Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 140149

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Measurement of total and free docetaxel concentration in human

plasma by ultra-performance liquid chromatographytandem mass
spectrometry
Ral Rigo-Bonnin a, , Sara Cobo-Sacristn b , Nria Gonzalo-Diego c , Helena Colom d ,
c

Carmen Munoz-Snchez
, Ander Urruticoechea e , Catalina Falo e , Pedro Ala a
a

Laboratori Clnic Department, IDIBELL, Hospital Universitari de Bellvitge, LHospitalet de Llobregat, Barcelona, Spain
Pharmacy Department, IDIBELL, Hospital Universitari de Bellvitge, LHospitalet de Llobregat, Barcelona, Spain
c
Pharmacy Department, IDIBELL, Catalan Institute of Oncology, Hospital Duran i Reynals, LHospitalet de Llobregat, Barcelona, Spain
d
Department of Biopharmaceutics and Pharmacokinetics, School of Pharmacy, University of Barcelona, LHospitalet de Llobregat, Barcelona, Spain
e
Catalan Institute of Oncology, IDIBELL, Hospital Duran i Reynals, LHospitalet de Llobregat, Barcelona, Spain
b

a r t i c l e

i n f o

Article history:
Received 14 April 2015
Received in revised form 18 August 2015
Accepted 19 August 2015
Available online 28 August 2015
Keywords:
Total docetaxel
Free docetaxel
Paclitaxel
Ultraltration
UPLCMS/MS

a b s t r a c t
Docetaxel is a semi-synthetic taxane with cytotoxic anti-neoplastic activity and, currently used as
anticancer agent in several types of cancer. Docetaxel is highly bound to plasma proteins, and this signicantly determines its clearance and activity. Therefore, measurement of free docetaxel in plasma
is pharmacologically important when pharmacokinetics is investigated. We developed and validated
chromatographic methods by ultra-performance liquid chromatography-tandem mass spectrometry to
measure total and free docetaxel concentration in human plasma. The nal validated methods involved
liquidliquid extraction followed by dryness under nitrogen evaporation. To measure free docetaxel
concentration, sample preparation was preceded by ultraltration. Chromatographic separation was
achieved using an Acquity UPLC BEHTM (2.1 100 mm id, 1.7 m) reverse-phase C18 column at a
ow rate of 0.4 mL/min, using isocratic elution mode containing ammonium acetate/formic acid in
water/methanol (30:70 v/v) as mobile phase. Docetaxel and its internal standard (paclitaxel) were
detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring
mode using mass-to-charge (m/z) transitions of 808.3 527.0 (quantier) and 808.3 509.0 (qualier);
and 854.3 569.0 (quantier) and 854,3 509,0 (qualier), respectively. The run time per sample was
3.5 min. The limits of quantication were 1,95 and 0.42 g/L and linearity was observed between 1.95 and
1000 and 0.42100 g/L for total and free docetaxel, respectively. Coefcients of variation and absolute
relative biases were less than 13.8% and 10.0%. Recovery values were greater than 79.4%. Evaluation of
the matrix effect showed ion suppression and no carry-over was observed. The validated methods could
be useful for both therapeutic drug monitoring and pharmacokinetic studies. They could be applied to
daily clinical laboratory practice to measure the concentration of total and free docetaxel in plasma.
2015 Published by Elsevier B.V.

1. Introduction

Abbreviations: DCX, docetaxel; PK, phamacokinetic; PD, pharmacodynamic;

HPLC, high-performance liquid chromatography; UV, ultraviolet; MS/MS, tandem
mass spectrometry; UPLC, ultra-performance liquid chromatography; MTBE, methyl
t-butyl ether; LC, liquid chromatography; MS, mass spectrometer; PCX, paclitaxel;
QC, quality control; LLOQ, lower limit of quantication; ULOQ, upper limit of quantication; ESI, electrospray ionization; MRM, multiple reaction monitoring; m/z,
mass-to-charge; EMA, European Medicines Agency; CLSI, Clinical and Laboratory
Standards Institute; IFCC, International Federation of Clinical Chemistry and Laboratory Medicine; LLOD, lower limit of detection; S/N, signal-to-noise; CV, coefcient
of variation; r , bias; Cmax , maximum concentration; CL, total clearance; AUC, area
http://dx.doi.org/10.1016/j.jpba.2015.08.025
0731-7085/ 2015 Published by Elsevier B.V.

Docetaxel (Fig. 1) is a semi-synthetic analog of paclitaxel

(Taxol ) with cytotoxic anti-neoplastic activity. Docetaxel (DCX)
is a promoter of microtubule polymerization leading to cell cycle

under the plasma concentrationtime curve; %D, percent deviation; TDM, therapeutic drug monitoring.
Corresponding author at: Laboratori Clnic Department, Hospital Universitari
de Bellvitge, Feixa Llarga s/n, 08907 LHospitalet de Llobregat, Barcelona, Spain.
Fax: +34 932607546
E-mail address: raulr@bellvitgehospital.cat (R. Rigo-Bonnin).

