Reviews in Analytical Chemistry 2022; 41: 146–157

Research Article

Erten Akbel*, Serdar Güngör, and İbrahim Bulduk

Alternative analytical methods for ibrutinib

quantification in pharmaceutical formulation:
A statistical comparison
https://doi.org/10.1515/revac-2022-0039 methods can be safely used in quality control tests for the
received February 28, 2022; accepted June 06, 2022 determination of the amount of ibrutinib in pharmaceutical
Abstract: Ibrutinib is a drug used for the treatment of products.
marginal zone lymphoma, mantle cell lymphoma, lympho- Keywords: ibrutinib, method, development, validation
cytic leukemia, chronic graft, and Waldenstrom macroglobu-
linemia. A simple, sensitive, and fast liquid chromatographic
and spectrophotometric method for the quantification of
ibrutinib in pharmaceutical forms and bulk was developed 1 Introduction
and validated. The chromatographic technique was devel-
oped using an ODS 3 C 18 (250 mm × 4.6 mm i.d., 5 µm) Ibrutinib belongs to a family of drugs known as kinase
column. The mobile phase was a mixture of 0.1% trifluoro- inhibitors. It is used in the treatment of marginal zone
acetic acid in water and acetonitrile (50/50, v/v) at a flow rate lymphoma, mantle cell lymphoma, Waldenstrom macro-
of 1.0 mL·min−1. Eluent detection was carried out at a wave- globulinemia, small lymphocytic lymphoma, chronic lym-
length of 260 nm using a ultraviolet detector. The retention phocytic leukemia, and chronic graft-versus-host disease.
time of ibrutinib was found to be 5.27. On the other hand, It works by stopping the function of the abnormal protein
Ibrutinib was determined using a spectrophotometric tech- that sends a proliferation signal to cancer cells. This slows
nique by measuring the absorbance of the solutions at a the spread of cancer cells. It was first approved by the FDA
wavelength of 260 nm. The developed techniques were for the treatment of mantle cell lymphoma (2013) and
validated in accordance with the protocols outlined in chronic lymphocytic leukemia (2014). It has also shown
International conference on harmonisation of technical efficacy in patients with Waldenström macroglobulinemia.
requirements for registration of pharmaceuticals for human It has a high rate of plasma protein binding, which has a
(ICH) guidelines Q2(R1). Correlation coefficients for both big impact on drug distribution and pharmacokinetics.
methods were greater than 0.999 in the concentration range This suggests that Ibrutinib has a reasonably high affinity
of 5–30 mg·mL−1. The relative standard deviation values for plasma proteins [1–5]. The chemical properties of
were low in intraday and interday precision tests. The accu- galantamine are given in Table 1 [6].
racy of the developed techniques ranged 99.74–100.23% for There are several articles on the detection of ibrutinib
the chromatographic method and 99.32–100.76% for the
in pharmaceutical formulation and biological fluids; the
spectrophotometric method. The limits of detection and
methodologies used herein include liquid chromatography-
quantitation were 0.90 and 2.80 µg·mL−1 for the chromato-
tandem mass spectrometry (LC-MS-MS) [7], liquid chroma-
graphic method and 1.10 and 3.20 µg·mL−1 for the spectropho-
tographic methods [8–13], and ultra-high-performance
tometric method. The developed and validated analytical
liquid chromatography coupled to photodiode array detection
method [14]. The ibrutinib monograph is not yet available in
pharmacopeias and has not been reported in the spectropho-

* Corresponding author: Erten Akbel, Department of Physiotherapy tometric method for the quantification of ibrutinib.
and Rehabilitation, Faculty of Health Sciences, Uşak University, However, these methods had limitations such as
Uşak, Turkey, e-mail: [email protected] sample preparation, gradient elution, and run time. These
Serdar Güngör: Department of Medical Microbiology, Faculty of
techniques also need the use of expensive equipment, spe-
Medicine, Uşak University, Uşak, Turkey
İbrahim Bulduk: Department of Occupational Health and Safety, cialized reagents, and a large amount of organic solvents.
Faculty of Health Sciences, Uşak University, Uşak, Turkey Some of these techniques are quite complex.

