Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 6570

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Journal of Pharmaceutical and Biomedical Analysis
j our nal homepage: www. el sevi er . com/ l ocat e/ j pba
A validated HPLC-MS method for quantication of the CCR5 inhibitor
maraviroc in HIV+ human plasma
Marco Simiele, Lorena Baietto, Alessio Audino, Mauro Sciandra, Stefano Bonora,
Giovanni Di Perri, Antonio DAvolio

Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy
a r t i c l e i n f o
Article history:
Received 3 August 2013
Received in revised form20 January 2014
Accepted 22 January 2014
Available online 31 January 2014
Keywords:
Maraviroc
HPLC-MS
HIV
Therapeutic drug monitoring
Plasma
a b s t r a c t
Maraviroc is a CCR5 inhibitor approved in 2007 for treatment of therapy experienced adult patients
infected with CCR5-tropic HIV-1 virus. International guidelines for HIV therapy indicate a plasma con-
centration cutoff of maraviroc for response. We developed and validated a new HPLC-MS method to
quantify maraviroc concentrations in human plasma. 6,7-Dimethyl-2,3-di(2-pyridyl)quinoxaline was
used as internal standard and added to 100 L of plasma. Samples were then treated with 500L of
acetonitrile for the protein precipitation procedure. An analytical T3 Atlantis column (150 mm 4.6 mm
i.d.) with a particle size of 5mwas used to separate the compounds and ions were detected at m/z 257.5
and 313.3 for maraviroc and quinoxaline respectively. The calibration curve was linear up to 2500 ng/mL.
The mean recovery of maraviroc was 89.1%. All validation data results were in accordance to Food and
Drug Administration and European Medicines Agency requirements. The HPLC-MS method reported here
could be used routinely to monitor plasma concentrations of maraviroc in HIV-infected patients.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Since Highly Active Antiretroviral Therapy (HAART) became
widely available in1996, the number of AIDS-relateddeaths deeply
decreased, and it led to improvement of quality of life to the peo-
ple living with HIV. Combination therapy includes at least one
protease inhibitor (PI) or a non nucleoside transcriptase inhibitor
(NNRTI) and/or one or more nucleoside or nucleotide transcrip-
tase inhibitors (NRTIs-NtRTI) and/or a fusion inhibitor (FI). HIV
virions might acquire mutations conferring cross resistance to
different compounds of each class, especially when the patient
does not intake the therapy correctly. Therefore, new therapeu-
tic compounds are needed that able to overcome the extensive
class-resistances observed in multi-drug treated patients [1].
New compounds as raltegravir (RGV), the rst integrase
inhibitor, and rilpivirine (TMC278) the last NNRTI approved, have
shown to be active against multidrug-resistant viral strains [26].
In contrast to other classes of drugs maraviroc has a target direct
of the cell host rather than viral components. Maraviroc (MVC;
Celsentri

