Journal of Chromatography B, 972 (2014) 117–123
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of cobimetinib in human plasma using protein precipitation extraction and high-performance liquid chromatography coupled to mass spectrometry Yuzhong Deng a,∗ , Luna Musib b , Edna Choo a , Matthew Chapple c , Sarah Burke c , James Johnson c , Steve Eppler b , Brian Dean a a
Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, CA 94080, United States Clinical Pharmacology, Genentech, South San Francisco, CA 94080, United States c Quintiles BioSciences, Ithaca, NY 14850, United States b
a r t i c l e
i n f o
Article history: Received 7 May 2014 Accepted 23 September 2014 Available online 7 October 2014 Keywords: LC–MS/MS Cobimetinib GDC-0973 Validation
a b s t r a c t Inhibition of MAP/ERK kinase (MEK) is a promising strategy to control the growth of tumors that are dependent on aberrant signaling in the MEK pathway. Cobimetinib (GDC-0973) (S)-[3,4-Difluoro-2(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone) inhibits proliferation of a variety of human tumor cell lines by inhibiting MEK1 and MEK2. A specific high performance liquid chromatography-mass spectrometric assay was developed and validated for the determination of cobimetinib in human plasma. The overall mean recovery using protein precipitation extraction with acetonitrile was found to be 54.1%. The calibration curve was ranged from 0.20 to 100 ng/mL. The LLOQ was sensitive enough to detect terminal phase concentrations of the drug. The intra- and inter-assay precision (%CV) was within 10.3% and 9.5% for cobimetinib. The assay accuracy (%RE) was within ±13.7% of the nominal concentration values for cobimetinib with the normal analytical QCs. The developed assay was successfully used to analyze the human plasma samples (for pharmacokinetic analysis) from clinical trials. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a worldwide clinical and economic problem. Conventional approaches to treating cancer include surgery, radiotherapy, and cytotoxic chemotherapy as single modalities or as combined therapies. Designing anticancer agents that selectively target molecules that are unique, mutated, or overexpressed in cancer cells is an approach made possible by recent advances in molecular and genomic methodologies. The MAPK signaling cascade transduces multiple proliferative and differentiating signals within tumor cells. Four major mammalian MAPK pathway modules have been identified: extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), p38 kinase, and ERK5 [1,2]. Each pathway responds to different extracellular signals that stimulate an intracellular pathway activator. Aberrant regulation of the MAPK pathway contributes to cancer cell survival including uncontrolled proliferation, invasion,
∗ Corresponding author. Tel.: +1 650 225 6239; fax: +1 650 467 3487. E-mail address: [email protected] (Y. Deng). http://dx.doi.org/10.1016/j.jchromb.2014.09.034 1570-0232/© 2014 Elsevier B.V. All rights reserved.
metastasis angiogenesis and evasion of apoptosis. Cobimetinib [3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone) (Fig. 1A) is an investigational drug which inhibits MEK1/2, a component of the mitogen-activated protein kinase (MAPK) pathway and therefore interferes with cellular processes that modulate tumor cell growth. In vivo xenograft efficacy is observed in BRAF and KRAS mutant cell lines both as a single agent cobimetinib and in combination with GDC-0941 [3–5]. Cobimetinib is currently being tested in multiple combinations, including a phase 3 clinical trial in combination with vemurafenib, in patients with metastatic melanoma (clinicaltrials.gov). chromatography–tandem mass spectrometry Liquid (LC–MS/MS) using triple-quadrupole mass spectrometers with an atmospheric pressure ionization (API) source and operated under selected reaction monitoring (SRM) mode has been used as an enabling technology for quantitative bioanalysis in drug development. A fast gradient using a short, narrow-bore reversed-phase column and a relative high flow rate has become the preferred choice for bioanalysis, and total cycle time has been shortened to less than 3 to 5 min while maintaining chromatographic
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Fig. 1. MS1 (A and C) and product ion scan (B and D) mass spectrometry spectra of cobimetinib and 13 C6 -cobimetinib and their chemical structures.
resolution [6]. In this manuscript, we report a validated LC–MS/MS method for the quantification of cobimetinib in human plasma samples to support the clinical trial of cobimetinib. The method validation process was fully compliant with regulatory guidance under Good Laboratory Practices [7]. This method utilized a stable-isotope labeled 13 C6 -cobimetinib as internal standard and protein precipitation extraction (PPE) to clean up plasma samples, which ensured good assay accuracy, precision and reproducibility. 2. Experimental 2.1. Materials and reagents Test compound cobimetinib (HCl salt, potency: 92%) was provided by Genentech (South San Francisco, CA, USA). The internal standards, 13 C6 -cobimetinib (HPLC purity: 97%) was provided by Ricerca Biosciences, LLC. (Concord, OH, USA) (Fig. 1B). Acetonitrile (ACN), methanol (MeOH), 0.1% formic acid in water and 0.1% formic acid in acetonitrile were purchased from Honeywell Burdick & Jackson (Morristown, NJ, USA). Formic acid was purchased from EM Science (Gibbstown, NJ, USA). Deionized water, purified