Open Access. © 2022 Erten Akbel et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0
International License.
 Alternative analytical methods for ibrutinib quantification  147

Table 1: Chemical properties of ibrutinib

Property Value

Chemical formula C25H24N6O2

Molecular weight 440.50
Melting point 149–158°C
Log P 3.63
pKa Strongest acidic 19.70
Strongest basic 6.58
Solubility It is practically insoluble in water, soluble in methanol, freely soluble in
dimethyl sulfoxide
Chemical structure

This study aims to develop simple, fast, inexpensive, 2.2 Analytical instruments
well-validated alternative chromatographic and spectro-
photometric methods for the quantification of ibrutinib in Chromatographic analyses were performed using an Agilent
pharmaceutical formulations. Also, these methods are less 1260 system consisting of a quaternary pump, autosampler,
time-consuming and cheaper compared to other published UV detector, and ChemStation software. Spectrophotometric
methods. The results obtained from these analytical techniques analyses were carried out using a Shimadzu UV 1800 spectro-
were compared statistically using the least-squares method. photometer with UV-Probe software and 1 cm quartz cuvette.
Furthermore, the applicability and reliability and of these
methods have been assessed by concentrating on routine
quality control analyses. Therefore, the current study can be
used for the quantification of drugs in bulk and phar-
2.3 Chromatographic separation
maceutical dosage forms, and successfully applied for
Chromatographic separation was carried out using an
routine quality control analysis. In addition, analytical
ODS 3 C 18 (250 mm × 4.6 mm i.d., 5 µm) column. Before
methods developed for ibrutinib quantification were
analysis, all solvents and solutions were sonicated and
assessed statistically.
filtered through 0.45 µm membrane filters. A mixture of
0.1% trifluoroacetic acid (TFA) in water and acetonitrile
(50/50, v/v) was used as the diluents at a flow rate of
1.0 mL·min−1. Eluent detection was carried out at a wave-
2 Materials and methods length of 260 nm using a ultraviolet (UV) detector.

2.1 Chemical and reagents

Ibrutinib reference standard (≥98.0%), trifluoroacetic acid 2.4 Standard solutions

(≥99.0%), acetonitrile (≥99.9%), and methanol (≥99.9%)
were purchased from Sigma-Aldrich Chemie GmbH. Imbruvica® To prepare a stock solution of ibrutinib (500 µg·mL−1),
extended-release capsule (140 mg) was purchased from 25 mg of reference standard was weighed precisely and
the local pharmacy. Ultrapure water was produced using transferred to a 50 mL volumetric flask, and a 15 mL
a water purification system (Merck Millipore, USA). diluent was added. The volumetric flask contents were
148  Erten Akbel et al.