, Selzentry

) is achemokineCCR5co-receptor antagonist

Corresponding author at: Laboratory of Clinical Pharmacology and Pharmaco-

genetics, Unit of Infectious Diseases, University of Turin, Department of Medical
Sciences, Amedeo di Savoia Hospital, CorsoSvizzera 164, 10149 Turin, Italy.
Tel.: +39 011 4393979; fax: +39 011 4393996.
E-mail address: antonio.davolio@unito.it (A. DAvolio).
[715] that is usedat present inexperiencedR5-tropic HIV-infected
patients, for whom previous antiretroviral regimens have failed.
MVC is administeredorallyat ausual doseof 300mgtwicedaily, but
can be taken at different doses (150mg or 600mg) mainly depend-
ing onother concomitant drugs [16]. MVC is a substrate for CYP3A4,
so potentially caninteract withseveral other antiretrovirals. There-
fore, it is administered at 150mg bid if co-administered with a
boosted protease inhibitor, at 300mg bid if co-administered with
nevirapine (NVP) or tipranavir/ritonavir (TPV/r), and at 600mg bid
if co-administered with etravirine(ETV) or efavirenz (EFV) [16,17].
MVC is also a substrate of P-glycoprotein [18]. Ritonavir, which
is commonly administered as a booster for protease inhibitors, is a
strong inhibitor of both CYP3A4 and P-glycoprotein, and can con-
sequently cause an increase of MVC plasma concentrations. On
the basis of the exposure-response analysis from the MOTIVATE
studies, an approximate maximal efcacy is achieved at a maravi-
roc trough concentration (C
trough
) above 50ng/ml determined in a
population predominantly receiving MVC (150mg) with boosted
protease inhibitors [19]. Although average plasma concentrations
(C
avg
) have a stronger correlationwithvirological efcacy, the mea-
surement of average plasma concentrations in the clinical setting
is difcult because of the requirement for multiple samples and the
associated cost. Therefore, C
trough
is more commonly used.
Accurate measurement of antiretroviral plasma concentra-
tions is crucial for pharmacokinetic/pharmacodynamic analyses,
drugdrug interaction studies, and therapeutic drug monitoring
(TDM) [20]. The latter is currently considered a useful tool for the
0731-7085/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jpba.2014.01.031
66 M. Simiele et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 6570
Table 1
Chromatographic conditions for gradient HPLCanalysis of maraviroc: Mobile phase:
Solvent A(HPLCgrade water +0.05%formic acid) andSolvent B(HPLCgrade acetoni-
trile +0.05% formic acid). Temperature of the column was set at 35

C.
Time (min) Flow(ml/min) Solvent A % Solvent B %
0 1 80 20
4 1 60 40
7.5 1 30 70
8.1 1 5 95
15 1 5 95
15.1 1 80 20
20 1 80 20
optimization of antiretroviral therapy in international guidelines
[2023]. An understanding of the pharmacokinetics (PK) of MVC
in the clinical setting, pharmacokinetic/pharmacodynamic proper-
ties and drug interaction proles, is still limited due to the recent
availability of this compound. Moreover, recent data suggest the
involvement of pharmacogenetic factors in the PK of MVC [24].
Therefore, PK studies of MVC are requested in order to dene
the possible role of TDM in the clinical context. Few methods for
MVC quantication in human plasma have been published to date
[2531].
The aim of our study was to develop and validate a cheap, fast
and reliable HPLC-MS analytical method for the quantication of
MVC in human plasma.
2. Experimental
2.1. Chemicals
Maraviroc was purchasedfromPzer Inc. (Groton, CT, USA). Ace-
tonitrile HPLC grade and methanol HPLC grade were purchased
from J.T. Baker (Deventer, Holland). HPLC grade water was pro-
duced with Milli-DI system coupled with a Synergy 185 system
by Millipore (Milan, Italy). Quinoxaline [6,7-dimethyl-2,3-di(2-
pyridyl)quinoxaline] (QX) and formic acid were obtained from
SigmaAldrich (Milan, Italy). Blank plasma from healthy donors
was kindly supplied by the Blood Bank of Maria Vittoria Hospital
(Turin, Italy).
2.2. Chromatographic and MS conditions
The HPLC-MS instrument used was a Waters system (Milan,
Italy), with binary pump model 1525, AF degasser, 717-plus
autosampler, and Micromass ZQ mass detector. LCMS Empower
2 Pro software (version year 2007, Waters; Milan, Italy) was used.
Chromatographic separation was performed at 35

C using a col-
umn oven, and an Atlantis T3 5m column (150mm 4.6mm
i.d.) (Waters, Milan, Italy), protected by a SecurityGuard with C18
(4.0mm3.0mm i.d.) pre-column (Phenomenex, CA). Runs were
performed with a gradient (Table 1), and the mobile phase was
composed of Solvent A (HPLC grade water +0.05% formic acid) and
Solvent B (HPLC grade acetonitrile +0.05% formic acid).
A T switch tube was applied post-column to introduce only
200L/minof total ow(1mL/min) intotheMS detector. Optimiza-
tion of the MS conditions has been performed by direct infusion of
reference standards (1g/mL) in mobile phase (solvent A:solvent
B (50:50)) at 10L/min; at the same time a ow of 1mL/min of
mobile phase (solvent A:solvent B (50:50)) was introduced in the
column to perform the infusion in combined mode. MS param-
eters were been optimized to maximize sensitivity as follows:
ESI, positive polarity ionization; capillary voltage, 3.5kV; source
temperature, 110