with a Milli-Q water purification system purchased from Millipore (Bedford, MA, USA), was generated in Quintiles BioSciences, Inc. (Ithaca, NY, USA). Human plasma with K2 EDTA anticoagulant, was purchased from Bioreclamation, Inc. (Hicksville, NY, USA) and Biological Specialty Corp. 2.2. Sample preparation for LC–MS/MS analysis Cobimetinib stock solutions for standard (STD) and quality control (QC), at nominal concentration of 1.0 mg/mL, were prepared in methanol. A cobimetinib standard working solution at 10,000 ng/mL was prepared by appropriate dilution of the 1.0 mg/mL stock solution with methanol. Internal standard stock solution of 13 C6 -cobimetinib was also prepared at 1 mg/mL in methanol. The 13 C6 -cobimetinib working solution at 15 ng/mL was prepared by appropriate dilution of the 1.0 mg/mL stock solution with methanol. The human plasma standards at nominal concentrations of 0.2, 0.4, 2.0, 5.0, 10, 25, 50, 75, 90 and 100 ng/mL were prepared by appropriate dilution of the 10,000 ng/mL standard working solution with control human plasma matrix. QC samples, from separate weighings, at nominal concentrations of 0.2, 0.6, 50, 80 and
Y. Deng et al. / J. Chromatogr. B 972 (2014) 117–123 Table 1 HPLC conditions for cobimetinib in the human plasma assay. Time (min)
Function
Value
0 0.9 1 2 2.01 2.01 2.5 2.5 2.8 2.8 3.01
B Conc. (%) B Conc. (%) B Conc. (%) B Conc. (%) Total flow B Conc. (%) B Conc. (%) Total flow Total flow B Conc. (%) Stop
45 55 65 65 0.8 (mL/min) 95 95 0.8 (mL/min) 0.5 (mL/min) 45 Not applicable
2000 ng/mL were prepared by appropriate dilution of the individual stock solutions or combined standard working solution. Aliquots of 50 L of plasma samples, STD and QC were transferred to 1.4 mL extraction tubes and then an equal volume of internal standard was added to each tube. After vortexing, 500 L of acetonitrile was added to each tube. The samples were vortexed for 1 min and centrifuged at 3200 rpm for 5 min. Supernatant, 450 L, was transferred to a clean 96-well plate and evaporated to dryness at 45 ◦ C. The dried extract was reconstituted with 200 L of 50:50 acetonitrile/water and 15 L was injected into the LC–MS/MS system for sample analysis. 2.3. LC–MS/MS instrumentation Analyst®
LC–MS/MS data were acquired by software (version 1.4) on a Sciex API 4000 tandem mass spectrometer (AB Sciex, Foster City, CA) in positive electrospray ionization (ESI) mode for Cobimetinib. The separation was achieved on a Phenomenex Luna PFP column (2 × 50 mm, 3 m) with a 0.5 m × 2 mm in-line frit in a column oven at 45 ◦ C with gradient elution using mobile phases of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Two Shimadzu (Tokyo, Japan) LC-10AD pumps and a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, NC, USA) were used as the delivery pumps and autosampler, respectively. The initial LC flow rate was 0.5 mL/min and the gradient condition is listed in Table 1. For quantitation, the mass spectrometer was operated in the positive selected reaction monitoring mode (SRM) for Cobimetinib. The following SRM transitions were monitored: m/z 532.1 → 249.1 for cobimetinib, m/z 538.1 → 255.1 for 13 C -cobimetinib. The dwell times were 200 ms for cobimetinib 6 and 100 ms for 13 C6 -cobimetinib, respectively. The ionspray voltages were + 4750 V, the declustering potential was 74 V and the collision energy was 53 eV for Cobimetinib. The retention time for cobimetinib and 13 C6 -cobimetinib was 0.6 min.
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blood samples were obtained from each subject and plasma harvested for the determination of cobimetinib plasma concentration over time. Individual subject PK parameter values for cobimetinib plasma concentration-time data were determined using noncompartmental methods (WinNonlin version 5.2.1, Pharsight Corp., Mountain View, CA). All AUC values were calculated using the linear trapezoidal method when the concentrations were rising and the logarithmic trapezoidal method when the concentrations were declining (Linear up/Log Down rule in WinNonlin® ). Concentrations below the limit of quantitation (BLQ) values were considered as zero for PK analysis. Actual blood collection time was used to calculate PK parameters. PK parameters were reported as their means ± standard deviations (SD). 3. Results and discussion 3.1. Mass spectrometry MS1 scan and product ion mass spectra of cobimetinib and are presented in Fig. 1. Based on the observed ion fragmentation, the ion transitions of m/z 532.1 → 249.1 and m/z 538.1 → 255.1 were utilized for monitoring cobimetinib and 13 C -cobimetinib, respectively. 6 13 C -cobimetinib 6
3.2. Selectivity Multiple lots of matrix were assessed to determine if any additional variability found during this method validation could be attributed to the analysis of multiple lots of matrix. Plasma inter-lot chromatographic selectivity was evaluated by preparing control blanks and zero samples (control blank plus internal standard) from six lots of human control plasma and comparing the peak area ratios (PAR) of each zero sample to the mean PAR of the LLOQ calibration standard samples analyzed within the same run. The results from the plasma inter-lot chromatographic
2.4. Pharmacokinetic assessments of cobimetinib in healthy volunteers The pharmacokinetics of cobimetinib were assessed in a single dose, randomized, cross-over absolute bioavailability study in 12 healthy subjects. The protocols were conducted in accordance with the guidelines on Good Clinical Practices and with ethical standards for human experimentation established by the Declaration of Helsinki. Each subject provided written informed consent before initiation of study procedures. Subjects received a 2 mg dose of cobimetinib administered as intravenous (IV) infusion (0.1 mg/mL in D5W) over approximately 30 min in the first study period and four 5 mg cobimetinib capsules with 240 mL of room temperature water in the second study period after at least an 8 h overnight fast. A minimum of 10 days separated the treatment periods. After drug administration, during an 8-day sampling period, fourteen serial
Fig. 2. Selected reaction monitoring chromatograms of cobimetinib (A) and its internal standard, 13 C6 -cobimetinib (B) in K2 EDTA control blank human plasma.