R. Rigo-Bonnin et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 140149

Fig.1. Molecular structure of docetaxel and paclitaxel (internal standard).

arrest at G2/M, apoptosis and cytotoxicity [1]. Docetaxel has significant activity in breast, non-small-cell lung, ovarian and head and
neck cancers [1].
The pharmacokinetic (PK) prole of DCX is characterized by
substantial interpatient variability. Causes of these inter-individual
variations may include age, gender, ethnicity, hepatic impairment
and genetic polymorphisms [2,3]. Another factor contributing to
this variation is protein binding. In plasma, DCX is extensively
bound to albumin and 1 -acid glycoprotein [1,4]. In cancer patients,
binding of DCX to 1 -acid glycoprotein might contribute to the
correlation between this glycoprotein and clearance of total DCX
by lowering concentrations of unbound DCX (free DCX) available
for metabolism [4]. Moreover, in population pharmacokinetic and
pharmacodynamic (PD) analysis of DCX using total drug concentrations, high plasma concentrations of 1 -acid glycoprotein have
been signicantly associated with low clearance of total DCX as
well as mild neutropenia and diminished antitumor efcacy [1,4].
Therefore, it might be important to measure concentrations of free
DCX when investigating its PK and PD.
Several high-performance liquid chromatography (HPLC) methods for the measurement of DCX in human plasma using ultraviolet
(UV) detection have been previously described [59], which
present low detection capabilities and are not very selective, owing
to the presence of endogenous interferences as well as the limited UV absorption characteristics of the taxane moiety and the
low wavelengths required to measure the concentration of DCX.
Greater detection capabilities and more selective HPLC methods
have been developed by the utilization of HPLC coupled with tandem mass spectrometry (MS/MS) [1021]. Nevertheless, some of
these methods present shortcomings. With regard to detection
capability, two of them presented lower limits of quantication
5 g/L [1011,14] which could be insufcient to perform DCX PK
studies. Furthermore, the analytical time of each run in these methods was between 4.518 min [1012,14,1621]. The relatively long

141

chromatographic run time could not satisfy the requirement of high

throughput measurement in PK studies and therapeutic drug monitoring in the daily practice of a clinical laboratory. Also, in some
cases they use protein precipitation extraction [14,15], very prone
to dirtiness, which often resulted in a signicant matrix effect due
to the presence of many residual matrix components. Others use
the cleaner but time-consuming and expensive solid phase extraction [11,19,21]. In addition, to our knowledge, only one of them
has been used to measure DCX concentrations in plasma using
ultra-performance liquid chromatography (UPLC)-MS/MS method,
in particular for measuring DCX from a lipid microsphere formulation in human plasma [13]. UPLC uses high ow and pressure
conditions with sub-2 m particles columns providing, in general, better performance than HPLC in reduced times even without
working under high ow and pressure conditions, due to a reduced
dwell and dead volumes (150 L). These characteristics provide
more resolution and shorter retention times [22,23].
Other analytical methods have been developed to measure free
DCX concentration in human plasma. Most of them use either
ultraltration or equilibrium dialysis to separate free drug from
the bound fraction [2428], two use ultraltration combined with
HPLC-MS/MS for direct measurement of free DCX concentrations
[24,25], but only one of them uses UPLC technology [24].
The aim of this work was to develop and validate an
UPLCMS/MS method for the measurement of total and free
DCX concentrations in human plasma within the same run. Furthermore, these validated methods were applied to support a
population pharmacokinetic model allowing dose modication of
docetaxel in pacients with breast cancer participating in a phase IV
clinical trial.
2. Material and methods
2.1. Chemicals and reagents
Certied reference standards with a 93.5% purity of DCX trihydrate and a 99.4% purity of paclitaxel (Fig. 1) were purchased
from European Pharmacopeia (European Directorate for the Quality
of Medicines-Council of Europe, Strasburg, France). LC-MS-grade
methyl t-butyl ether (MTBE), formic acid and ammonium acetate
were obtained from SigmaAldrich (Steinheim, Germany). Glacial
acetic acid, LCMSgrade methanol and water and Centrifree
Ultraltration Device (catalog number 4104) were supplied by
Merck Millipore Group (Darmstadt, Germany). Drug-free human
plasma was obtained from patients not treated with DCX or paclitaxel (PCX) in our hospital.
2.2. Plasma sample collection
Approximately, three milliliters of blood were collected in an
EDTA-K3 tube (Vacuette, Kremsmnster, Austria) and centrifuged
at 1600 g for 10 min at (4 1) C. Plasma samples obtained were
aliquoted and stored at (75 5) C until analysis.
2.3. Ultraltration sample collection
To measure free DCX concentration in plasma a separation of the
protein-bound fraction of DCX had to be achieved. For this purpose,
one milliliter of plasma sample was subjected to ultraltration
using Centrifree Ultraltration Device (with an Ultracel YM-T
regenerated cellulose membrane 30,000 NMWL units) by centrifugation at 1500 g for 45 min at room temperature (25 3 C) to
provide about 200 L of the ultraltrate. Ultraltrate samples were
processed on the same day of the analysis.