C; desolvation temperature, 350

C; nitrogen
desolvation ow, 400l/h; nitrogen cone ow, 50L/h. The ion tran-
sitions and cone voltages were: 513.3m/z 257.5 with a cone
voltage of 35V for MVC and 312.3m/z 313.3 with a cone voltage
of 50V for QX (IS).
2.3. Stock solutions, standards (STD) and quality controls (QC)
MVC and QX stock solutions were made in a solution of
methanol and HPLC grade water (90:10, v/v) to obtain a nal con-
centration of 1mg/mL; all stock solutions were then refrigerated
at 4

C until use, within 1 month. A working solution of Inter-

nal Standard (IS) was made for every validation analyses with QX
(3g/mL) inmethanol andHPLCgrade water (50:50, v/v). The high-
est calibration standard (STD 10) and four quality controls (QCs)
were prepared adding a stock solution to blank plasma; the others
STDs were prepared by serial dilution fromSTD10 (2500ng/mL) to
STD 1 (4.9ng/mL) with blank plasma, to obtain 10 different spiked
concentrations plus ablanksample(STD0). QCconcentrations were
2000ng/mL, 500ng/mL, 50ng/mL and 12.5ng/mL for QC-High, QC-
Medium, QC-Lowand QC-Low-Low, respectively.
The range of MVC concentrations was established in accord
with relevant clinical studies [812,1416] in order to include all
described concentrations. As for patient samples, STDs and QCs
were inactivated for 35min at 58

C to provide HIV-free plasma,

prior to storing them at 20

C for no more than three months.

They were thawed in the beginning of analysis avoiding more then
two freezethawcycles.
2.4. STD, QC and samples preparation
Patients receiving standard dosing of MVC (150mg, 300mg or
600mg) underwent bloodsamplingfor the measurement of plasma
drug concentrations. The study was conducted in compliance with
the Declaration of Helsinki and with the local ReviewBoard regula-
tions; all patients gave written informed consent according to the
local ethic committee standards. Blood samples were collected in
lithium heparin tubes (7mL), and plasma was obtained after cen-
trifugation at 1400g for 10min at +4

C (Jouan Centrifuge, Model

BR4i; Saint-Herblain, France) andthenunderwent heat inactivation
as described above.
A protein precipitation procedure was performed for extrac-
tion of maraviroc fromplasma. Fifty L of IS working solution was
added to 100L of sample (STDs, QCs or patient samples) in a PTFE
microfuge tube, then 500L of protein precipitation solution (ace-
tonitrile 100%) was added. The tube was vortexed for 10s and then
centrifuged at 13,000g for 10min at 4