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Table 2 Extraction recovery of cobimetinib from human plasma. Peak area ratio (Cobimetinit/IS)
LQC 0.6 ng/mL
Mean CV (%) MQC 50 ng/mL
Mean CV (%) HQC 80 ng/mL
Mean CV (%)
Pre-extract
Post-extract
0.037063 0.035695 0.036764 0.035425 0.035667 0.036523 0.03619 1.9
0.075223 0.073398 0.072976 0.073909 0.071975 0.072492 0.073329 1.6
2.724512 2.780922 2.829437 2.784637 2.76756 2.823851 2.785153 1.4
4.614785 4.518034 4.616525 4.738739 4.646028 4.565513 4.616604 1.6
4.551582 4.620164 4.547155 4.696027 4.501753 4.538161 4.575807 1.5
8.670785 8.686135 8.828592 8.674721 8.594046 8.62756 8.680307 0.9
Overall Mean
Recovery (%)
validation of cobimetinib in human plasma over the range of 0.20 to 100 ng/mL. The correlation coefficients from five independent curves were ≥0.99 for cobimetinib.
3.5. Lower limit of quantification and carryover assessment
49.4
60.3
The lower limit of quantification (LLOQ) of cobimetinib was determined to be 0.2 ng/mL by extracting and analyzing six replicates of LLOQ QC samples in the five accuracy and precision (A&P) runs. The representative chromatograms of cobimetinib at LLOQ (0.2 ng/mL) and IS, 13 C6 -cobimetinib at 15 ng/mL are shown in Fig. 3. The signal to noise ratio of the LLOQ QC was greater than 7, which was sufficient for the reliable detection of cobimetinib in the human plasma samples. Carryover was evaluated during the validation run by injecting at least one carryover blank sample after the highest STD. The analyte response from the blank sample was found to be lower than 20% of the LLOQ response.
3.6. Accuracy and precision 52.7 54.1
CV = (SD/Mean) × 100. %Recovery = (Mean pre-extract/Mean post-extract) × 100.
selectivity experiment for cobimetinib from six different lots of human plasma with K2 EDTA showed no response for cobimetinib in the control plasma. The selectivity of the method was also evaluated during each validation run by LC–MS/MS to monitor for possible interfering peaks at the same chromatographic retention time as the analyte and its internal standard. No significant chromatographic interferences were detected at the retention time of the analyte or its internal standard (Fig. 2). The assay selectivity was also tested with its metabolite, EXEL-0382 and co-administered compounds in the clinical trials, such as GDC-0941, GDC-0068 and vemurafenib, by spiking these compounds into the low and high QCs of cobimetinib and analyzed by the assay. The results showed no interference of these compounds with the cobimetinib analysis.
The accuracy and precision of the method was assessed using six replicates of each LLOQ QC, low quality control sample (LQC), medium quality control sample (MQC), high quality control sample (HQC) and diluted quality control sample (Dil QC) over five separate A&P runs for each analyte. The accuracy and precision information, based on the five QC levels, are listed on Table 3. The repeatability (within-run precision) and intermediate precision (between-run precision) calculated using ANOVA method was within 5.7% and 9.04% for cobimetinib, respectively. The assay accuracy (%RE) was within ±13.7% of the nominal concentration values for cobimetinib with the normal analytical LQC, MQC and HQC and within ±16.7% of the nominal concentration values for cobimetinib with the LLOQ QC.
3.3. Extraction recovery The extraction recovery of cobimetinib from human plasma was determined by comparing the PAR for cobimetinib (0.6, 50, and 80 ng/mL)/13 C6 -cobimetinib (IS) (15 ng/mL) in samples spiked with cobimetinib after extraction (post-extract) with the PAR of samples spiked with cobimetinib before extraction (pre-extract). In both of the post-extract and pre-extract samples, 13 C6 -cobimetinib was spiked after extraction. The extraction recovery (%recovery) was determined by dividing the mean pre-extract values by the mean post-extract values and expressing the result as a percentage. The CV of mean PAR at each concentration level was also calculated. Six replicates were used at each concentration. The average extraction recoveries of cobimetinib at low, medium and high QCs were 54.1% (Table 2). 3.4. Linearity Calibration curves were determined by plotting the concentration versus analyte-to-IS peak area ratio. Quadratic regression with 1/x2 weighting was determined to provide the best curve fit for the
Fig. 3. Selected reaction monitoring chromatograms of cobimetinib (A) and its internal standard, 13 C6 -cobimetinib (B) from the LLOQ sample in human K2 EDTA plasma (0.2 ng/mL).
Y. Deng et al. / J. Chromatogr. B 972 (2014) 117–123 Table 3 Precision and accuracy of cobimetinib in human plasma for QC samples from five validation runs.
