cancer targeting mutations in the ErbB
family. Bosutinib, imatinib, dasatinib, nilotinib, and ponati-

clinically available and have contributed signiﬁcantly to

nib are used to treat leukemia by inhibiting BCR-ABL
activity. Axitinib and ibrutinib exert their eﬀects by inhibit-* Corresponding author
Masafumi Kikuchi

ing the vascular endothelial growth factor receptor and Bru-

Faculty of Pharmaceutical Sciences, Tohoku University, 6‒3
Aoba, Aramaki, Aoba-ku, Sendai 980‒8578, Japan

ton's tyrosine kinase, respectively. In recent years, the useof multikinase inhibitors that inhibit multiple tyrosine

Tel: ＋81‒22‒717‒7541, Fax: ＋81‒22‒717‒7545
E-mail: masafumi.kikuchi.b2@tohoku.ac.jp

kinases such as pazopanib, sorafenib, sunitinib, lenvatinib,

Epub April 26, 2022.

vandetanib, regorafenib, and nintedanib, has also increased.
Dose reduction and the temporal or persistent discontinua-

DOI: 10.24508/mms.2022.06.006

tion of drugs are required due to serious side eﬀects caused

Received: December 30, 2021. Accepted: March 24, 2022.

52

June 2022

Medical Mass Spectrometry Vol. 6 No. 1

by the administration of kinase inhibitors. Furthermore,
there are large inter-individual variations in the concentra-

can be used for the simultaneous analysis of the concentrations of drugs and their metabolites with diﬀerent calibra-

tions of the oral kinase inhibitors in blood, and these con-tion curve ranges

centrations are correlated with therapeutic eﬀects and sidetion curves was investigated by adjusting the following

eﬀects. Therapeutic drug monitoring (TDM) of oral kinase

parameters: collision energy defect (CED), which shifts the19）collision energy (CE) from the optimized value ; second-

inhibitors has been reported to be useful for improving their
3,4）
therapeutic eﬀects . Therefore, a consideration of the con-14‒18). The linear range shift of the calibra-

ary product ion selected reaction monitoring (s-SRM),
20）
which selects low-intensity product ions ; and isotopo-

centration of the oral kinase inhibitors is important for
5‒10）. In addiensuring the eﬃcacy and safety of these drugs

logue SRM (i-SRM), which sets precursor and product ionstion, cancer patients often have multiple therapeutic targets

to those containing isotope atoms (Fig. 1).

and may be combined with oral kinase inhibitors. Due tothe importance of administration design based on blood lev-In this study, we investigated whether the linear range
shifts by adjusting CED, in-source CID, s-SRM, and i-SRM

els of many oral kinase inhibitors, a rapid simultaneousfor the measurement of the plasma concentration of 20 oral
kinase inhibitors and active metabolite of sunitinib: afati-21）

measurement system is required in clinical practice.
Many methods have been reported for the quantiﬁcationof drug concentrations in human plasma using liquid chro-nib, axitinib, bosutinib, dacomitinib, dasatinib, erlotinib,

matography-electrospray ionization tandem mass spectrom11‒13). Altering the measurement conetry (LC-ESI-MS/MS)

nintedanib, osimertinib, pazopanib, ponatinib, regorafenib,
sorafenib, sunitinib, N-desethyl sunitinib, and vandetanib

ditions for analyzing a patient sample requires considerable(Fig. 2).

time and eﬀort, and the eﬃciency of the routine of TDM issigniﬁcantly reduced. Therefore, a method for the simulta-

Materials and Methods

neous evaluation of the oral kinase inhibitors is required.