C. Supernatant was trans-

ferredintoglass tubes andtreatedby vortexvacuumevaporationto
dryness at 60

C and then reconstituted with 125L of HPLC grade

water and acetonitrile solution (70:30, v/v). Fifty L were injected
intothe HPLCcolumn. All QCs samples were performedinduplicate
during validationsessions, and all procedure steps were carried out
at roomtemperature.
During every routine analysis session, the patients samples have
been processed together with STDs from 0 to 10 (STD0 was a
blank plasma) and QCs (High, Medium, Low, Low-Low). Before
the injection of these samples a series of blank (water and ace-
tonitrile solution (70:30, v/v)) was injected for the conditioning of
the HPLC-MS system. A fresh mix with the analytes (10g/mL in
water:acetonitrile solution (70:30, v/v)) was also injected to verify
the retention times. Concentrations data of patients samples were
considered reliable if standard deviation of all QCs from nominal
values were below15%.
2.5. Specicity and selectivity
Interference from endogenous compounds was investigated
by analysis of six different blank plasma samples. Potential
interference by antiretroviral drugs concomitantly administered
M. Simiele et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 6570 67
to the patients was also evaluated by spiking blank plasma.
These included zidovudine (AZT), didanosine (ddI), stavudine
(d4T), lamivudine (3TC), abacavir (ABV), tenofovir (TDF), emtric-
itabine (FTC), and enfuvirtide (T-20). Other possible concomitant
drugs were also investigated, including amodiaquine, desacety-
lamodiaquine, amoxicillin, caspofungin, ceftazidime, ciprooxacin,
clavulanic acid, ethambutol, furosemide, insulin, isoniazid, levo-
oxacin, nimesulide, omeprazole, pravastatin, and ribavirin. An
interfering drug was considered to be a molecule that exhibits a
retention time within 0.3min of the analyte, and with the potential
capability to cause ion suppression.
2.6. Matrix effect
The matrix effect was investigated on six lots of blank plasma
from individual donors, as requested by guidelines [32,33]. Peak
areas from blank extracts spiked with all analytes at three QC
concentrations werecomparedwithpeakareas fromstandardsolu-
tions (water and acetonitrile, 70:30, v/v) spiked with analytes in
the some way, as described by Taylor [34]. The possible matrix
effect was calculated, as deviation %, by comparing the peak area
obtained fromthe plasma extract with the peak area obtained from
the standard solution.
2.7. Accuracy, precision, limit of quantication and limit of
detection
Intra-day and inter-day accuracy and precision were deter-
mined by assaying ten spiked plasma samples at four different
concentrations (QCs). Accuracy was calculated as the percent devi-
ation from the nominal concentration. Inter-day and intra-day
precision was expressed as the standard deviation at each QC
concentration. Each calibration curve was obtained using ten cali-
bration points, ranging from4.9 to 2500ng/mL. Calibration curves
were created by plotting the peak area ratios of each drugs rela-
tive to the IS against the various drugs concentrations in the spiked
plasma standards. A 1/X weighted quadratic regression was used
for all curves in order to obtain the best t for all calibration points
and particularly for low concentration points, close to theoretical
range andcut-off of activity(50ng/mL) [35] of C
trough
concentration
of maraviroc. The limit of detection (LOD) in plasma was dened
as the lowest concentration that yields a signal-to-noise ratio of at
least 3/1. The lowest concentration levels that could be determined
with a percent deviation from the nominal concentration and rel-
ative standard deviation <20%, was considered the lowest limit of
quantication (LOQ), as requested by FDA guidelines [33].
2.8. Recovery
Recovery fromplasma, was assessed by comparing the average
peak area obtainedfrommultiple analyses (n=6) of spikedsamples
(QCs) extracted with the procedure in 2.4, with the average peak
area fromstandard solution of all analytes in HPLC grade water and
acetonitrile (60:40, v/v) solution at the same concentrations.
2.9. Stability
MVC pre-assay stability was not evaluated, and we relied on
manufacturers data [15] and on previously published articles
[27,28]. Therefore, stability studies were performedonly after inac-
tivation procedure and post extraction.
Stabilityof MVC ininactivatedQCs was evaluatedcomparingthe
peak areas with not inactivated QCs. Moreover, stability of MVC
in plasma extracts at room temperature (2025