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2.4. Preparation of stock and working solutions

Two stock solutions of DCX were prepared in methanol at a concentration of 5 mg/L for total DCX and 500 g/L for free DCX. Stock
solutions were used for the preparation of working calibrator samples and working quality control (QC) samples. Various quantities
from a set of the DCX stock solutions were diluted with 0.1% (v/v)
acetic acid in methanol according to Richheimer et al. [9], resulting
in seven working calibrators samples (0, 10, 25, 100, 250, 1000 and
2500 g/L for total DCX and 0.0, 1.0, 2.5, 10, 25, 100 and 250 g/L
for free DCX). The other set of DCX stock solution was diluted with
0,1% (v/v) acetic acid in methanol to obtain three working QC samples (50, 500 and 1500 g/L for total DCX and 5.0, 50 and 150 g/L
for free DCX). The working calibrators and QC samples were stored
light-protected for up to 6 months at (75 5) C as 150 L aliquots
in 1.5 mL-polypropylene microcentrifuge tubes, and thawed on the
day of analysis.
Similarly, an IS (PCX) stock solution (10 mg/L) was prepared in
methanol and diluted with 0.1% (v/v) acetic acid in methanol to give
an IS working solution (100 g/L).
All solutions were stored at (75 3) C until use.
2.5. Preparation of calibrators and quality control samples in
plasma and ultraltrate
Drug-free pooled human plasma and ultraltrate plasma were
used as a biological matrix for calibrators and QC samples.
According to Mortier et al. [25], for the preparation of plasma
or ultraltrate calibrators, 250 L of water, 50 L of IS working
solution (PCX at 100 g/L) and 50 L of working calibrator samples were added to 125 L of drug-free plasma or ultraltrate into
5 mL-glass tubes, resulting in calibrator nominal concentrations
ranging between 4.01000 g/L for total DCX and 0.4100 g/L
for free DCX. After vortexing for 5 s, 1.5 mL of MTBE was added
for liquidliquid extraction. Tubes were vortexed for 45 s and centrifuged at 1500 g for 5 min at room temperature. Subsequently,
1 mL of upper organic layer was transferred to a new 5 mL-glass
tube and evaporated to dryness under nitrogen at 40 C. The
residues were redissolved in 200 L of methanol/water containing
0.1% acetic acid; 50/50, v/v. After vortexing for 5 s, all volume was
transferred into HPLC vials and placed in the autosampler ready for
injection.
Similary, plasma or ultraltrate QC sample were prepared in
order to obtain nominal concentrations of 20, 200 and 600 g/L for
total DCX and 2.0, 20 and 60 g/L for free DCX.
2.6. Sample preparation
For the preparation of plasma or ultraltrated samples, 50 L of
IS working solution (PCX at 100 g/L), 50 L of 0.1% (v/v) acetic acid
in methanol and 250 L of water were added to 125 L of plasma
(for total DCX) or 125 L of ultraltrate (for free DCX), into 5 mLglass tubes. After that, the same liquidliquid extraction procedure
described above for plasma or ultraltrate calibrators was applied.
2.7. Instrumentation
Analyses were conducted using an Acquity UPLC integrated
measurement system (Waters, Milford, MA, USA) consisting of a
thermostatic autosampler, a binary solvent delivery manager and
a column over a thermostated compartment. Separation was performed on an Acquity UPLC BEHTM C18 reverse-phase column,
2.1 100 mm, packed with 1.7 m particles (Waters, Milford, MA,
USA), protected by a pre-column frit (0.2 m 2.1 mm). The column chamber was held at a temperature of 35 C.

The chromatographic separation was performed using two

mobile phases isocratically. Mobile phase A consisted of 2 mM
ammonium acetate with 0.1% formic acid (v/v) in water (pH 4.2),
and was also used as a weak wash solvent, while mobile phase B
consisted of 2 mM ammonium acetate with 0.1% formic acid (v/v)
in methanol, and was also used as a strong wash solvent. The isocratic program was 30% A: 70% B at a 0.4 mL/min ow rate. The
sample injection volume was 20 L in a 50-L loop (partial loop
with needle overll injection mode) and the autosampler was held
at a temperature of (4 1) C.
Detection was carried out using an Acquity TQD tandemquadrupole MS equipped with a Z-spray electrospray ionization
(ESI) source (Waters, Milford, MA, USA) operating in positive mode.
Nitrogen was used as the nebulizing and desolvation gas, and argon
was used as collision gas. DCX and PCX were detected by multiple reaction monitoring (MRM) using the following transitions
of mass-to-charge (m/z): DCX, 808.3 527.0 (quantier ion) and
808.3 509.0 (qualier ion); PCX, 854.3 569.0 (quantier ion)
and 854.3 509.0 (qualier ion). The quantier to qualier ratio
was used for peak identication based on criteria set forth by the
CLSI C50-A guideline [29]. The scan dwell time was set at 0.2 s for
every channel. The other optimized MS settings employed for DCX
and PCX were capillary potential 3.5 kV, extractor voltage 3 V, RF
lens voltage 0.1 V, source temperature 120 C, desolvation temperature 400 C, desolvation gas ow rate 850 L/h, cone gas ow rate
150 L/h, collision gas ow 0.13 mL/min, cone voltage 18 V for DCX
and 24 V for PCX and collision energy 10/14 eV for DCX and PCX as
quantier/qualier ions, respectively.

2.8. Validation procedure

Validation was performed according to the European Medicines
Agency (EMA) and Clinical and Laboratory Standards Institute
(CLSI)-International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) guidelines [2931].

2.8.1. Calibration
Eight-level calibration standards containing DCX were processed daily before total and free DCX concentration measurement.
Integration of smoothed peak areas and calculation of DCX concentrations was performed with TargetLynxTM v 4.1 software (Waters,
Milford, MA, USA).
According to the EMA guideline [30], calculated concentrations
of the calibration standards were all within 15% of the nominal
value, except for the lower limit of quantication (LLOQ) for which
it was within 20%.
The calibration curves were generated by linear t of the
DCX/PCX standard area response ratio versus DCX concentration
(1/X2 weighting; excluding the option to force through the point of
origin).