Chemical and reagents

geﬁtinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib,

However, both the therapeutic range and linearity of theAfatinib, bosutinib, dacomitinib, geﬁtinib, ibrutinib, imati-

calibration curve must be considered when designing asimultaneous LC-ESI-MS/MS method. Therapeutic ranges

nib, lapatinib, lenvatinib, nilotinib, nintedanib, osimertinib,
2
ponatinib, regorafenib, vandetanib, afatinib- H6 (internal

standard (IS)), bosutinib- H8, dasatinib- H8, geﬁtinib- H6,
2
2
2
13 2
ibrutinib- H5, lapatinib- H4, nilotinib- H6, nintedanib- C- H3,

for every drug vary with diﬀerences in their pharmacokinetic and pharmacodynamic parameters. In fact, the thera-

2

2

2

and pazopanib- H6 were purchased from Cayman Chemicalpeutic concentration range of ibrutinib (0.3‒300 ng/mL)
and pazopanib (30‒50,000 ng/mL) varies more than

2

Company (Ann Arbor, MI). Axitinib, erlotinib, sorafenib,
2
2
sunitinib, N-desethyl sunitinib, axitinib- H3, erlotinib- H6,

100,000 times.
In addition, the linearity range of the calibration curvefor each drug was speciﬁed using its physicochemical prop-2
2
2
Imatinib- H8, sorafenib- H3, and sunitinib- H4 were purchased from Toronto Research Chemicals (Toronto, Can-erties based on the chemical structure. Previously, weada). Dasatinib was purchased from LC Laboratories
2
2(Woburn, MA). Dacomitinib- H10, lenvatinib- H5, osimerti-

demonstrated the utility of the linear range shift techniqueusing in-source collision-induced dissociation (CID), which

Fig. 1.

13 2
2
13 2
nib- C- H3, ponatinib- H8, regorafenib- C- H3, and vandetan-

Ion abundance adjustment method in LC/ESI-MS/MS.
53

June 2022

Fig. 2.

Medical Mass Spectrometry Vol. 6 No. 1

Chemical structures of afatinib, axitinib, bosutinib, dacomitinib, dasatinib, erlotinib, geﬁtinib, ibrutinib, imatinib,
lapatinib, lenvatinib, nilotinib, nintedanib, osimertinib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib,
N-desethyl sunitinib, and vandetanib.

13 2
ib- C- H6 were purchased from Alsachim (Illkirch-Graﬀen-

staden, France). Pazopanib was purchased from Synkinase

solution was prepared by diluting the stock solutions to
100 ng/mL in acetonitrile-methanol (9 : 1, v/v).

(Melbourne, Victoria, Australia). High performance liquid
chromatography (HPLC)-grade ammonium formate, formic

Sample pre-treatmentacid, and methanol were purchased from the Fujiﬁlm Wako
Pure Chemical Corporation (Osaka, Japan). HPLC-grade

Eighty microliters each of IS and acetonitrile-methanol(9 : 1, v/v) solution were added to 40 μL of the plasma sam-acetonitrile was purchased from the Kanto Chemical Cor-ple contained in a 1.5 mL microcentrifuge tube. The mix-

poration (Tokyo, Japan). Ultrapure water was preparedusing a Puric- α apparatus and was used for the LC-ESI-MS/

ture was vortexed for 5 min and centrifuged at 14,000 × g

MS analysis. All other chemicals used were of the highest

injected into the analytical system.

for 10 min. After centrifugation, 2 μL of supernatant wascommercially available purity. Heparinized human plasma

LC-ESI-MS/MS conditions

was supplied by Cosmo Bio Co. Ltd. (Tokyo, Japan).

Preparation of calibration standards and IS solutions

The LC-ESI-MS/MS analysis was performed using an
LCMS-8050 triple quadrupole mass spectrometer coupled

Each calibration standard was prepared by diluting thewith a Nexera X2 UHPLC system (Shimadzu, Kyoto,stock solutions to 0.1, 0.3, 1.0, 3.0, 10, 30, 100, 300, 1,000,

Japan). All data acquisition and processing was performed

3,000, and 10,000 ng/mL in plasma. Calibration standards
were stored at −80°
C in light-protected containers. Each IS

using LabSolutions (Shimadzu). The Nexera X2 UHPLC
system consisted of a vacuum degasser, two solvent deliv54

June 2022

Medical Mass Spectrometry Vol. 6 No. 1

ery systems, an autosampler, and a column oven. Chromatographic separation was achieved using a YMC-Triart

CED

C18 metal-free column (2.1 mm i.d. × 50 mm, 3 μm; YMC,

The collision energy was changed from −5 to −50 V
under the optimized SRM conditions, and the peak intensi-

Kyoto, Japan). The column temperature was maintained atties of each 10 pg analyte were plotted against voltages.