Table 4 Matrix effect test results of cobimetinib in human plasma. Matrix lot
Cobimetinib concentration (ng/mL)
Run 1
Mean CV (%) RE (%) Run 2
Mean CV (%) RE (%) Run 3
Mean CV (%) RE (%) Run 4
Mean CV (%) RE (%) Run 5
Mean CV (%) RE (%) Overall Mean Between run precision CV (%) Within run precision CV (%) Overall CV (%) Overall RE (%)
LLOQ QC
LQC
MQC
HQC
0.2 0.200 0.193 0.207 0.184 0.186 0.194 0.194 4.4 −3
0.6 0.590 0.607 0.581 0.583 0.588 0.570 0.587 2.1 −2.3
50 50.0 49.0 48.8 48.6 49.3 50.2 49.3 1.3 −1.4
80 78.9 79.2 79.1 77.5 78.4 77.3 78.4 1.1 −2
Dil QC (25-fold) 2000 2150 2160 2070 2140 2020 2310 2140 4.6 7.1
0.196 0.206 0.187 0.209 0.200 0.193 0.199 4.1 −0.8
0.527 0.535 0.530 0.502 0.518 0.496 0.518 3.1 −13.7
46.2 46.5 45.0 45.2 47.1 46.9 46.2 1.9 −7.7
73.1 74.1 73.9 77.2 74.0 75.7 74.7 2 −6.7
1900 1910 1910 1920 1980 1960 1930 1.7 −3.5
0.222 0.242 0.237 0.228 0.235 0.236 0.233 3.1 16.7
0.653 0.697 0.658 0.636 0.663 0.644 0.659 3.2 9.8
53.5 54.4 54.2 53.7 52.9 53.8 53.8 1 7.5
84.8 83.7 83.2 84.3 84.7 84.7 84.2 0.8 5.3
2230 2300 2260 2280 2270 2260 2270 1 13.3
0.173 0.210 0.197 0.186 0.179 0.177 0.187 7.5 −6.5
0.505 0.566 0.582 0.601 0.562 0.575 0.565 5.8 −5.8
47.5 48.0 46.9 47.2 47.6 44.8 47 2.4 −6
79.5 75.0 76.4 74.4 76.8 76.0 76.4 2.3 −4.6
2070 2010 2020 2060 2010 2030 2030 1.3 1.7
0.189 0.199 0.185 0.199 * 1.35 0.196 0.194 3.3 −3.2
0.553 0.576 0.547 0.567 0.550 0.703 0.583 10.3 −2.9
44.7 46.0 45.2 47.0 46.4 46.1 45.9 1.8 −8.2
69.5 69.0 68.1 70.4 72.5 72.6 70.4 2.6 −12.1
2040 2010 2060 1950 1890 1950 1980 3.2 −0.8
0.202 9.04
0.582 8.38
48.4 6.71
76.8 6.6
2070 6.39
4.65
5.7
1.72
1.84
2.74
9.4 0.8
9.5 −3
6.3 −3.2
6.3 −4
6.4 3.6
CV = (SD/mean) × 100; RE = [(mean − nominal)/nominal] × 100. * Excluded from statistics; possible contamination.
3.7. Matrix effect The matrix influence on the ionization of cobimetinib was determined by extracting blank control plasma from six individual lots. Post-extraction, the analytes (0.6, 50, and 80 ng/mL) and its internal standard (15 ng/mL) are added to the extracted blank samples (matrix present). Additionally, the analyte and its internal standard at those concentrations listed were added to empty wells of the injection block (matrix absent). The samples were then analyzed by the validated method. The IS normalized matrix
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LQC 0.6 ng/mL
PAR (Cobimetinib/IS) Matrix absent (neat spike)
1 2 3 4 5 6
0.077450 0.075949 0.078004 0.077050 0.079822 0.077164
0.079949 0.081583 0.080485 0.078823 0.077335 0.077946 0.079354
0.98 0.96 0.98 0.97 1.01 0.97 0.98 1.66
1 2 3 4 5 6
4.751258 4.872861 4.838011 4.742248 4.794225 4.704802
4.837975 4.764476 4.809272 4.925102 4.823232 4.819331 4.829898
0.98 1.01 1.00 0.98 0.99 0.97 0.99 1.32
1 2 3 4 5 6
8.833995 8.719925 8.773085 8.779580 8.652599 8.807475
8.703801 8.712858 8.745092 8.763914 8.773859 8.814204 8.752288
1.01 1.00 1.00 1.00 0.99 1.01 1.00 0.75
Mean CV (%) MQC 50 ng/mL
Mean CV (%) HQC 80 ng/mL
Matrix factor
Matrix present (post-extract)
Mean CV (%) Overall mean matrix factor
0.99
CV = (SD/mean) × 100. Normalized matrix factor = (peak area ratio in presence of matrix)/(mean peak area ratio in absence of matrix).
factor was calculated as the PAR (analyte/IS) for the analyte in each individual sample with matrix ions present to the mean PAR (analyte/IS) for the analyte in samples (n = 6 at each concentration) with matrix ions absent. The IS normalized matrix factor results are listed in Table 4. The %CV at each concentration level was ≤15%, indicating that there is acceptable variability in matrix effects among six lots of control human plasma. In addition, the matrix effect was also evaluated by comparing the peak area of cobimetinib from the post-extract and neat spike. The results (not shown) show the matrix factor is close to 1.0. 3.8. Stability The stock and working solution stability, freeze–thaw stability, bench-top stability, room temperature stability, long-term storage stability (−70 ◦ C), reinjection reproducibility and whole blood stability were assessed in this validation and the results are summarized in Table 5. Cobimetinib was shown to be stable in human plasma after five freeze–thaw cycles and stable on the bench-top at room temperature for 24 h prior to extraction. Cobimetinib was also shown to be stable in human plasma after storage between −60 ◦ C and −80 ◦ C for 400 days. The reinjection reproducibility test showed an acceptable run could be repeated after 103 h storage at room temperature. The whole blood stability test showed that cobimetinib was stable for at least 1 h at room temperature and in an ice bath. Both stock and working solutions of cobimetinib were stable at room temperature for at least 24 h when compared to solutions stored at 4 ◦ C. 3.9. Dilution capability In order to measure the concentration of cobimetinib above the calibration curve range, a 25-fold dilution was evaluated in this validation. Dilution QC was made at the 2000 ng/mL level.