sonicated for 5 min, and the volume was filled up to were obtained. The mobile phase was then acidified with
the mark with diluent. The standard solution series tetrahydrofuran, and different flow rates were used to deter-
(5–30 µg·mL−1, n = 6) was prepared by diluting the stock mine the assay method. Columns with many different prop-
solution with diluent. erties were tested, and good peak shapes (sharp peaks)
and good resolution were obtained with the ODS 3 C 18
(250 mm × 4.6 mm i.d., 5 µm) column. A long column was
used both to see impurities that could interfere with an
2.5 Sample solution ibrutinib peak in sample solutions and to ensure that there
were no matrix components left in the column for much
The contents of ten capsules were weighed precisely and longer under the specified conditions, and the sample solu-
ground into powder in a mortar. The capsule powder tion was analyzed for 60 min. However, continuing the ana-
containing 50 mg of ibrutinib was weighed precisely into lysis after 10 min will increase both the analysis time and
a 100 mL beaker, 75 mL of diluent was added to it and the cost of the analysis. As a result of the sample analysis,
dissolved. The contents of the beaker were transferred which was injected into the system consecutively with a
to a 100 mL volumetric flask and sonicated for 10 min 10-min analysis time, no interference peaks were found.
in a sonicator, then the diluent was added to make up Due to all this, the analysis time was determined to be
the volume to 100 mL. By diluting this solution with 10 min. The chromatographic analysis was carried out at
diluent, a sample solution was prepared at a concen- 25°C, which has several advantages, including superior chro-
tration of 20 µg·mL−1 and filtered through a 0.45 µm matographic peak shape, increased column efficiency, and
filter. reduced column pressure, in addition to being cost-effective.
The reasonable retention time and best separation were
obtained with a mobile phase ratio of 0.1% TFA in water
2.6 Method development and acetonitrile (50:50, v/v), a flow rate of 1.0 mL·min−1, a
column temperature of 25°C, and an injection volume of 20 µL.
Chromatographic conditions were optimized by various The spectral pattern and maximum absorbance values
factors such as the composition of the mobile phase, flow of ibrutinib were extensively analyzed. Different solvents
rate of the mobile phase, pH of the mobile phase, and (ultrapure water, methanol, ethanol, and isopropyl alcohol)
column type. Optimization of chromatographic condi- were used for UV spectrophotometric analysis. The best
tions was carried out to obtain good peak parameters, spectra of ibrutinib were obtained in methanol, and this
such as a good peak shape, a short retention time, and solvent was used in UV spectrophotometric analyses.
queuing factor, as well as the theoretical number of
plates. Different ratios of water:methanol, water:aceto-
nitrile, and methanol:acetonitrile were tested as mobile 2.7 Method validation
phase. Columns of different lengths of C18 and C8 were
tested. However, both defective peak shape and weak Selectivity, linearity, accuracy, precision, robustness, limit
system compliance parameters were obtained. The reten- of detection (LOD), and limit of quantification (LOQ) are
tion time rose as the water content of the mobile phase all validation parameters. The International conference on
increased, resulting in a higher tailing factor and the harmonisation of technical requirements for registration of
creation of asymmetric peaks. To develop and validate pharmaceuticals for human (ICH) guidelines were used as
an efficient method for quantification of ibrutinib in phar- a guide for the validation of analytical methods [15–22].
maceutical formulations, chromatographic conditions were Parameters such as selectivity, linearity, accuracy, preci-
optimized. First, the wavelength at which the ibrutinib stan- sion, robustness, and LOD and LOQ were validated.
dard solution absorbs UV rays to the maximum was deter-
mined. A reference solution at a concentration of 25 µg·mL−1
was scanned in the 200–800 nm region for this purpose. A 2.8 System suitability test
wavelength of 260 nm was chosen to reduce baseline noise
at maximum absorption of ibrutinib; there was no interfer- Six replicates of ibrutinib standard solution (20 µg·mL−1)
ence from solvents, excipients, or contaminants in the phar- were injected to determine system suitability. Peak area,
maceutical formulation at this wavelength. The mobile retention time, theoretical plate number, and tailing factor
phase consisted of various acetonitrile and water composi- were measured. The RSD% values of six injections were
tions, initially kept as 20–80, v/v. Very long analysis times calculated. Chemstation software was used to calculate
 Alternative analytical methods for ibrutinib quantification  149

system suitability parameters (tailing factor <2.0, theoret- on the same day to determine repeatability. The results
ical plate number >2,000, and RSD% <2.0). were reported in the form of an average, standard deviation,
and RSD% values.