40°
C, and the samples were kept at 4°
C. The ﬂow rate was

Triplicate analyses were performed for every voltage for all

set at 0.45 mL/min, and the injection volume was 2 μL for

analytes.

analysis. Mobile phases A and B comprised 10 mM aqueous
ammonium formate (A) adjusted to pH 3.6 and 10 mM ofammonium formate-methanol (B). The gradient program

In-source CID

was as follows: elution was initiated with 5% B, followed

to 180 V under the optimized SRM conditions, and the peakintensities of each 10 pg analyte were plotted against volt-

The Q-array bias in SRM analyses was changed from 0

by a linear gradient to 100% B in 1 to 3 min, held at 100%

ages. Triplicate analyses were performed for each voltage
for all analytes. Although the Q-array bias could be set to

B for 1 min, and then immediately returned to the initialconditions, which were maintained for 1 min until the endof the run. The LCMS-8050 was equipped with an ESI

240 V, the upper limit was set to 180 V because the repro-

source operating in the positive-ion detection mode. The

ducibility deteriorated at high voltages.

conditions for mass spectrometry (MS) analysis were as
follows: probe voltage, 4,000 V; desolvation line tempera-s-SRM
Three product ions with higher peak intensities obtainedby a product-ion scan during the optimization of ESI-MS/

ture, 250°
C; block heater temperature, 400°
C; interface
temperature, 300°
C; nebulizing gas ﬂow, 3 L/min; drying
gas, 10 L/min; and heating gas ﬂow, 10 L/min. Table 1 sum-

MS conditions were selected, and the optimum voltage
value was set for each analyte (data not shown). The prod-

marizes the optimized SRM conditions for each analyte.

uct ions in SRM analyses were changed under optimized
SRM conditions, and the peak intensities of each 10 pg anaTable 1.
AnalyteOptimized SRM conditions

SRM Transitions

CE [V]

Q1 pre bias [V]Q3 pre bias [V]Afatinib

486.0＞371.2

−29

−10

−18

Axitinib

387.1＞356.1

−20

−23

−27

Bosutinib

530.1＞141.3

−24

−10

−28

Dacomitinib

470.0＞385.1

−27

−8

−14

Dasatinib

488.0＞401.2

−29

−6

−30

Erlotinib

394.1＞278.2

−35

−14

−30

Geﬁtinib447.2＞128.2

−25

−5

−22

Ibrutinib

442.1＞138.3

−27

−22

−28

Imatinib494.1＞394.3

−28

−12

−28

Lapatinib

581.0＞365.2

−39

−20

−26

Lenvatinib

427.1＞370.1

−31

−16

−18

Nilotinib

530.1＞289.3

−32

−5

−20

Nintedanib

540.1＞113.3

−26

−16

−46

Osimertinib

500.2＞72.2

−28

−10

−12

Pazopanib

438.2＞341.4

−48

−28

−16

Ponatinib

533.2＞260.1

−31

−8

−46

Regorafenib

483.1＞270.2

−33

−14

−28

Sorafenib

465.1＞252.2

−30

−5

−12

Sunitinib
N-Desethyl Sunitinib

399.4＞283.3

−25

−26

−30

370.9＞283.1

−22

−46

−50

Vandetanib

475.2＞112.2

−22

−14

−12

SRM, selected reaction monitoring; CE, collision energy.