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Table 5 Stability assessments for cobimetinib. Nominal conc. (ng/mL)
Cobimetinib Stability after 5 freeze–thaw cycles (at −70 ◦ C)
0.6 80
Mean observed Conc. Accuracy (%RE)
CV (%)
(ng/mL, n = 4)
(n = 4)
(n = 4)
0.575 77.1
−4.3 −3.7
1.4 0.4
Bench-top stability at room temperature for 24 h
0.6 80
Mean observed Conc. Accuracy (%)
CV (%)
(ng/mL, n = 4)
(n = 4)
(n = 4)
0.583 71.6
−2.9 −10.6
3 0.9
Storage at −60 to −80 ◦ C for 400 days
0.6 80
Mean observed Conc. Accuracy (%RE)
CV (%)
(ng/mL, n = 6)
(n = 6)
(n = 6)
0.531 72
−11.6 −10.1
2.3 1.3
Injection repordocibility at room temperature for 103 h
0.6 50 80
Mean observed Conc. Accuracy (%)
CV (%)
(ng/mL, n = 6)
(n = 6)
(n = 6)
0.531 49.2 79.4
−11.5 −1.6 −0.7
4.7 1.5 4.2
Fig. 4. Selected reaction monitoring chromatograms of cobimetinib (A) and its internal standard, 13 C6 -cobimetinib (B) from one of the patient samples in human K2 EDTA plasma.
Whole blood stability (ng/mL, n = 6)
Mean CV (%) %Dev from baseline
Baseline
1 h (room temp) 1 h (ice bath)
54.2 7.9
49.2 3.7 −9.19
55.3 1.7 2
Stock solution (1 mg/mL) at RT for 24 h (PAR (analyte/IS), n = 6) Stored at 4 ◦ C Mean 0.923 CV (%) 1.67 %Dev from stored at 4 ◦ C
Stored at RT 0.974 0.73 5.6
Working solution (10 g/mL) at RT for 24 h (PAR (analyte/IS), n = 6) Stored at RT Stored at 4 ◦ C Mean 8.476 5.5 CV (%) ◦ %Dev from stored at 4 C
7.96 2.1 −6.1 Fig. 5. Mean ± SD cobimetinib plasma concentration versus time profiles after a single 20 mg oral dose and 2 mg IV dose.
The accuracy (%RE) of the dilution QCs was within ±13.3% for cobimetinib (Table 3).
t1/2 = 59.5 (32.5) h. The 2 mg iv infusion results of this study and the metabolism information were reported previously [8,9].
3.10. Pharmacokinetics Validation of the LC–MS/MS assay was performed over the calibration curve range 0.20–100 ng/mL, which was sufficient to detect cobimetinib human plasma concentration throughout the course of the 8-day study in healthy human volunteers (n = 12). Fig. 4 showed the selected reaction monitoring chromatograms from one of the patient samples and Fig. 5 showed the mean cobimetinib human plasma concentrations versus time profile from the 12 subjects that were administered a single 20 mg oral dose. Cobimetinib plasma concentration of the 20 mg oral dose ranged from 0.253 to 24.4 ng/mL, with Cmax ranging from 6.5 to 24.4 ng/mL. Mean (SD) pharmacokinetic parameters for the twelve subjects were: Cmax (%CV) = 13.8 (35.6) ng/mL, AUC0-inf = 716 (38.0) ng/mL h, and
4. Conclusions A LC–MS/MS assay for the analysis of cobimetinib in human plasma has been developed and validated in the present study. This validated assay was proven to be rugged, accurate and sensitive and it has been utilized for the analysis of human plasma samples generated from multiple clinical studies. Acknowledgement The authors would like to thank the Medicinal Chemistry Groups in Genentech, Inc. for providing compounds for the validation.
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of cobimetinib in human plasma using protein precipitation extraction and high-performance liquid chromatography coupled to mass spectrometry Yuzhong Deng a,∗ , Luna Musib b , Edna Choo a , Matthew Chapple c , Sarah Burke c , James Johnson c , Steve Eppler b , Brian Dean a a
Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, CA 94080, United States Clinical Pharmacology, Genentech, South San Francisco, CA 94080, United States c Quintiles BioSciences, Ithaca, NY 14850, United States b
a r t i c l e
i n f o
Article history: Received 7 May 2014 Accepted 23 September 2014 Available online 7 October 2014 Keywords: LC–MS/MS Cobimetinib GDC-0973 Validation
a b s t r a c t Inhibition of MAP/ERK kinase (MEK) is a promising strategy to control the growth of tumors that are dependent on aberrant signaling in the MEK pathway. Cobimetinib (GDC-0973) (S)-[3,4-Difluoro-2(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone) inhibits proliferation of a variety of human tumor cell lines by inhibiting MEK1 and MEK2. A specific high performance liquid chromatography-mass spectrometric assay was developed and validated for the determination of cobimetinib in human plasma. The overall mean recovery using protein precipitation extraction with acetonitrile was found to be 54.1%. The calibration curve was ranged from 0.20 to 100 ng/mL. The LLOQ was sensitive enough to detect terminal phase concentrations of the drug. The intra- and inter-assay precision (%CV) was within 10.3% and 9.5% for cobimetinib. The assay accuracy (%RE) was within ±13.7% of the nominal concentration values for cobimetinib with the normal analytical QCs. The developed assay was successfully used to analyze the human plasma samples (for pharmacokinetic analysis) from clinical trials. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a worldwide clinical and economic problem. Conventional approaches to treating cancer include surgery, radiotherapy, and cytotoxic chemotherapy as single modalities or as combined therapies. Designing anticancer agents that selectively target molecules that are unique, mutated, or overexpressed in cancer cells is an approach made possible by recent advances in molecular and genomic methodologies. The MAPK signaling cascade transduces multiple proliferative and differentiating signals within tumor cells. Four major mammalian MAPK pathway modules have been identified: extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), p38 kinase, and ERK5 [1,2]. Each pathway responds to different extracellular signals that stimulate an intracellular pathway activator. Aberrant regulation of the MAPK pathway contributes to cancer cell survival including uncontrolled proliferation, invasion,
∗ Corresponding author. Tel.: +1 650 225 6239; fax: +1 650 467 3487. E-mail address: [email protected] (Y. Deng). http://dx.doi.org/10.1016/j.jchromb.2014.09.034 1570-0232/© 2014 Elsevier B.V. All rights reserved.