2.8.2. Selectivity
Ten different batches of plasma and its plasma ultraltrate from
patients not treated with DCX or PCX but receiving, in combination
or alone, other drugs as anticonvulsants, antibiotics, immunosuppressants, methotrexate or digoxin were used. Two patients
received cyclosporine A, mycophenolic acid and everolimus; two,
valproic acid and tazobactam; one, ceftriaxone and tacrolimus; one,
pheytoin and metotrexate; one, sirolimus and mycophenolic acid;
one, digoxin; one, carbamazepine; and one, cefepime. Drug concentrations were in the respective therapeutic intervals or maintained
above the minimal inhibitory concentration of the pathogen.
According to the EMA guideline [30], the absence of interfering
components is accepted when the peak area response of interfering

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peak at the retention time of analyte (DCX) is less than 20% of the
LLOQ for the analyte (DCX) and 5% for the internal standard (PCX).
2.8.3. Limit of detection and limit of quantication
The CLSI-IFCC C50-A guideline [29] denes the lower limit of
detection (LLOD) as the lowest concentration where the signalto-noise (S/N) ratio is three or more, and the LLOQ as the lowest
concentration at which the S/N ratio is 10 or more and that could
be estimated with an acceptable inter-day imprecision (coefcient
of variation of 20%).
To estimate the LLOD and the LLOQ, the lowest working calibrator samples (10 g/L for total DCX and 1.0 g/L for free DCX) were
either not diluted, diluted 2-fold or 5-fold with 0.1% (v/v) acetic
acid in methanol. These dilutions were treated with the previously
described procedure for calibrators to obtain nominal concentration in plasma of 4.0, 2.0 and 0.8 g/L for total DCX and 0.40, 0.20
and 0.08 g/L for free DCX. Each working sample was processed
repeatedly 15 times in one day and in a single series per day.
2.8.4. Carry-over
In accordance to the EMA guideline [30], carry-over was
assessed by injecting a blank sample (0.0 g/L) after a highconcentration sample at the upper limit of quantication (nominal
concentration in plasma of 1000 g/L and 100 g/L for total and
free DCX, respectively). Carry-over is acceptable if the peak area
response in the blank sample obtained after measurement of the
high-concentration sample is not greater than 20% of the analyte (DCX) peak area response at the LLOQ, and 5% the peak area
response of the internal standard (PCX).
2.8.5. Imprecision and bias
Four samples, three QC and one LLOQ, were used to estimate
intra- and inter-day imprecision and bias according to the following
equations:
CV(%) =

s
x

.100

r (%) =

 x  


100

where CV, s,x, r and  are coefcient of variation, standard deviation, mean, relative bias and the conventional value, respectively.
The conventional value of the QC samples was assigned by weighting procedures.
For intra- and inter-day imprecision and bias, 15 aliquots of each
concentration were tested repeatedly in 1 day and in a single series
per day, for 30 days, respectively. Coefcient of variation and r
results were analyzed following the EMA acceptance criteria (15%
for QC materials and 20% for LLOQ).
2.8.6. Measurement interval (linearity)
According to the EMA and the CLSI EP6-A guidelines [30,31], to
calculate the measurement interval, the highest working calibrator
samples (2500 g/L for total DCX and 250 g/L for free DCX) were
either diluted with 0.1% (v/v) acetic acid in methanol at ratios of
4:0, 3:1, 2:2, 1:3 and 0:4. These dilutions were treated with the
previously described procedure for calibrators to achieve theoretical nominal concentrations in plasma of 1000, 750, 500, 250, 0 g/L
(total DCX) and 100, 75, 50, 25 and 0 g/L (free DCX). According to
the EMA guideline [30], each dilution samples was processed in
triplicate, and imprecision and bias should be 15%.
The measured values were plotted on the y-axis versus the
expected or known values on the x-axis. The measured values
were considered to be linear following criteria from the CLSI document EP6-A [31], as assessed in the Analyse-it statistical software
(Analyse-it Software, Ltd., Leeds, UK).

143

2.8.7. Recovery
A post-extraction spiking experiment was performed to determine the recovery of the DCX and IS (PCX) in plasma and
ultraltrate following extraction with MTBE. The extraction recovery of DCX and free DCX was measured by comparing the mean peak
areas obtained from blank plasma or ultraltrate samples spiked
with DCX before extraction (A) with those from blank plasma or
ultraltrate to which DCX was added after extraction (B). The ratio
A/B was used to evaluate extraction recovery. The recovery was
calculated in six different lots of matrix plasma or ultraltrate at
three different concentrations (20, 200 and 600 g/L for total DCX
and 2.0, 20 and 60 g/L for free DCX). The recovery of PCX was
similarly studied at the concentration 100 g/L.
According to the CLSI-IFCC C50-A guideline [29], the variation
in recovery between all concentrations should be less than 15%.
2.8.8. Matrix effect
According to the EMA guideline [30] and Viswanathan et al. [32],
the quantitative measure of the matrix effect can be termed as the
matrix factor and dened as the ratio of the peak area response in
the presence of the matrix (measured by analyzing a blank matrix
spiked after extraction with analyte) to the peak area response in
the absence of the matrix (pure solution of analyte):
Matrix Factor =