55Internal standard
Afatinib- H6
2
Axitinib- H3
2

2
Bosutinib- H82
Dacomitinib- H10
2
Dasatinib- H82
Erlotinib- H62
Geﬁtinib- H62
Ibrutinib- H5

2
Imatinib- H82
Lapatinib- H4

2
Lenvatinib- H5
2
Nilotinib- H6

13 2
Nintedanib- C- H3
13 2
Osimertinib- C- H3
2
Pazopanib- H6
2
Ponatinib- H8,

13 2
Regorafenib- C- H3
2
Sorafenib- H3
2
Sunitinib- H4
2
Sunitinib- H4

13 2
Vandetanib- C- H6

June 2022

Medical Mass Spectrometry Vol. 6 No. 1

lyte were plotted against m/z. Triplicate analyses were per-ion intensities even at high energies. As a result, the linear-formed for each product ion for all analytes.

ity of the calibration curve shifted to a higher concentration
range only when excessive energy was applied. In addition,

i-SRM

the decrease in ion intensities of vandetanib was also small,

Isotopologue transitions were selected from the optimized SRM transitions, and the SRM conditions were opti-

the linearity of the calibration curve did not shift to a higher

mized for each transition (data not shown). The isotopo-

Considering these results, CED can be widely used

logue transitions in SRM analyses were changed underregardless of the resolution of MS and the structure of theanalytes. However, the eﬀect of CID owing to CE was con-concentration range (Fig. 3 and Table 2).

optimized SRM conditions, and the peak intensities of each10 pg analyte were plotted against m/z. Triplicate analyses

sidered to be less eﬀective when the m/z of product ions
was small, as seen for osimertinib (m/z 72.2) and vande-

were performed for each SRM transition for all analytes.

tanib (m/z 112.2). Because the product ions of these ana-

Linear range shift of calibration curves

lytes have already been observed as small fragments, it is

According to the previous study of Ishii and Maekawa,

expected that further fragmentation will be diﬃcult even if

14,17）

we deﬁne linear range shift of calibration curves. The

the CE is increased.

averages of the triplicates at each calibration standard wereused to plot the calibration curves, which were generated
2
using the analyte-to-IS peak area ratios by weighted (1/x )Reduction of the peak intensity and linear range shiftof calibration curves by in-source CID

least squares linear regression. Additional rules for the

Next, the ion abundance of the analytes was investigatedby changing the in-source CID conditions. We changed the

preparation of the calibration curves were as follows: (1)Q-array bias, which is the ion-guide voltage set immediately after the oriﬁce, and attempted ion reduction to quad-calibration points with saturated MS intensities were
excluded; (2) the lower limits of quantiﬁcation were set atthe levels with a relative standard deviation of ＜15% wererupoles by in-source CID. Although afatinib was stable up
to a Q-array bias of 120 V, the ion intensity gradually

included as calibration points; (4) the levels within
85‒115% of the relative error were used as calibration

decreased after 120 V. Consequently, the linearity of the calibration curve shifted to the widest and highest concentra-

points; and (5) calibration curves were generated using

tion range at Q-array biases of 130 and 140 V (0.3‒300 to
3.0‒10,000 ng/mL). Bosutinib, dacomitinib, erlotinib, geﬁ-

levels less than 20% of the relative standard deviation; (3)more than four calibration points.

tinib, ibrutinib, imatinib, lenvatinib, nintedanib, osimerti-

Results and Discussionnib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib,
N-desethyl sunitinib, and vandetanib showed similar trends

Reduction of the peak intensities and linear range
shift of calibration curves by CEDFirst, we changed the CE parameters aﬀecting the CID of

as afatinib. In contrast, dasatinib, lapatinib, and nilotinib
were stable against an increase in Q-array bias, and no sig-

the collision cell and investigated the ion abundance of

niﬁcant shift in the linearity of the calibration curve waseach analyte. Afatinib showed the maximum intensity at aobserved (Fig. 4 and Table 2).
Considering these results, in-source CID can be used for

CE of −29 V. The peak intensities of afatinib gradually
decreased as they increased or decreased from the opti-

most analytes without contaminating MS with excess ions.

