Pharmacokinetic Methods
Samples were periodically obtained from patients enrolled in both arms of the study to
monitor the steady-state minimum concentration of hydroxychloroquine (HCQ) in whole
blood and plasma. Patients were instructed to take the HCQ dosage at approximately the
same time every day, at a time in the morning that would allow them to arrive at the
outpatient clinic for collection of a predose sample. Blood specimens were obtained
before initiating treatment, at the beginning of weeks 2, 3, and 4 of cycle 1, and once a
month during regularly scheduled visits to the clinic for study evaluations as long as the
patient was receiving treatment. Sampling was also performed during cycle 1 to define
the plasma concentration-time profile of erlotinib over a single 24-h dosing interval after
steady-state conditions had been achieved when given alone and together with HCQ.
Blood specimens were obtained before initiating treatment, immediately before taking the
seventh daily dose of single agent erlotinib, at 0.5, 1, 2, 3, 4, 6 and 8-h after dosing, and
before dosing on the following day. Sampling was also performed according to this same
schedule after six days of combined treatment with erlotinib and HCQ.
Blood samples (6-mL) were drawn from a peripheral arm vein into collection tubes
containing sodium heparin and placed in crushed ice until processed for storage within 15
min. Actual sample collection times were recorded. For the HCQ pharmacokinetic
samples, 2-mL of whole blood was transferred from the collection tube, after inverting it
several times to disperse the blood cells, directly into a polypropylene cryotube. The
remaining blood was centrifuged (1,100-1,300 g, 4°C, 10 min) to afford plasma which
was transferred into a cryotube. Plasma was similarly harvested from the erlotinib
samples. The blood and plasma samples were stored at -70ºC until assayed.
HCQ in whole blood and plasma was determined by liquid chromatography/mass
spectrometry. Study samples were thawed at room temperature and mixed by vortexing
before removing a 100-µL aliquot for analysis. After spiking with 5-µL of internal
standard (IS) working solution (10-µg/mL chloroquine in acetonitrile), the sample was
vigorously mixed with 300-µL of acetonitrile, then centrifuged (10,000-g, 5-min) to
pellet the lysed cellular fragments and precipitated protein. The supernatant (250-µL)
was combined with 200-mM triethylamine/272 mM-formic acid in water (25-µL) and
100-µL of this solution was injected onto a 150 x 4.6-mm Luna 5-µm CN HPLC column
(Phenomenex, Torrance, CA) eluted with a binary mobile phase composed of 30%
acetonitrile and 70% 10-mM triethylamine/13.6-mM formic acid in water (pH 4.0)
delivered at 1.0-mL/min. An Agilent 1100 Series LC/MSD system with an electrospray
ionization interface (Agilent Technologies, Palo Alto, CA) was used for detection.
Nitrogen was used as the nebulizing gas (60-p.s.i.) and as the drying gas (10-L/min,
350°C). The single-quadrupole mass spectrometer was operated in the selected-ion
monitoring mode to measure positive ions corresponding to [M+H]+ ions of HCQ and the
IS at m/z 336.2 and 320.2, respectively. Additional operating parameters were: capillary
voltage, 1,500-V; fragmentor voltage, 150-V; peak width, 0.1-min; dwell time, 114-msec.
Extracted ion chromatograms were integrated to provide peak areas.