metastasis angiogenesis and evasion of apoptosis. Cobimetinib [3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)3-hydroxy-3-piperidin-2-yl-azetidin-1-yl)-methanone) (Fig. 1A) is an investigational drug which inhibits MEK1/2, a component of the mitogen-activated protein kinase (MAPK) pathway and therefore interferes with cellular processes that modulate tumor cell growth. In vivo xenograft efficacy is observed in BRAF and KRAS mutant cell lines both as a single agent cobimetinib and in combination with GDC-0941 [3–5]. Cobimetinib is currently being tested in multiple combinations, including a phase 3 clinical trial in combination with vemurafenib, in patients with metastatic melanoma (clinicaltrials.gov). chromatography–tandem mass spectrometry Liquid (LC–MS/MS) using triple-quadrupole mass spectrometers with an atmospheric pressure ionization (API) source and operated under selected reaction monitoring (SRM) mode has been used as an enabling technology for quantitative bioanalysis in drug development. A fast gradient using a short, narrow-bore reversed-phase column and a relative high flow rate has become the preferred choice for bioanalysis, and total cycle time has been shortened to less than 3 to 5 min while maintaining chromatographic
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Fig. 1. MS1 (A and C) and product ion scan (B and D) mass spectrometry spectra of cobimetinib and 13 C6 -cobimetinib and their chemical structures.
resolution [6]. In this manuscript, we report a validated LC–MS/MS method for the quantification of cobimetinib in human plasma samples to support the clinical trial of cobimetinib. The method validation process was fully compliant with regulatory guidance under Good Laboratory Practices [7]. This method utilized a stable-isotope labeled 13 C6 -cobimetinib as internal standard and protein precipitation extraction (PPE) to clean up plasma samples, which ensured good assay accuracy, precision and reproducibility. 2. Experimental 2.1. Materials and reagents Test compound cobimetinib (HCl salt, potency: 92%) was provided by Genentech (South San Francisco, CA, USA). The internal standards, 13 C6 -cobimetinib (HPLC purity: 97%) was provided by Ricerca Biosciences, LLC. (Concord, OH, USA) (Fig. 1B). Acetonitrile (ACN), methanol (MeOH), 0.1% formic acid in water and 0.1% formic acid in acetonitrile were purchased from Honeywell Burdick & Jackson (Morristown, NJ, USA). Formic acid was purchased from EM Science (Gibbstown, NJ, USA). Deionized water, purified
with a Milli-Q water purification system purchased from Millipore (Bedford, MA, USA), was generated in Quintiles BioSciences, Inc. (Ithaca, NY, USA). Human plasma with K2 EDTA anticoagulant, was purchased from Bioreclamation, Inc. (Hicksville, NY, USA) and Biological Specialty Corp. 2.2. Sample preparation for LC–MS/MS analysis Cobimetinib stock solutions for standard (STD) and quality control (QC), at nominal concentration of 1.0 mg/mL, were prepared in methanol. A cobimetinib standard working solution at 10,000 ng/mL was prepared by appropriate dilution of the 1.0 mg/mL stock solution with methanol. Internal standard stock solution of 13 C6 -cobimetinib was also prepared at 1 mg/mL in methanol. The 13 C6 -cobimetinib working solution at 15 ng/mL was prepared by appropriate dilution of the 1.0 mg/mL stock solution with methanol. The human plasma standards at nominal concentrations of 0.2, 0.4, 2.0, 5.0, 10, 25, 50, 75, 90 and 100 ng/mL were prepared by appropriate dilution of the 10,000 ng/mL standard working solution with control human plasma matrix. QC samples, from separate weighings, at nominal concentrations of 0.2, 0.6, 50, 80 and
Y. Deng et al. / J. Chromatogr. B 972 (2014) 117–123 Table 1 HPLC conditions for cobimetinib in the human plasma assay. Time (min)
Function
Value
0 0.9 1 2 2.01 2.01 2.5 2.5 2.8 2.8 3.01
B Conc. (%) B Conc. (%) B Conc. (%) B Conc. (%) Total flow B Conc. (%) B Conc. (%) Total flow Total flow B Conc. (%) Stop
45 55 65 65 0.8 (mL/min) 95 95 0.8 (mL/min) 0.5 (mL/min) 45 Not applicable
2000 ng/mL were prepared by appropriate dilution of the individual stock solutions or combined standard working solution. Aliquots of 50 L of plasma samples, STD and QC were transferred to 1.4 mL extraction tubes and then an equal volume of internal standard was added to each tube. After vortexing, 500 L of acetonitrile was added to each tube. The samples were vortexed for 1 min and centrifuged at 3200 rpm for 5 min. Supernatant, 450 L, was transferred to a clean 96-well plate and evaporated to dryness at 45 ◦ C. The dried extract was reconstituted with 200 L of 50:50 acetonitrile/water and 15 L was injected into the LC–MS/MS system for sample analysis. 2.3. LC–MS/MS instrumentation Analyst®
LC–MS/MS data were acquired by software (version 1.4) on a Sciex API 4000 tandem mass spectrometer (AB Sciex, Foster City, CA) in positive electrospray ionization (ESI) mode for Cobimetinib. The separation was achieved on a Phenomenex Luna PFP column (2 × 50 mm, 3 m) with a 0.5 m × 2 mm in-line frit in a column oven at 45 ◦ C with gradient elution using mobile phases of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Two Shimadzu (Tokyo, Japan) LC-10AD pumps and a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, NC, USA) were used as the delivery pumps and autosampler, respectively. The initial LC flow rate was 0.5 mL/min and the gradient condition is listed in Table 1. For quantitation, the mass spectrometer was operated in the positive selected reaction monitoring mode (SRM) for Cobimetinib. The following SRM transitions were monitored: m/z 532.1 → 249.1 for cobimetinib, m/z 538.1 → 255.1 for 13 C -cobimetinib. The dwell times were 200 ms for cobimetinib 6 and 100 ms for 13 C6 -cobimetinib, respectively. The ionspray voltages were + 4750 V, the declustering potential was 74 V and the collision energy was 53 eV for Cobimetinib. The retention time for cobimetinib and 13 C6 -cobimetinib was 0.6 min.