Peak Area response in presence of matrix components

Peak Area response in absence of matrix components

A matrix factor greater than 1 may be due to ion enhancement

and that less than 1 may be due to ion suppression. Similarly, the
internal standard can also experience ion enhancement or ion suppression.
To take into account the matrix effects of the internal standard
(in our case, PCX), a PCX-normalized matrix factor was calculated by
dividing the matrix factor of the DCX by the matrix factor of the PCX.
The PCX-normalized matrix factor was calculated in six different
lots of matrix plasma or ultraltrate at three different concentrations (20, 200 and 600 g/L for total DCX and 2.0, 20 and 60 g/L
for free DCX) to determine the variability of the matrix effect in
samples from different individuals.
According to the CLSI-IFCC C50-A and the EMA guidelines
[29,30], the variability in matrix effect as measured by the coefcient of variation should be less than 15% and the variation in
matrix effect between all concentrations should be less than 15%.
2.8.9. Stability study
Stability studies included stock solution stabilities of DCX and
PCX, extracted samples in-autosampler stability and short- and
long-term stabilities for DCX and free DCX.
To evaluate the stability of stock solutions, the peak area
response of the stock solutions refrigerated at (5 3) C for 1, 3
and 7 days and at (75 5) C for six months were compared with
fresh stock at room temperature.
The stability of samples in the autosampler was tested, reinjecting them after a 6, 12 and 24 h storage at (4 1) C.
Short- and long-term stabilities for DCX and free DCX were conducted in various storage conditions at three plasma or ultraltrate
QC concentration levels (20, 200 and 600 g/L for total DCX and
2.0, 20 and 60 g/L for free DCX), and 10 replicates of each concentration was used. To evaluate short-term stability, the QC samples
were rst stored at (5 3) C for 1, 3 and 7 days and then equilibrated to room temperature and extracted and tested against their
fresh counterparts. For long-term stability evaluation, the QC samples were rst frozen at (75 5) C for 6 months and then thawed
before extraction and tested against fresh calibration and QC samples.
The EMA guideline [30] denes stable samples as having a mean
concentration at each level within 15% of the nominal concentration.

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3. Application to biological samples

The UPLCMS/MS methods here described were developed to
measure total and free DCX in human plasma for PK investigations, from pacients with operable nonmetastatic breast carcinoma
receiving neoadjuvant chemotherapy, and it is currently applied in
a phase IV trial research protocol. This study was carried out according to the principles of the Declaration of Helsinki and was approved
by the local Ethics Committee of Catalan Institute of Oncology.
Written informed consent according to local practice was obtained
for every patient.

of the DCX peak area response at the LLOQ for DCX and free DCX,
respectively. On the other hand, peak area responses were 2.6% and
3.8% of the peak area response of the PCX.
4.4. Imprecision and bias
Data for intra-day and inter-day imprecision and relative bias
are summarized in Table 1. Imprecision and relative bias were
lower than the maximum permissible requirements for these
metrological characteristics (15% for quality control materials and
20% for LLOQ).

3.1. Patients and docetaxel administration

4.5. Measurement interval (linearity)

Women who had primary operable breast cancer receiving

neoadjuvant chemotherapy are being included in the clinical trial,
namely patients with tumors >20 mm (T2, T3, T4, N0, N1 and N2)
and the absence of metastatic disease. Chemotherapy consisted of
4 cycles of intravenously 1h-infusion of 100 mg/m2 dose of DCX
every 21 days, followed by 4 cycles of intravenously doxorubicin
plus cyclophosphamide.

The measurement interval was found linear between 1.95 and

1000 g/L and 0.42 and 100 g/L for DCX and free DCX, respectively. The resulting mean measured values of each sample were
compared to predicted concentrations and yielded a linear regression of y = 0.939 x + 15.4 (r 2 = 0.9909) and y = 0.934 x + 3.88
(r2 = 0.9915) for DCX and free DCX, respectively. Dilution integrity
of the samples achieved acceptance criteria for imprecision and bias
(15%).

3.2. Sample collection and pharmacokinetics parameters

4.6. Recovery
Pharmacokinetic sampling occurred for the rst and the fourth
cycles. Approximately, ve milliliters of blood were collected in
plasma tubes with EDTA-K3 (Vacuette, Kremsmnster, Austria)
immediately before starting the drug infusion and at 0.5, 1.0, 2.0,
4.0, 8.0, 12 and 24 h post-dose. Blood samples were quickly centrifuged at 1600 g for 10 min at (4 1) C, aliquoted and stored at
(75 5) C until analysis.
Plasma concentrationtime data were analyzed using Phoenix
WinNonlin software v.7.3 (Certara USA Inc, Princeton, NJ, USA).
Non-compartmental model was utilized to calculate the pharmacokinetic parameters. The total clearance (CL) and the area under
the plasma concentrationtime curve (AUC024 ) were estimated
using the linear trapezoidal method from time 0 to 24 h.
4. Results
Under the chromatographic conditions described above for
UPLCMS/MS methods, the retention times for DCX and PCX were
1.94 and 1.74 min, respectively. Typical MRM chromatograms are
shown in Fig. 2. The UPLCMS/MS run time was 3.5 min, including
the time necessary to return to baseline conditions before the next
injection.
4.1. Selectivity
No interfering peaks were present in any plasma or ultraltrate sample from patients receiving other drugs. The peak area
responses observed in all plasma or ultraltrate batches at DCX
retention time were 1.8% of the LLOQ of total and free DCX, being
less than 0.9% at the PCX retention time.
4.2. Limit of detection and limit of quantication
The LLOD were 0.96 g/L (S/N ratio of 7.8) and 0.21 g/L (S/N
ratio of 3.2) for DCX and free DCX, respectively. The LLOQ were
1.95 g/L (S/N ratio of 17.5, CV of 12.4%) and 0.42 g/L (S/N ratio of
10.2, CV of 19.6%) for DCX and free DCX, respectively.
4.3. Carry-over
Peak area responses observed in the blank samples after measurement of the high-concentration samples were 5.9% and 7.7%