mized CE value. As a result, the linearity of the calibration

In addition, the calibration standards for axitinib, imatinib,
sorafenib, sunitinib, and N-desethyl sunitinib showed simi-

curve shifted to the highest concentration range at a CE of−5 V (100‒10,000 ng/mL). Axitinib, bosutinib, dacomi-lar eﬀects as the in-source CID either with plasma or aceto-tinib, dasatinib, erlotinib, geﬁtinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, nintedanib, pazopanib, ponati-

nitrile in their preparation . In contrast, the eﬀect of
in-source CID was considered to be less eﬀective for ana-nib, regorafenib, sorafenib, sunitinib, and N-desethyl
sunitinib showed similar trends to those observed for afati-lytes stable to a Q-array bias of 180 V, such as dasatinib,
lapatinib, and nilotinib. Although setting the Q-array bias tonib. In contrast, osimertinib, which showed the maximum

190 V or higher may be eﬀective, we considered that the
number of ions could not be adjusted by in-source CID for

17）

intensity at a CE of −28 V, had relatively little change in56

June 2022

Fig. 3.

Medical Mass Spectrometry Vol. 6 No. 1

Peak intensities plot and linearity range of calibration curves for each collision energy value: afatinib, axitinib, bosutinib, dacomitinib, dasatinib, erlotinib, geﬁtinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, nintedanib, osimertinib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, N-desethyl sunitinib, and vandetanib.

The columns represent the linear range of the calibration curves (left y-axis). The white dots represent the change in peak intensitiesof each 10 pg analyte (right y-axis).

57

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Table 2.

Medical Mass Spectrometry Vol. 6 No. 1

Results of ion adjustment methods for each compound

Compound

CED

concentration range in the transition at m/z 387.1＞221.2(0.1‒30 to 1.0‒3,000 ng/mL). Bosutinib, dasatinib, erlo-

IS-CID

s-SRM

i-SRM

tinib, ibrutinib, imatinib, lapatinib, nilotinib, osimertinib,

Afatinib

〇

○

×

○

pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, andAxitinib

○

○

○

○

Bosutinib

○

○

○

○

vandetanib showed similar trends to those exhibited byDacomitinib

○

○

×

○

Dasatinib

○

×

○

○

Erlotinib

○

○

○

○

Geﬁtinib

○

○

×

○

Ibrutinib

○

○

○

○

Imatinib○○○

Lapatinib

○

×

○

×
×

Lenvatinib

○

○

○

〇

nintedanib for the secondary product ions (m/z 470.0＞
319.1 and m/z 540.1＞70.2) were comparable to the opti-

Nilotinib

○

×

○

○

Nintedanib

○

○

○

mized transitions, and the linearity of their calibration

×Osimertinib

×

○

○

○

Pazopanib

○

○

○

○

Ponatinib

○

○

○

○

Regorafenib

○

○

○

○

Sorafenib

○

○

○

○

Sunitinib
N-Desethyl Sunitinib

○

○

○

○

○

○

×

○

considered to be less eﬀective when the intensities of product ions, such as afatinib, geﬁtinib, N-desethyl sunitinib,

Vandetanib

×

○

○

○

dacomitinib, and nintedanib, were used.

axtinib. In contrast, the ion intensities of afatinib, geﬁtinib,
and N-desethyl sunitinib for the secondary product ions (m/z
486.0＞100.1, m/z 447.2＞231.2, and m/z 370.9＞210.5)
were very small, and the linearity of their calibration curves
shifted to relatively high concentrations in a very narrow
range. In addition, the ion intensities of dacomitinib andcurves did not shift to the higher concentration range (Fig.
5 and Table 2).
Considering these results, s-SRM can be used for various
analytes because it is easy to predict the change in peakintensities from the product scan. In contrast, s-SRM was〇 , adjustable; ×, not adjustable.