C, autosampler
temperature) was evaluated. Processed QCs, at four different con-
centrations were analyzedimmediately after preparation, andafter
Table 2
Accuracy and precision data for validation of maraviroc LCMS method. Accuracy
(%), intraday, and interday precision of the four quality control (QC), expressed as
percent relative standard deviation (RSD%).
MVC Accuracy % Precision (RSD%)
Intra-day Inter-day
QC H 0.37% 2.37% 3.55%
QC M 1.03% 3.48% 7.51%
QC L 6.55% 2.01% 7.35%
QC LL 8.47% 6.68% 9.24%
standingfor 24hat roomtemperature, bycomparingthepeakareas
of drugs in the two sample injections.
The stability of IS was not performed because it was extensively
evaluated and used in similar condition in many previous articles
[26,3647].
2.10. Carry over
Carry-over was investigated in triplicate by injecting extracted
blank plasma samples immediately after samples containing target
analytes at ten-fold concentration of STD 10. A value 20% of the
lower limit of quantication (LLOQ) and a value 5% for IS were
considered as absence of carry over.
3. Results
Retention times of MVC and QX are shown in Fig. 1. A represen-
tative chromatogramof blank human plasma overlapped to LOD is
shown in Fig. 2. Mean regression coefcient (r
2
) of all calibration
curves was 0.999. A quadratic through zero regression was chosen
respect to a linear regression due to better signal-response.
3.1. Specicity and selectivity
The assay did not show any signicant interference with other
potentially concomitant drugs (see Section 2.5). The retention time
for MVC and QX were 6.75min and 10.40min respectively (Fig. 1).
The tested six-blank plasma also did not show any interference in
the retention time windows for both compounds (Fig. 2). An exam-
ple of sample from patient in therapy with maraviroc is shown in
Fig. 3.
3.2. Accuracy, precision, limit of quantication, limit of detection
Results of the validation of the method are listed in Table 2. All
observed data [intraday and interday precision (RSD%) and accu-
racy] were below 15.0, according to FDA guidelines [33]. The LOQ
and LOD for MVC were 4.9ng/mL (STD1) and 2.5ng/mL, respec-
tively (data not shown).
3.3. Recovery
Multiple aliquots (n=6) at each different concentration were
assayed. Mean recovery of MVC was 89.1% (85.195.3%) and for IS
was 95.2%(92.398.7%). The meanrecovery for eachquality control
was: 89.7% for QC high, 92.3% for QC medium, 85.7% for QC Lowand
88.7% for QC lowlow(data not shown).
3.4. Matrix effect
The deviation % of the peak area at the three concentrations for
both analytes (MVC and IS) was comparable, always below 15%,
showing absence of matrix effect.
68 M. Simiele et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 6570
Fig. 1. HPLCMS chromatogramof STD 10, containing a MVC concentration of 2500ng/mL and an IS QX concentration of 3g/mL. The retention time for MVC and QX were
6.75 min and 10.40min. Molecules were monitored in SIR mode (single ion recording): MVC m/z 257.5 and IS QX m/z 313.3.
Fig. 2. Overlapping chromatograms of LOD of maraviroc and STD 0 (blank extracted plasma). The ratio between the areas of LOD (2.5ng/ml of maraviroc) and STD 0 peaks
is greater than 3 times as required by FDA guidelines.
3.5. Stability
After inactivation procedure, 35min at 58

C, the MVC plasma

concentration of QCs showed a degradation less than 5% respect
to QCs not inactivated. Mover all STDs, QCs and patients samples
underwent to inactivation step, normalizing the data.
The post extraction stability studies (2025