Erlotinib in plasma was determined by liquid chromatography-tandem mass
spectrometry, as previously reported, with minor modifications (1). Plasma (100-µL)
was spiked with 5-µL of IS working solution (2.4-µg/mL N-ethylerlotinib in acetonitrile)
and vigorously mixed with 300-µL of acetonitrile. The mixture was centrifuged (10,000g, 5-min) and the resulting supernantant (100-µL) was diluted with an equivalent volume
of 0.1% (v/v) formic acid in water. The final sample solution (10-µL) was injected onto
a 150 x 4.6-mm Luna 5-µm C18(2) HPLC column (Phenomenex, Torrance, CA) eluted
with a mobile phase composed of methanol/0.1% (v/v) formic acid in water (47:53, v/v)
at 1.0-mL/min. Upon completing the run at 6.0-min, the column was flushed with
methanol/0.1% (v/v) formic acid in water (95:5, v/v) for 5-min and reequilibrated with
the original composition for 3-min. An Agilent 1100 series XCT ion trap mass
spectrometer with an atmospheric pressure ionization-electrospray interface (Agilent
Technologies, Palo Alto, CA) was used for detection. Operating parameters were
established to individually maximize the response for the most abundant product ions
resulting from isolation and fragmentation of the [M+H]+ ions for the IS (m/z 422.2) and
erlotinib (m/z 394.2), which were monitored in separate time segments of 2.0-4.0-min and
4.0-6.0-min, respectively. Nitrogen was used as the nebulizing gas (50-psi) and drying
gas (12-L/min, 350°C). Time programmed parameters for ion transport and focusing
were as follows (2.0-4.0 min/4.0-6.0 min): capillary potential (V), -1,598/-2,366;
skimmer (V), 16.4/19.2; capillary exit offset (V), 50.0/50.0; octopole 1 DC (V),
13.4/16.1; octopole 2 DC (V), 2.5/2.8; trap drive (rel %), 51.3/52.1; octopole rf (Vpp),
181.2/193.4; lens 1 (V), -6.7/-3.5; lens 2 (V), -100.0/-67.5. Ion Charge Control was used
with a 250-ms maximum accumulation time and maximum abundance of 200,000 ions.
Helium was used as the collision gas to fragment the parent ions with an amplitude of
1.02-V and m/z 155.0 cutoff for the IS and an amplitude of 1.00-V and m/z 140.9 cutoff
for erlotinib. Product ions were monitored by scanning from m/z 250-450 with data
averaging over every 5 scans. Extracted ion chromatograms for the m/z 422.2→364.2
and m/z 394.2→336.1 transitions for the IS and erlotinib, respectively, were integrated to
provide peak areas.
The analytical methods were validated according to current recommendations (2). Peaks
that interfered with detection of either drug or IS were not evident in chromatograms of
pretreatment plasma or blood samples obtained from patients participating in this clinical
trial. Calibration standards of HCQ in human donor plasma or whole blood and erlotinib
in plasma had concentrations ranging from 10-1,000 ng/mL. Study samples were
independently assayed in duplicate, on different days, together with a set calibration
standards, drug-free plasma or blood prepared for analysis with and without addition of
the IS, and quality control samples. In each case, the calibration curves were best
described by the equation, y = a + bxc, where y is the analyte/IS peak area ratio and x is
the known analyte concentration in each calibration standard. Nonlinear regression was
performed using WinNonlin Professional version 5.0 software (Pharsight Corp., Cary,
NC), with weighting in proportion to the reciprocal of the drug concentration, normalized
to the number of calibration standards, to determine the y-intercept (a), coefficient (b)
and exponent (c), of the best-fit curve. Values of the parameters describing the best-fit
line were used to calculate the analyte concentration in study samples. Specimens with
concentrations exceeding the upper range of the standard curve were reassayed upon
appropriate dilution with drug-free human plasma or whole blood. The average of the
two initial determinations of each study sample was calculated. Samples were reassayed
in cases where the individual determinations differed from their average by more than
15%. During the analysis of samples from this study, the calibration curves exhibited
correlation coefficients ranging from 0.995 to 1.000 and the interday accuracy was within
±11.1% of the known concentration of the calibration standards and quality control
solutions with a precision ≤10.5%.
Actual sample times were calculated relative to the time that the prior dose of either drug
was taken. Observations were excluded for samples collected after the daily dose had
already been taken, if the sample was not collected within 24 ± 2 h of taking the prior
dose, or determined to be an outlier by application of Dixon's test. Cminss was not
calculated if there were fewer than three acceptable determinations.
References
1. Zhao M, He P, Rudek MA, Hidalgo M, Baker SD. Specific method for determination
of OSI-774 and its metabolite OSI-420 in human plasma by using liquid
chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed
Life Sci. 2003 Aug 15;793(2):413-20.
2. Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ, et al.
Bioanalytical method validation--a revisit with a decade of progress. Pharm Res. 2000
Dec;17(12):1551-7.