2.9 Selectivity
2.12 Accuracy
The selectivity of the analytical methods was evaluated
by comparing the chromatograms and spectra obtained The method’s accuracy was determined by adding three
from the analysis of standard and sample solutions. different quantities of the ibrutinib standard to the sample
To evaluate the selectivity of the chromatographic method, solution and amplifying it. The amounts added were 80%,
a sample and a standard solution were prepared and 100%, and 120% of the target concentration (20 mg·mL−1),
injected into the chromatographic system. The obtained respectively. At each concentration, these samples were
chromatograms were compared, and the presence of inter- made in triplicate, and the % recovery quantities were
fering peak(s) was examined. To evaluate the selectivity of calculated.
the spectrophotometric method, a sample and a standard
solution were prepared, and spectra of both solutions were
taken in the spectrophotometer device in the wavelength
range of 200–800 nm. The obtained spectrums were com- 2.13 Sensitivity
pared, and the presence of interfering bands was examined.
To assess the sensitivity of chromatographic and spectro-
photometric techniques, the detection and quantification
2.10 Linearity limits were utilized. They were calculated separately
depending on the standard deviation of the slope and
Stock standard solutions (500 μg·mL−1) were prepared in intercept of the calibration curve using the equations:
triplicate. Six standard solutions were diluted from stock stan- LOD = 3.3 × σ / S (1)
dard solutions in the concentration range of 5–30 µg·mL−1 for
both methods. These solutions were injected into the chroma- LOQ = 10 × σ / S (2)
tography column in triplicate; 20 µL of the volume of injection where S is the slope of the calibration curve and σ is the
remained constant. Methanol was utilized as a solvent for standard deviation of the y-intercept.
the spectrophotometric analyses. UV spectrum of standard
solutions was recorded between 200 and 800 nm, and
absorbance values were measured on the spectrophot- 2.14 Robustness
ometer. The linearity was examined by analyzing six stan-
dard solutions (n = 3) in the range of 5–30 µg·mL−1 for both To evaluate the robustness of the chromatographic method,
methods. The calibration curve was created for standard minor changes at a flow rate (0.9 and 1.1), acetonitrile content
solutions by plotting the responses against concentrations. of the mobile phase (48:52 and 52:48 v/v), column tempera-
Regression analysis was carried out using the least-squares ture (20°C and 30°C), and detection wavelength (257 and
method with the data obtained from both analytical methods. 263 nm) were made. The effect of these changes on the results
of the analysis was investigated. Minor changes in organic
solvent (ethanol and isopropyl alcohol) and detection wave-
2.11 Precision length (257 and 263 nm) were made to evaluate the robust-
ness of the spectrophotometric method. The effect of these
The precision of analytical methods was assessed both in changes on the results of the analysis was investigated.
terms of method and system precision. Method precision
was evaluated in two different ways, both intra-day pre-
cision and inter-day precision. System precision was 2.15 Analysis of pharmaceutical formulation
evaluated by analyzing six commercial ibrutinib sample
solutions on the same day and under the same chromato- Recommended methods were used for quantification of
graphic conditions. The RSD% values of peak area were ibrutinib in the capsule dosage form. The sample solution
computed. Six test results from three consecutive days was prepared from capsules and processed as a test solu-
were analyzed to determine intermediate decisiveness. tion. The obtained peak area (n = 6) and absorbance
Six test solutions at the same concentration were analyzed value were compared with an ibrutinib standard solution
150  Erten Akbel et al.

at the same concentration level and the amount specified

on the drug package.

2.16 Solution stability

Throughout 24 h, the stability of the standard solution

(20 µg·mL−1) was examined. During the stability research,
standard solutions were kept at room temperature (25°C)
and shielded from light. For this purpose, the standard
solution was analyzed by chromatographic method with
8-h periods, and the peak area was determined and
recorded. The standard solution was analyzed using the
spectrophotometric method with 8-h periods, and the
absorbance values were determined and recorded. RSD%
values were calculated for the absorbance values of
Figure 1: UV spectrum of ibrutinib standard solution (25 µg·mL−1).
ibrutinib standard solutions.