The recoveries of DCX at 20, 200, 600 g/L and PCX at 100 g/L
were (79.4 9.6)%, (83.1 8.2)%, (81.5 6.2)% and (87.1 9.0)%,
respectively. In the case of free DCX at 2.0, 20, 60 g/L and
PCX at 100 g/L the recoveries were (86.2 8.4)%, (89.3 7.2)%,
(90.0 6.9)% and (93.4 8.1)%, respectively.
4.7. Matrix effect
Values for matrix factor, variabilities of matrix effect and PCXnormalized matrix factor in different lots of plasma are summarized
in Table 2. Evaluation of the matrix effect showed ion suppression
for DCX and free DCX. The variation in matrix effect between all
concentrations was less than 15%. As shown in Table 2, the matrix
effects were well compensated by the internal standard, PCX.
4.8. Stability study
Total and free DCX concentrations in plasma were stable during
storage at (5 3) C for a period of 7 days with an absolute percent
deviation of the nominal concentration (%D) lower than 13.1%. In
plasma sample extracts, total and free DCX concentrations were
stable in autosampler at (4 1) C for 24 h (absolute%D 9.7%).
Also, plasma total and free DCX concentrations at (75 5) C were
stable for at least 6 months (absolute%D 5.9%).
Stock solutions of DCX and PCX stored at (5 3) C were stable
for 7 days (absolute%D of 11.9% and 10.8%, respectively). Also, stock
solution of DCX at (75 5) C was stable for 6 months (absolute%D
of 6.9%).
Percent deviations were in all cases negative indicating a loss of
DCX or PCX concentration in all matrices with regard to the nominal
value, i.e., a decomposition or degradation of DCX or PCX occurred.
5. Clinical application
The UPLCMS/MS method here described was developed to
measure total and free DCX concentrations in human plasma for
pharmacokinetic investigations and it is currently applied in a
research protocol.
Total and free DCX concentration values in human plasma
are being used for developing a population PK/PD model in
women who had primary operable breast cancer receiving

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145

Fig. 2. A representative MRM total ion chromatogram obtained from human (A) plasma patient sample at concentration of 161,7 g/L and (B) ultraltrate plasma patient
sample at concentration of 11,0 g/L.

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R. Rigo-Bonnin et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 140149

Table 1
Intra-day and inter-day imprecision and bias values obtained in UPLCMS/MS for docetaxel and free docetaxel concentration in human plasma.
Quantity/theorical concentration in g/L (Sample type)

Intra-day (n = 15)
x s (g/L)

Inter-day (n = 15)
CV (%)

x s (g/L)

r (%)

CV (%)

r (%)

PDocetaxel; mass c.
2.0 (LLOQ)
20.0 (QC1)
200 (QC2)
600 (QC3)

1.92 0.19
19.0 1.31
196 9.8
592 24.3

10.0
6.9
5.0
4.1

4.0
5.0
2.0
1.3

1.95 0.24
18.0 1.51
192 11.7
589 30.0

12.4
8.4
6.1
5.1

2.5
10.0
4.0
1.8

PDocetaxel(free); mass c.
0.40 (LLOQ)
2.0 (QC1)
20.0 (QC2)
60.0 (QC3)

0.38 0.07
1.97 0.23
18.9 1.49
58.5 3.22

18.1
11.6
7.9
5.5

5.0
1.5
5.5
2.5

0.42 0.08
1.94 0.27
18.6 1.82
57.9 3.47

19.6
13.8
9.8
6.0

5.0
3.0
7.0
3.5

n: number of samples processed;x: mean values; s: standard deviation; CV: coefcients of variation; r : relative bias; LLOQ: low limit of quantication; QC1: internal quality
control 1; QC2: internal quality control 2; QC3: internal quality control 3. According to IUPAC and IFCC: P: plasma; mass c.: mass concentration.

Table 2
Matrix factor and PCX-normalized matrix factor for docetaxel in plasma and free docetaxel in ultraltrate plasma.
Matrix lot

Matrix factor (%)

PDocetaxel; mass c.

20 mg/L

200 mg/L

600 mg/L

PCX-normalized matrix factor (%)

20 mg/L

200 mg/L

600 mg/L

1
2
3
4
5
6

84.3
77.3
83.4
88.2
69.8
74.2

93.4
88.6
85.9
92.7
77.6
81.1

96.5
92.2
87.6
94.4
85.5
83.3

94.2
96.6
94.6
94.0
90.5
87.8

96.7
100.5
97.7
97.2
88.2
91.7

99.0
98.2
95.1
99.0
94.5
93.9

x (%)
s (%)
CV (%)

79.5
6.9
8.7

86.6
6.3
7.3

89.9
5.2
5.8

93.0
3.2
3.4

95.3
4.5
4.7

96.6
2.4
2.4

Matrix lot

Matrix factor (%)

PDocetaxel(free); mass c.