C, autosampler
temperature) showed MVC stable, with degradation less than 5%.
Fig. 3. Chromatogram of a C
trough
patient sample, treated with celsentry (300mg MVC twice daily). Maraviroc concentration was calculated with a quadratic through zero
curve built on ten standard points (range STD 10 2500ng/mL STD1 4.9ng/mL).
M. Simiele et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 6570 69
3.6. Carry over
The mean MVC concentration of blank plasma samples injected
immediately after samples containing high concentration of ana-
lytes was lower than LOD value (2.5ng/mL). The mean IS area of
blank plasma samples was lower than 1% of IS area of plasma
samples containing IS. These data have showed the absence of car-
ryover.
4. Discussion
The efcacy of existing antiretroviral drugs is constantly at risk
from emerging drug-resistant HIV-1 strains. For this reason, the
needtodevelopnewcompounds andnewdrugclasses is of increas-
ing interest. All of these newantiretroviral drugs have been shown
tobe highlyeffective inmultidrug-treatedpatients andhave shown
to be well tolerated [6,12,48].
TDM has become an essential tool for the management of
HIV-positive patients. Measurement of antiretroviral plasma con-
centrations can be useful in several clinical settings, such as
management of side effects, optimization of efcacy, monitor-
ing metabolic impairment, and detecting drugdrug interaction
[2023,35]. It is noteworthy that MVC has a high potential for drug
interactions, being a substrate of CYP3A4 [15,16]. Moreover, MVC
has been identied as a substrate of OATP1B1, has an association
with a single nucleotide polymorphism(SLCO1B1 521 C>T), and its
MVC C
trough
has been described [24].
The clinical value of MVC TDMhas yet to be fully evaluated, and
a method for the quantication of MVC will be an essential tool for
PK studies and management of special populations. For this pur-
pose, a novel analytical method for quanticationof MVC inplasma
of HIV-infected patients was developed using protein precipita-
tion extraction, and liquid chromatography with mass detection.
Other methods, for measuring MVC using liquid chromatography
coupled with UV or mass spectrometry, have been reported pre-
viously [2531,37]. Our assay extraction is simpler than the solid
phase extraction (SPE) described by Notari et al. [30] and the iso-
cratic run described, without a wash step, appears to be unsuitable
in clinical routine practice. Moreover mass spectrometry detection
is more sensitive than UV-detection, and facilitates the measure-
ment of analytes at very low concentrations. Other authors used
very expensive instrumentation like HPLC-MS/MS, and other pre-
viously described methods are limited to a narrow concentration
range for MVC that appears inadequate for quantifying the C
max
of
this drug [25,2729].
Only one known method uses our same type of instrumenta-
tion (HPLC coupled with mass spectrometry detection) to quantify
MVC in human plasma [31]. But this other method, with respect
our procedure, has a longer chromatographic run time (40min vs
20min) and requires a ve-fold higher volume of plasma (500 vs
100L). While the run time is only 20min, it still ensures a good
separationof drugs andavoids potential matrixeffects. Our method
has been fully validated and has been proven to be precise and
accurate. Calibration curves cover a wide range of MVC concentra-
tions that correspond to the lowest and highest values reported in
clinical settings and PK studies [816]. A quadratic through zero
regression was chosen respect to a linear regression due to bet-
ter signal-response, because the highest STDs of the curves were
slightly in saturation and the response was not a linear function of
the concentration of the analyte.
The method was optimized to achieve low levels of quanti-
cation (see Section 3.2), as requested from HIV guidelines [19,35].
The evaporation step to concentrate the analytes, after protein pre-
cipitation with acetonitrile, allows to obtain a high signal also for
concentration of maraviroc very low. All analytes were adequately
retained; ensuring a good capacity factor.
Reliability, cost, simply to perform, and reproducibility are
key points for the measurement of drug plasma concentrations.
Our assay extraction is cheaper than SPE extraction. Likewise,
our instrumentation is very inexpensive with regard to liquid
chromatography coupled with tandem-mass spectrometry; even
though it is more expensive than HPLC-UV instruments [26,30].
The use of QX as IS, an inexpensive and easy to purchase sub-
stance, improvedreliability andreproducibility of our assay. QXhas
beenusedby our laboratory inmany other works [26,3647,4951]
and it was fully used for maraviroc in a previous UV method [26],
moreover it showed a chemical and physical behavior similar to
the different class of antiretroviral drugs. Relative error, in intra-
day and interday precision (Table 2) demonstrate acceptable the
accuracy and precision of our procedure. Absence of matrix effects
at the retention times of the analytes of interest allowed an accu-
rate measurement of MVC plasma concentrations, even in samples
frompatients administered with several concomitant drugs.
5. Conclusion
This method has been developed and validated following FDA
andEMA guidelines [32,33] andaccording to ENUNI ISO9001:2008
certication planning rules of our laboratory [52,53]. TDMof MVC
could be a useful tool for clinicians to verify correct plasma expo-
sure in drug-drug interactions or special populations. This method,
based on a simple precipitation extraction and HPLC-MS instru-
mentation, for quantify plasma concentration of MVC is accurate
and reproducible and can be used in a wide range of PK, clinical
studies and routinely in HIV infected patients.
Conicts of interest
The authors disclose no conicts.
Funding
This study was not supported.
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