2.17 Comparative analysis a short run time. After various tests, the method was
optimized as a mixture of 0.1% TFA in water and aceto-
After validation, the developed analytical techniques were nitrile (50:50, v/v) at a flow rate of 1.0 mL·min−1 at 260 nm
determined to be suitable for quantifying ibrutinib in phar- for a run time of 5.17 min. An ODS 3 C 18 (250 mm × 4.6 mm
maceuticals. The recovery percentages were statistically i.d., 5 µm) column was used in an isocratic mode These
compared when both analytical techniques were employed chromatographic conditions provided a reasonable retention
on commercial pharmaceuticals. For this, the F-test and the time and good tailing factor for ibrutinib. On the other hand,
t-test were utilized. Ibrutinib was determined by a spectrophotometric technique
by measuring the absorbance of the solutions at a wave-
length of 260 nm. Chromatograms obtained by the HPLC
method are given in Figure 2. Spectra obtained by the
3 Results spectrophotometric method are given in Figure 3.

3.1 Determination of detection wavelength

3.3 Validation of the chromatographic
To determine the λmax value, the standard solution (25 µg·mL−1) method
was scanned on a spectrophotometer device in the spectral
mode in the range of 200–800 nm. Measurements were After the chromatographic method was successfully devel-
obtained against methanol as a blank. As shown in oped, it was validated by ICH guidelines. Selectivity, accu-
Figure 1, it was observed that the ibrutinib standard racy, precision, sensitivity, and robustness were identified
solution had maximum absorption at 260 nm wavelength. as validation parameters [15–22]. Verification was also per-
Therefore, a wavelength of 260 nm was chosen for ibrutinib formed to ensure that the established approach may pro-
quantification. The absorbance values of the standard solu- duce consistent and trustworthy results when employed
tion series at the wavelength of 260 nm were recorded, and by laboratories of varying sizes.
it was shown that the absorbance values were proportional
to the concentration of standard solutions.
3.4 System suitability

3.2 Development of methods Six standard solutions (20 µg·mL−1) were injected into the
chromatographic system to assess system suitability. The
The goal in developing the chromatographic method was peak area, retention time, tailing factor, and theoretical
to achieve good performance of the analytical method in plate number were all taken into account. RSD% was
 Alternative analytical methods for ibrutinib quantification  151

Figure 2: (a) HPLC chromatogram of blank, (b) standard solutions (5–30 µg·mL−1), (c) pharmaceutical product (20 µg·mL−1), and (d) standard
solution (30 µg·mL−1).

calculated for six injections and was less than 2.0%. obtained from the standard and sample solution were
System suitability test results are given in Table 1. compared and no interference bands were detected.

3.6 Linearity
3.5 Selectivity
The linearity of the methods was established using linear
To test the selectivity of the chromatographic method, regression analysis and least-squares method. The con-
chromatograms obtained from the standard and sample centration ranged between 5 and 30 µg·mL−1. Regression
solution were compared in the area of the ibrutinib peak, analysis revealed a strong correlation coefficient (0.999).
and no interfering peaks were detected. To test the The RSD% value for each point (n = 3) was less than 2%;
selectivity of the spectrophotometric method, the spectra the results are shown in Table 2.
152  Erten Akbel et al.

Figure 3: (a) UV spectrum of blank, (b) standard solutions (5–30 µg·mL−1), (c) pharmaceutical product (20 µg·mL−1), and (d) standard
solution (25 µg·mL−1).

3.7 Precision
Table 2: The results of regression analysis
Both precisions had RSD% values less than 2.0%. The
No. Concentration Chromatographic Spectrophotometric precision research results demonstrate that the suggested
(μg·mL−1) method method
methods are precise. Table 3 displays the results of the
(peak area) (absorbance)
method precision and system precision research.
1 5 235.0 0.302
2 10 463.0 0.581
3 15 697.0 0.882
4 20 940.0 1.194
5 25 1185.0 1.491 3.8 Accuracy
6 30 1415.0 1.767
Slope 47.09 0.0592 The mean recovery percentages for the standard addi-
Intercept 0.0000 0.0000 tions of 80%, 100%, and 120% to the sample solution
Correlation 0.9999 0.9999
were 99.40%, 99.83%, and 99.78%, respectively. This
coefficient (r2)
suggests that the described methods are appropriate for
 Alternative analytical methods for ibrutinib quantification  153