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blood samples were obtained from each subject and plasma harvested for the determination of cobimetinib plasma concentration over time. Individual subject PK parameter values for cobimetinib plasma concentration-time data were determined using noncompartmental methods (WinNonlin version 5.2.1, Pharsight Corp., Mountain View, CA). All AUC values were calculated using the linear trapezoidal method when the concentrations were rising and the logarithmic trapezoidal method when the concentrations were declining (Linear up/Log Down rule in WinNonlin® ). Concentrations below the limit of quantitation (BLQ) values were considered as zero for PK analysis. Actual blood collection time was used to calculate PK parameters. PK parameters were reported as their means ± standard deviations (SD). 3. Results and discussion 3.1. Mass spectrometry MS1 scan and product ion mass spectra of cobimetinib and are presented in Fig. 1. Based on the observed ion fragmentation, the ion transitions of m/z 532.1 → 249.1 and m/z 538.1 → 255.1 were utilized for monitoring cobimetinib and 13 C -cobimetinib, respectively. 6 13 C -cobimetinib 6
3.2. Selectivity Multiple lots of matrix were assessed to determine if any additional variability found during this method validation could be attributed to the analysis of multiple lots of matrix. Plasma inter-lot chromatographic selectivity was evaluated by preparing control blanks and zero samples (control blank plus internal standard) from six lots of human control plasma and comparing the peak area ratios (PAR) of each zero sample to the mean PAR of the LLOQ calibration standard samples analyzed within the same run. The results from the plasma inter-lot chromatographic
2.4. Pharmacokinetic assessments of cobimetinib in healthy volunteers The pharmacokinetics of cobimetinib were assessed in a single dose, randomized, cross-over absolute bioavailability study in 12 healthy subjects. The protocols were conducted in accordance with the guidelines on Good Clinical Practices and with ethical standards for human experimentation established by the Declaration of Helsinki. Each subject provided written informed consent before initiation of study procedures. Subjects received a 2 mg dose of cobimetinib administered as intravenous (IV) infusion (0.1 mg/mL in D5W) over approximately 30 min in the first study period and four 5 mg cobimetinib capsules with 240 mL of room temperature water in the second study period after at least an 8 h overnight fast. A minimum of 10 days separated the treatment periods. After drug administration, during an 8-day sampling period, fourteen serial
Fig. 2. Selected reaction monitoring chromatograms of cobimetinib (A) and its internal standard, 13 C6 -cobimetinib (B) in K2 EDTA control blank human plasma.
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Table 2 Extraction recovery of cobimetinib from human plasma. Peak area ratio (Cobimetinit/IS)
LQC 0.6 ng/mL
Mean CV (%) MQC 50 ng/mL
Mean CV (%) HQC 80 ng/mL
Mean CV (%)
Pre-extract
Post-extract
0.037063 0.035695 0.036764 0.035425 0.035667 0.036523 0.03619 1.9
0.075223 0.073398 0.072976 0.073909 0.071975 0.072492 0.073329 1.6
2.724512 2.780922 2.829437 2.784637 2.76756 2.823851 2.785153 1.4
4.614785 4.518034 4.616525 4.738739 4.646028 4.565513 4.616604 1.6
4.551582 4.620164 4.547155 4.696027 4.501753 4.538161 4.575807 1.5
8.670785 8.686135 8.828592 8.674721 8.594046 8.62756 8.680307 0.9
Overall Mean
Recovery (%)
validation of cobimetinib in human plasma over the range of 0.20 to 100 ng/mL. The correlation coefficients from five independent curves were ≥0.99 for cobimetinib.
3.5. Lower limit of quantification and carryover assessment
49.4
60.3
The lower limit of quantification (LLOQ) of cobimetinib was determined to be 0.2 ng/mL by extracting and analyzing six replicates of LLOQ QC samples in the five accuracy and precision (A&P) runs. The representative chromatograms of cobimetinib at LLOQ (0.2 ng/mL) and IS, 13 C6 -cobimetinib at 15 ng/mL are shown in Fig. 3. The signal to noise ratio of the LLOQ QC was greater than 7, which was sufficient for the reliable detection of cobimetinib in the human plasma samples. Carryover was evaluated during the validation run by injecting at least one carryover blank sample after the highest STD. The analyte response from the blank sample was found to be lower than 20% of the LLOQ response.
3.6. Accuracy and precision 52.7 54.1
CV = (SD/Mean) × 100. %Recovery = (Mean pre-extract/Mean post-extract) × 100.
selectivity experiment for cobimetinib from six different lots of human plasma with K2 EDTA showed no response for cobimetinib in the control plasma. The selectivity of the method was also evaluated during each validation run by LC–MS/MS to monitor for possible interfering peaks at the same chromatographic retention time as the analyte and its internal standard. No significant chromatographic interferences were detected at the retention time of the analyte or its internal standard (Fig. 2). The assay selectivity was also tested with its metabolite, EXEL-0382 and co-administered compounds in the clinical trials, such as GDC-0941, GDC-0068 and vemurafenib, by spiking these compounds into the low and high QCs of cobimetinib and analyzed by the assay. The results showed no interference of these compounds with the cobimetinib analysis.