Reduction of the peak intensity and linear range shiftof calibration curves by i-SRM

these three analytes because it is diﬃcult to precisely control the Q-array bias under high voltage. In particular, nilotinib has diﬀerent fragment ions produced by a high Q-ar-

Finally, we investigated the ion abundance of the analytes under the i-SRM conditions. We changed the SRM

ray bias and low-energy CID (data not shown). Therefore, ittransition with the highest ion intensity to three isotopo-

was suggested that the reaction under high pressure withnitrogen gas led to a high threshold of the Q-array bias inm/z M1＋2＞M3＋2) and investigated the ion abundance of

logue transitions (m/z M1＋1＞M3, m/z M1＋1＞M3＋1, andthe in-source CID.

each analyte. Three isotopologue transitions (m/z 487.0＞
371.2, m/z 487.0＞372.2, and m/z 488.0＞373.2) were

Reduction of the peak intensity and linear range shiftof calibration curves by s-SRM

obtained for afatinib, and the ion intensities decreased at all
isotopologue transitions compared with the optimized transition m/z 386.0＞371.2. As a result, the linearity of the calibration curve was obtained in the widest and highest con-We then investigated the ion abundance of the analytes
under the s-SRM conditions. We changed the product ion
with the highest ion intensity to one with a relatively lower
intensity and investigated the ion abundance of each ana-

centration range in the transition at m/z 487.0＞372.2(0.3‒30 to 1.0‒3,000 ng/mL). Axitinib, bosutinib, dasatinib,
erlotinib, geﬁtinib, ibrutinib, lenvatinib, nilotinib, nin-lyte. Contaminant ions were excluded during analysis, andvandetanib did not contain the corresponding product ions.
Three product ions (m/z 387.1＞356.1, m/z 387.1＞221.2,

tedanib, osimertinib, pazopanib, ponatinib, regorafenib,
sorafenib, sunitinib, N-desethyl sunitinib, and vandetanib

and m/z 387.1＞355.2) were obtained for axtinib, and theshowed similar trends as afatinib. In contrast, although theion intensity gradually decreased in transitions at m/z 387.1

ion intensities of lapatinib at the isotopologue transitions＞221.2 and m/z 387.1＞355.2, compared to the optimized(m/z 582.0＞365.2, m/z 582.0＞366.2, and m/z 583.0＞

transition m/z 387.1＞356.1. As a result, the linearity of the367.2) and imatinib at the isotopologue transitions (m/zcalibration curve was obtained in the widest and highest

494.1＞394.3, m/z 495.1＞395.3, and m/z 496.1＞396.3)
58

June 2022

Fig. 4.

Medical Mass Spectrometry Vol. 6 No. 1

Peak intensities plot and linearity range of calibration curves for each Q-array bias: afatinib, axitinib, bosutinib, dacomitinib, dasatinib, erlotinib, geﬁtinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, nintedanib, osimertinib,
pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, N-desethyl sunitinib, and vandetanib.

The columns represent the linear range of the calibration curve (left y-axis). The white dots represent the change in peak intensitiesof each 10 pg analyte (right y-axis). When the peak intensity was not obtained, the white dot was not described.

59June 2022

Fig. 5.

Medical Mass Spectrometry Vol. 6 No. 1

Peak intensities plot and linearity range of calibration curves for each product ion: afatinib, axitinib, bosutinib, dacomitinib, dasatinib, erlotinib, geﬁtinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, nintedanib, osimertinib,
pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, and N-desethyl sunitinib.

The columns represent the linear range of the calibration curve (left y-axis). The white dots represent the change in peak intensitiesof each 10 pg analyte (right y-axis). When the peak intensity was not obtained, the white dot was not described.

60

June 2022

Fig. 6.

Medical Mass Spectrometry Vol. 6 No. 1

Peak intensities plot and linearity range of calibration curves for optimized transitions and the isotopologue transition:
afatinib, axitinib, bosutinib, dacomitinib, dasatinib, erlotinib, geﬁtinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib,
nintedanib, osimertinib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, N-desethyl sunitinib, and vandetanib.
The columns represent the linear range of the calibration curve (left y-axis). The white dots represent the change in peak intensitiesof each 10 pg analyte (right y-axis). When the peak intensity was not obtained, the white dot was not described.

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Medical Mass Spectrometry Vol. 6 No. 1

decreased compared to the optimized transitions m/z 581.0

EGFR/ErbB family of protein-tyrosine kinases in human＞365.2, and 493.1＞394.3, and no signiﬁcant shift wascancers. Pharmacol Res 139: 395‒411, 2019.
3) Mueller-Schoell A, Groenland SL, Scherf-Clavel O, vanobserved in the linearity of the calibration curves (Fig. 6
and Table 2).