2.0 mg/L

20 mg/L

60 mg/L

2.0 mg/L

20 mg/L

60 mg/L

1
2
3
4
5
6

87.7
80.9
90.1
95.4
71.1
75.4

91.1
78.8
91.3
97.7
77.7
78.8

95.3
92.3
90.2
94.7
79.4
83.3

93.8
96.1
97.9
101.3
86.6
97.0

97.6
93.4
95.6
101.2
94.5
99.9

96.5
100.1
100.1
96.9
95.4
98.7

x (%)
s (%)
CV (%)

83.4
9.2
11.1

85.9
8.5
9.9

89.2
6.5
7.2

95.4
5.0
5.2

97.0
3.1
3.2

98.0
2.0
2.0

PCX-normalized matrix factor (%)

x : mean values; s: standard deviation; CV: coefcients of variation. According to IUPAC and IFCC: P: plasma; mass c.: mass concentration.

neoadjuvant chemotherapy. Pharmacokinetic/pharmacodynamic

modeling could be guide DCX dosage optimization, thus
increasing the clinical effectiveness. Because of its multicompartmental behavior and large volume of distribution, a
method with sufcient detection capability is of great importance.
The plasma concentrationtime curve of total and free DCX in a
breast cancer patient after i.v. 1h-infusion of 100 mg/m2 dose of
DCX for the rst and the fourth cycles are shown in Fig. 3. The
results indicated that the described methods had a sufcient capability of detection to measure total and free DCX concentration in
human plasma for up to 16 h after the administration of a dose of
100 mg/m2 . The values of CL and AUC0-t in a breast patient for total
DCX were 85.67 L/h (44.92 L/h/m2 ) and 1.86 mg h/L at the rst cycle
and 92.6 L/h (48.56 L/h/m2 ) and 1.72 mg h/L at the fourth cycle,
respectively. For free DCX, the CL and AUC0t were 1343 L/h and
118.34 g h/L at the rst cycle and 1204 L/h and 132.03 g h/L at
the fourth cycle, respectively.
The pharmacokinetics of DCX has been investigated in several
phase I and phase II trials [14,34]. The maximum tolerated dose
is 80115 mg/m2 and the main dose-limiting toxicity is neutropenia. A relationship has been observed between systemic exposure

to DCX, as measured by the AUC, and neutropenia [1]. However,

to date, a widely accepted AUC value to establish the best relationship between efcacy and toxicity of DCX, is still lacking.
Engels et al. [33] suggested to use a weighted mean for the target AUC (4.90 mg h/L) that was based on several representative
DCX PK studies, including a total of 806 patients treated with DCX
100 mg/m2 .
The results of our patient were comparable with previous
reports in PK studies that used similar doses of DCX. Bruno R.
et al. [3] reported a CL mean value of 36.5 L/h (percentile 595%:
17.559.3) and AUC mean value of 4.81 mg h/L (percentile 595%:
2.939.52) with a considerable interpatient PK variability; and Rosing H. et al. [34] (who used 1-h intravenous infusion at a dose level
of 100 mg/m2 with PK monitoring) reported a CL mean value of
34 L/h/m2 (range: 19.253.8) and a AUC mean value of 3.1 mg h/L
(range: 1.45.2).
Given that DCX metabolization is mainly carried out by CYP450
enzymes, there is a potential for altered pharmacokinetics and
therapeutic effect due to high protein-binding in the plasma and
drugdrug interaction. For this representative patient, the percentage free fraction of drug in each sample in cycles 1 and 4 were
(6.01 1.34)% and (7.12 2.24)%, respectively and did not appear

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147

Fig.3. Free and total concentration-time curve of docetaxel in a typical patient receiving 1 h-infusion of 100 mg/m2 docetaxel i.v. every 21 days. Pharmacokinetic data showed
are for the rst and the fourth cycles.

to vary with time. These free fraction data agree with all previous
reports [1,4,24,25].
6. Discussion
UPLCMS/MS methods were developed and validated for the
measurement of DCX and free DCX concentrations in human
plasma and are being currently applied in a research protocol.
These methods could support the therapeutic drug monitoring
(TDM) of DCX in different patients, particularly in women with
breast cancer receiving neoadjuvant chemotherapy. Intrinsic characteristics of the UPLCMS/MS technique let us achieve similar
[13,15,24] or shorter [1012,14,1621,2526] retention times than
those reached using other methods.
6.1. Method development
Various combinations of mobile phase and reverse-phase UPLC
columns were tested to achieve a good resolution and symmetric peaks, a high response, a short retention time and better
peak shape. Different mobile phases were evaluated to improve
UPLC separation and to enhance MS sensitivity. Several experiments were performed testing different mobile phases consisting
of water, on one hand, and acetonitrile and methanol as organic
phases on the other hand. All these mobile phases were combined
with ammonium acetate (2 mM), with formic acid at 0.1% (v/v) or
with both additives. From all the possible combinations, that composed of methanol and water and both additives offered the highest
MS response. Two kinds of Bridget Ethyl Hybrid UPLC columns
(Acquity UPLC BEHTM C18 reverse-phase columns) with the same
particle size (1.7 m) and internal diameter (2.1 mm) but with different length (50 mm vs 100 mm) were evaluated jointly. With the
50 mm-lenght BEH column shorter retention times were obtained
(0.62 min for PCX and 0.73 min for DCX) but it presented wider
peaks and worst peak shapes, probably, because it was near to the
elution front. It was found that the use of an Acquity UPLC BEHTM
C18 reverse-phase column, 2.1 100 mm; 1.7 m, in combination