Table 3: The results of the precision study

No. Concentration (μg·mL−1) Chromatographic method Spectrophotometric method

Retention time (min) Peak area Assay (%) Absorbance Assay (%)

Method precision 1 20 5.268 941.30 100.08 1.197 100.11

2 5.270 940.10 99.97 1.193 99.78
3 5.271 941.21 100.09 1.196 100.03
4 5.271 940.32 100.00 1.195 99.94
5 5.274 939.86 99.95 1.196 100.03
6 5.267 940.35 100.00 1.197 100.11
Average 5.270 940.52 100.02 1.196 100.00
SD 0.0025 0.5942 0.0587 0.0015 0.1259
RSD (%) 0.0480 0.0632 0.0632 0.1259 0.1259
System precision 1 20 5.273 940.80 100.08 1.194 100.04
2 5.268 939.30 99.92 1.192 99.87
3 5.269 941.21 100.13 1.196 100.21
4 5.274 939.32 99.93 1.190 99.71
5 5.276 939.15 99.91 1.196 100.21
6 5.278 940.35 100.03 1.193 99.96
Average 5.273 940.02 100.00 1.194 100.00
SD 0.0039 0.8830 0.0939 0.0023 0.1965
RSD (%) 0.0754 0.0939 0.0939 0.1965 0.1965

Table 4: The results of the accuracy study

Spiked level Added (μg·mL−1) Recovered (μg·mL−1) Recovery (%) Average (%) SD RSD (%)

80% 16 15.96 99.75 99.40 0.344 0.346

16 15.90 99.38
16 15.85 99.06
100% 20 19.98 99.90 99.83 0.076 0.077
20 19.97 99.85
20 19.95 99.75
120% 24 23.96 99.83 99.78 0.064 0.064
24 23.93 99.71
24 23.95 99.79

quantifying ibrutinib in pharmaceutical formulations.

Table 4 displays accuracy data.

3.9 Detection and quantitation limit

The detection and quantitation limit values were calcu-

lated separately depending on the standard deviation of
the slope and intercept of the calibration curves. The
detection and quantitation limit values for the chromato-
graphic method were calculated as 0.90 and 2.80 µg·mL−1,
respectively. In spectrophotometric analysis, the detection
and quantitation limit values were calculated as 1.40 and
4.30 µg·mL−1, respectively. The chromatogram of the Figure 4: HPLC chromatogram of the sample solution at a concen-
sample solution at a concentration close to the tration close to the quantification limit of the analyte (2.5 mg·mL−1).
154  Erten Akbel et al.

Table 5: The results of the robustness study

Method Method conditions Retention time (min) Tailing Plate count RSD (%)

Chromatographic Control (no change) 5.270 1.204 7,694 0.82

method Flow rate of the mobile phase 1.1 mL·min−1 4.755 1.182 7,350 0.91
0.9 mL·min−1 5.810 1.225 8,309 0.98
Organic content of mobile phase 48:52 5.085 1.198 7,662 0.86
52:48 5.433 1.214 7,724 0.75
Column temperature 22°C 5.268 1.204 7,693 0.83
28°C 5.271 1.204 7,694 0.81
Detection wavelength 257 nm 5.268 1.205 7,691 0.83
263 nm 5.271 1.204 7,698 0.82
Absorbance (20 µg·mL−1, n = 3) RSD (%)
Spectrophotometric Control (no change) 1.194 1.007
method Organic solvent Ethanol 1.193 1.012
Isopropyl alcohol 1.192 1.014
Detection wavelength 257 nm 1.191 1.010
263 nm 1.192 1.013

Table 6: Analysis results of the marketed pharmaceutical formulation

Label claim (mg·capsule−1) Spectrophotometric method, Chromatographic method, found

found ibrutinib (mg·capsule−1) ibrutinib (mg·capsule−1)

140 139.21 ± 0.51 139.74 ± 0.27

Table 7: The results of standard solution stability (n = 3, 20 μg·mL−1)

Time (h) Chromatographic method Spectrophotometric method

Peak area Mean SD RSD (%) Absorbance Mean ± SD SD RSD (%)