Dyk M, Huisinga W, et al: Therapeutic drug monitoring

Considering these results, i-SRM can be used for mostof oral targeted antineoplastic drugs. Eur J Clin Pharmacol 77: 441‒464, 2021.

analytes because it is easy to predict changes in the peakintensities from the isotope abundance ratio. On the con-4) Verheijen RB, Yu H, Schellens JHM, Beijnen JH, Steeghs

trary, i-SRM was considered to be less eﬀective in atomic
compositions such as lapatinib (C29H26ClFN4O4S) and ima-

N, et al: Practical recommendations for therapeutic drug
monitoring of kinase inhibitors in oncology. Clin Pharmacol Ther 102: 765‒776, 2017.

tinib (C29H31N7O). Lapatinib, in particular, was not aﬀectedby i-SRM because it contains Cl atoms in the structure of5) Takasaki S, Kikuchi M, Kawasaki Y, Ito A, Arai Y, et al:product ions and has a high natural abundance ratio, evenA case of renal cell carcinoma with high everolimus

in m/z M1＋2＞M3＋2 (m/z 583.0＞367.2). Furthermore, theblood concentrations and hyperglycemia due to everolim-

eﬀect of stable isotopes ( H) is shown by overlapping peaks

us-induced hepatic dysfunction. Gan To Kagaku Ryoho(similar m/z ions) from the same/similar retention times. It
is necessary to investigate the possibility of aﬀecting quan-

44: 87‒89, 2017.

26) Takasaki S, Kikuchi M, Kawasaki Y, Ito A, Arai Y, et al:
Severe toxicity induced by accumulation of active suni-

tiﬁcation depending on the compound.

tinib metabolite in a Japanese patient with renal cell carci-

Conclusion

noma: A case report. J Med Case Rep 11: 28, 2017.

In this study, we investigated the linear range shifts ofthe calibration curve by adjusting the CED, in-source CID,

7) Takasaki S, Kawasaki Y, Kikuchi M, Tanaka M, Suzuka

s-SRM, and i-SRM for 20 oral kinase inhibitors and thetration and clinical outcomes in Japanese patients withactive metabolite of sunitinib. It was found that althoughmetastatic renal cell carcinoma. Int J Clin Oncol 23: 936‒

these four techniques can easily adjust the number of ions

943, 2018.

M, et al: Relationships between sunitinib plasma concen-

in MS for many analytes, it is diﬃcult to adapt 21 analytes

8) Yamaguchi H, Takasaki S, Kikuchi M, Kawasaki Y, Araiusing only one technique. Therefore, to develop a method

Y, et al: Toward personalized cancer therapy with oral

for the simultaneous quantiﬁcation of oral kinase inhibitors
and the active metabolite in molecular-targeted therapy, it ismolecular-targeted agents. Yakugaku Zasshi 139: 911‒

important to utilize multiple techniques considering the9) Takasaki S, Adachi H, Kawasaki Y, Kikuchi M, Ito A, et915, 2019.

therapeutic concentration range of each drug.

al: Importance of therapeutic drug monitoring to detect
drug interaction between pazopanib and warfarin: A caseAcknowledgments

report. J Pharm Pharm Sci 23: 200‒205, 2020.

This work was supported by JSPS KAKENHI (grant10) Takasaki S, Kawasaki Y, Kikuchi M, Ito A, Yamaguchi H,

number JP21K06708). We would like to thank Editage

et al: Clinical importance of blood drug concentration of(www.editage.com) for the English language editing.

oral molecular targeted drugs for renal cell carcinoma. J
Pharm Pharm Sci 24: 127‒136, 2021.

Conﬂict of Interest11) Takasaki S, Tanaka M, Kikuchi M, Maekawa M, Kawasa-

The authors declare no conﬂicts of interest associatedki Y, et al: Simultaneous analysis of oral anticancer drugs

with this manuscript.

for renal cell carcinoma in human plasma using liquid
chromatography/electrospray ionization tandem mass

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