with isocratic elution mode based on 2 mM ammonium acetate

with 0.1% formic acid in methanolwater (70:30, v/v), let us achieve
the chromatographic conditions mentioned above. Other parameters such as column temperature, ow rate and injection volume
were studied in order to get a fast and reliable separation, and the
best results were obtained when 35 C was used as column temperature (versus 25 C, 45 C or 55 C), 0.4 mL/min as ow rate (better
than 0.3 mL/min or 0.5 mL/min) and 20 L were injected (versus
5 L or 10 L). Under all these conditions, retention times of DCX
and PCX were constant and reproducible.
All MS parameters were optimized by direct injection of 10 mg/L
of DCX and PCX in a methanol/water solution containing 0.1%
acetic acid (50/50 v/v) into the mass spectrometer at a ow
rate of 20 L/min. In our case, the most abundant ions obtained
were the [MH]+ adducts. The choice of the monitored ions was
made after studying the MS/MS fragmentation pattern of DCX and
PCX. DCX and PCX were quantied using the MRM mode due
to its high-sensitivity data acquisition when the precursor and
the product ions are monitored. To prevent analytes misidentication, and specically to conrm the presence of the analytes
and the absence of false contributions from similar components
coeluted in the samples, two MRM transitions were followed for
DCX and PCX. One transition was used for quantication (the quantier): 808.3 527.0 for DCX and 854.3 569.0 for PCX, and the
other transition was monitored for identication (the qualier):
808.3 509.0 and 854.3 509.0 for DCX and PCX, respectively.
The quantier to qualier ratio was used for peak identication
based on criteria set forth by the CLSI C50-A guideline [29]. Results
report peak ratios of the two peaks which did not deviate from the
average ratio in the standards by more than the 20%, indicating that
there is no analyte misidentication.
Although liquidliquid extraction is not probably the more adequate method to prevent the matrix effects, in our evaluation,
a liquidliquid with MTBE followed by dryness under nitrogen
evaporation and further redissolution of the residues in mobile
phase simplied the extraction procedures published by others
[11,15,19,21], and provided acceptable recoveries and matrix effect

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R. Rigo-Bonnin et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 140149

results. The evaluation and the variability of the matrix effect

in samples from different individuals are a crucial aspect. These
two issues are often not properly studied and could compromise
the analysis performance. An ideal internal standard should be a
structural analog or a stable labeled compound, and should track
the analyte during the extraction and compensate for any analyte
on the column and any inconsistent response. Due to problems
of availability and the high price of stable labeled compounds, a
chemical structural analog of DCX with similar physico-chemical
properties, PCX, was the rst choice for the analysis as other
authors [10,12,13,1618]. Although, in our case, DCX and PCX did
not elute simultaneously and this could lead to a lack of compensation for matrix effect and other characteristics, for example,
related to extraction efciency, in our evaluation of the matrix
effect we observed that the concentration of the three samples
assayed showed a steady value, given that the use of PCX as an
internal standard compensates for the ion suppression observed
in the DCX. Besides, PCX is unlikely to be co-administered with
DCX and will therefore be subject to similar matrix effects. For
all these reasons, we considered PCX as an adequate internal
standard.
The major advantage of our method is the possibility of measuring total and free DCX concentrations in a same run. Another
advantage is a chromatographic run time of only 3.5 min per sample, which is considerably shorter than that of all other methods
previously reported [1012,14,1621,2526]. Although the combination of sample preparation and global chromatographic run
time can achieve just a moderate throughput, TDM of DCX could
be combined with TDM of other drugs, such as immunosuppressants, on the same instrument, which in our case also use the same
chromatographic solvents. Hence, delays due to priming and equilibration of solvents are minimized.

6.2. Validation procedure

In the validation procedure, different analytical characteristics
and studies were evaluated. No interfering peaks were present in
any plasma or ultraltrate sample from patients receiving other
drugs indicating that the proposed UPLCMS/MS methods provide
acceptable selectivity. Also, no signicant carry-over was observed.
Recovery values from samples studied were above 79.4% and PCXnormalized matrix factors ranged between 93.0 to 98.0%. The
imprecision and bias values obtained, for each concentration, were
found to neither exceed the 15% of the CV for QC samples nor the 20%
for LLOQ, thus conforming to the EMA criteria [30], and were better
or similar to those of previous publications [12,1518,20,24]. These
results indicate that the proposed UPLCMS/MS methods provide
acceptable precision and trueness. Lower limits of quantication
for total and free DCX were better or the same that some methods reported [10,11,13,14,16,25]. UPLCMS/MS methods presented
linearity within the concentration range of 1.95 and 1000 g/L and
0.42 and 100 g/L for total and free DCX, respectively. Linearity
and the LLOQ obtained were acceptable considering the expected
concentrations in the PK study.

7. Conclusions
In conclusion, the simple UPLCMS/MS methods that we have
developed and validated for measurement of total and free concentrations of DCX in human plasma could be useful for both PK studies
and TDM in the daily practice of the clinical laboratory, considering their time of analysis, practicability and analytical performance
characteristics as selectivity, precision, trueness, capability of
detection, linearity, recovery and matrix effect. In addition, these

methods allow measurement of both total and free DCX concentrations within the same run.
Conict of interest
The authors declare no conict of interest.
Acknowledgments
The authors thank staff members of Pharmacy Department of
Catalan Institute of Oncology, IDIBELLs Clinical Research Unit (UCICEC) and the Biobank HUB-ICO-IDIBELL. This work was supported
by a grant from Ministerio de Sanidad y Consumo (EC11-4054).
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