0 937.3 938.3 1.323 0.141 1.194 1.193 0.004 0.349

939.8 1.196
937.8 1.188
8 941.7 940.1 1.650 0.176 1.189 1.193 0.005 0.378
940.1 1.198
938.4 1.193
16 938.4 940.4 1.955 0.208 1.192 1.193 0.004 0.302
940.6 1.197
942.3 1.190
24 941.7 941.4 2.563 0.272 1.196 1.194 0.006 0.491
943.8 1.187
938.7 1.198

Table 8: Statistical comparison (n = 6)

Statistical data Chromatographic method Spectrophotometric method

Value on average 99.89 99.67

SD 0.32 0.63
RSD (%) 0.32 0.63
Standard error 0.34 0.75
F-Test 0.27/0.51
Fcalculation/Ftable
t-Test 1.40/2.62
tcalculation/ttable
Table 9: Characteristics of previously reported HPLC methods and the proposed method

Mobile phase Column Concentration range Correlation Precision LOD/LOQ Retention Application Reference
coefficient (R2) (RSD, %) time (min)

Potassium dihydrogen Symmetry C8 × Terra 5–30 mg·mL−1 0.9994 Intraday: 0.64 mg·mL−1 3.40 Bulk and [8]
phosphate buffer and column (150 mm × 0.134 pharmaceutical
acetonitrile (40:60 v/v) pH 3.0 4.6 mm, 5 µm Interday: 1.95 mg·mL−1 formulations
particle size) 0.243
A mixture (40:60) of 0.1% Kromasil C18 column 35–210 mg·mL−1 0.9990 Intraday: 0.394 mg·mL−1 3.06 Bulk and [9]
ortho phosphoric acid and (150 mm × 4.6 mm, 5 µm 1.01 pharmaceutical
acetonitrile particle size) Interday: 1.194 mg·mL−1 dosage forms
1.50
A mixture of acetonitrile-0.1% Agilent ZORBAX SB-C18 5.0–500 ng·mL−1 0.9996 Intraday: 5 ng·mL−1 5.07 Rabbit plasma [10]
trifluoroacetic acid water (4.6 mm × 125 mm, 5 μm) 7.30
(43:27:30) Interday: 14 ng·mL−1
7.60
0.1% Orthophosphoric acid Inertsil ODS (100 mm × 3.5–21.0 mg·mL−1 0.9997 Intraday: 0.03 mg·mL−1 2.52 Pharmaceutical [11]
buffer and acetonitrile in the 4.6 mm, 5 μm) column 1.06 dosage form
ratio 70:30 %v/v Interday: 0.10 mg·mL−1
1.73
A mixture of 0.1% TFA in water ODS C18 (250–4.6 mm, 5–30 mg·mL−1 0.9999 Intraday: 0.90 mg·mL−1 5.17 Bulk and Proposed
and 0.1% TFA in acetonitrile particle size 5 µm) 0.05 pharmaceutical method
(50/50, v/v) Interday: 2.80 mg·mL−1 dosage forms
0.08
Alternative analytical methods for ibrutinib quantification

155
156  Erten Akbel et al.

quantification limit of the analyte (2.5 µg·mL−1) is pre- ibrutinib in pharmaceutical formulations. The statistical
sented in Figure 4. comparison results of described methods are presented in
Table 8.

3.10 Robustness

The robustness of both techniques was verified by each 4 Discussion

variable condition. Table 5 shows that the obtained values
are within the acceptable limit. Spectrophotometric and chromatographic methods have
been developed for the quantification of ibrutinib in phar-
maceutical formulations. Various analytical methods have
3.11 Analysis of the commercial formulation been reported in the literature to determine the amount of
ibrutinib in pharmaceutical products. Some of these tech-
The newly prepared sample solution was filtered using a niques are quite complex, requiring long analysis times,
filter with a pore size of 0.45 µm and then analyzed by expensive apparatus, special reagents, and large amounts
chromatographic and spectrophotometric methods. Ibrutinib of organic solvents. Furthermore, the approaches described
quantification in pharmaceutical formulations has been suc- in the literature involve lengthy and difficult sample pre-
cessfully applied using both developed and validated paration procedures. In all these studies, there is no study
methods. The analysis results of the pharmaceutical for- yet on the comparison of two different analysis techniques
mulation sold commercially in pharmacies are presented and their results. The developed methods are very simple in
in Table 6. terms of both the detection (UV) of the analyte and the
elution of the mobile phase (isocratic). The characteristics
of the previously reported HPLC methods and the proposed
method are presented in Table 9.
3.12 Solution stability

Throughout 24 h, the stability of ibrutinib standard solu- 5 Conclusion

tion (20 µg·mL−1) was examined. For this purpose, the
standard solution was analyzed using the chromato- The spectrophotometric method has advantages over the
graphic method with 8-h periods and the peak area was chromatographic method because the spectrophotometric
determined and recorded. The standard solution was method generally does not require detailed processes
analyzed by the spectrophotometric method, and the and procedures as in the chromatographic method. The
absorbance value was determined and recorded. Table 7 spectrophotometric technique is more economical and
shows the results of the stability study. The RSD% was consumes less time than the chromatographic technique.
determined as 0.272 for peak area using the chromato- However, the statistical evaluation of both methods shows
graphic method and as 0.491 for absorbance using the that the chromatographic method is more precise and
spectrophotometric method. The concentration of ibru- accurate than the spectrophotometric method. The results
tinib in the reference solution did not vary significantly. show that the chromatographic and spectrometric methods
are sufficient methods for the quantification of ibrutinib in
pharmaceutical formulations. No interfering peaks were
observed during the retention time of ibrutinib in the chro-
3.13 Statistical comparison of methods matographic method, and no interfering absorption bands
were observed at 260 nm in the spectrophotometric method.
The F-test and the t-test were used to compare developed These analytical techniques may be used effectively for reg-
and validated procedures statistically. Statistical analyses ular quality control analysis of ibrutinib in pharmaceutical
revealed no statistically significant differences between the preparations because they are quick, simple, precise, spe-
results obtained from the analyzes using both methods. cific, and accurate.
The F-value and t-value were calculated, and they
were found to be lower than the table values of both Acknowledgments: The authors are also grateful to Uşak
techniques at the 95% confidence range. It is clear that University for providing the necessary facilities to per-
the analytical methods described can be used to measure form the research study.
 Alternative analytical methods for ibrutinib quantification  157

Funding information: Authors state no funding is involved. bulk and pharmaceutical dosage form. World J Pharmacy
Pharm Sci. 2016;5(5):868–74.
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Min Z. A simple HPLC method for the determination of Ibrutinib
writing – review and editing; İbrahim Bulduk: chemical
in rabbit plasma and its application to a pharmacokinetic
analysis; Serdar Güngör: writing– original draft, writing – study. Latin Am J Pharmacy. 2016;35(1):130–4.
review and editing. This work was carried out in collabora- [11] Chintala R, Golkonda R, Kapavarapu S. Validation of stability
tion among all authors. All authors read and approved the indicating RP-HPLC method for the assay of ibrutinib in phar-
final manuscript. maceutical dosage form. Ana Chem. 2016;16(1):7–19.
[12] de Vries R, Huang M, Bode N, Jejurkar P, Jong Jd,
Sukbuntherng J, et al. Challenge, Bioanalysis of ibrutinib and its
Conflict of interest: Authors state no conflict of interest. active metabolite in human plasma: selectivity issue,
impact assessment and resolution. Bioanalysis.
Data availability statement: The files used to support the 2015;7:2713–24.
data findings of this study are available from the corre- [13] Veeraraghavan S, Viswanadha S, Thappali S, Govindarajulu B,
Vakkalanka S, Rangasamy M. Simultaneous quantification of
sponding author on request.
lenalidomide, ibrutinib and its active metabolite PCI-45227 in
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