The accuracy and precision of the method was assessed using six replicates of each LLOQ QC, low quality control sample (LQC), medium quality control sample (MQC), high quality control sample (HQC) and diluted quality control sample (Dil QC) over five separate A&P runs for each analyte. The accuracy and precision information, based on the five QC levels, are listed on Table 3. The repeatability (within-run precision) and intermediate precision (between-run precision) calculated using ANOVA method was within 5.7% and 9.04% for cobimetinib, respectively. The assay accuracy (%RE) was within ±13.7% of the nominal concentration values for cobimetinib with the normal analytical LQC, MQC and HQC and within ±16.7% of the nominal concentration values for cobimetinib with the LLOQ QC.
3.3. Extraction recovery The extraction recovery of cobimetinib from human plasma was determined by comparing the PAR for cobimetinib (0.6, 50, and 80 ng/mL)/13 C6 -cobimetinib (IS) (15 ng/mL) in samples spiked with cobimetinib after extraction (post-extract) with the PAR of samples spiked with cobimetinib before extraction (pre-extract). In both of the post-extract and pre-extract samples, 13 C6 -cobimetinib was spiked after extraction. The extraction recovery (%recovery) was determined by dividing the mean pre-extract values by the mean post-extract values and expressing the result as a percentage. The CV of mean PAR at each concentration level was also calculated. Six replicates were used at each concentration. The average extraction recoveries of cobimetinib at low, medium and high QCs were 54.1% (Table 2). 3.4. Linearity Calibration curves were determined by plotting the concentration versus analyte-to-IS peak area ratio. Quadratic regression with 1/x2 weighting was determined to provide the best curve fit for the
Fig. 3. Selected reaction monitoring chromatograms of cobimetinib (A) and its internal standard, 13 C6 -cobimetinib (B) from the LLOQ sample in human K2 EDTA plasma (0.2 ng/mL).
Y. Deng et al. / J. Chromatogr. B 972 (2014) 117–123 Table 3 Precision and accuracy of cobimetinib in human plasma for QC samples from five validation runs.
Table 4 Matrix effect test results of cobimetinib in human plasma. Matrix lot
Cobimetinib concentration (ng/mL)
Run 1
Mean CV (%) RE (%) Run 2
Mean CV (%) RE (%) Run 3
Mean CV (%) RE (%) Run 4
Mean CV (%) RE (%) Run 5
Mean CV (%) RE (%) Overall Mean Between run precision CV (%) Within run precision CV (%) Overall CV (%) Overall RE (%)
LLOQ QC
LQC
MQC
HQC
0.2 0.200 0.193 0.207 0.184 0.186 0.194 0.194 4.4 −3
0.6 0.590 0.607 0.581 0.583 0.588 0.570 0.587 2.1 −2.3
50 50.0 49.0 48.8 48.6 49.3 50.2 49.3 1.3 −1.4
80 78.9 79.2 79.1 77.5 78.4 77.3 78.4 1.1 −2
Dil QC (25-fold) 2000 2150 2160 2070 2140 2020 2310 2140 4.6 7.1
0.196 0.206 0.187 0.209 0.200 0.193 0.199 4.1 −0.8
0.527 0.535 0.530 0.502 0.518 0.496 0.518 3.1 −13.7
46.2 46.5 45.0 45.2 47.1 46.9 46.2 1.9 −7.7
73.1 74.1 73.9 77.2 74.0 75.7 74.7 2 −6.7
1900 1910 1910 1920 1980 1960 1930 1.7 −3.5
0.222 0.242 0.237 0.228 0.235 0.236 0.233 3.1 16.7
0.653 0.697 0.658 0.636 0.663 0.644 0.659 3.2 9.8
53.5 54.4 54.2 53.7 52.9 53.8 53.8 1 7.5
84.8 83.7 83.2 84.3 84.7 84.7 84.2 0.8 5.3
2230 2300 2260 2280 2270 2260 2270 1 13.3
0.173 0.210 0.197 0.186 0.179 0.177 0.187 7.5 −6.5
0.505 0.566 0.582 0.601 0.562 0.575 0.565 5.8 −5.8
47.5 48.0 46.9 47.2 47.6 44.8 47 2.4 −6
79.5 75.0 76.4 74.4 76.8 76.0 76.4 2.3 −4.6
2070 2010 2020 2060 2010 2030 2030 1.3 1.7
0.189 0.199 0.185 0.199 * 1.35 0.196 0.194 3.3 −3.2
0.553 0.576 0.547 0.567 0.550 0.703 0.583 10.3 −2.9
44.7 46.0 45.2 47.0 46.4 46.1 45.9 1.8 −8.2
69.5 69.0 68.1 70.4 72.5 72.6 70.4 2.6 −12.1
2040 2010 2060 1950 1890 1950 1980 3.2 −0.8
0.202 9.04
0.582 8.38
48.4 6.71
76.8 6.6
2070 6.39
4.65
5.7
1.72
1.84
2.74
9.4 0.8
9.5 −3
6.3 −3.2
6.3 −4
6.4 3.6
CV = (SD/mean) × 100; RE = [(mean − nominal)/nominal] × 100. * Excluded from statistics; possible contamination.
3.7. Matrix effect The matrix influence on the ionization of cobimetinib was determined by extracting blank control plasma from six individual lots. Post-extraction, the analytes (0.6, 50, and 80 ng/mL) and its internal standard (15 ng/mL) are added to the extracted blank samples (matrix present). Additionally, the analyte and its internal standard at those concentrations listed were added to empty wells of the injection block (matrix absent). The samples were then analyzed by the validated method. The IS normalized matrix
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LQC 0.6 ng/mL
PAR (Cobimetinib/IS) Matrix absent (neat spike)
1 2 3 4 5 6
0.077450 0.075949 0.078004 0.077050 0.079822 0.077164
0.079949 0.081583 0.080485 0.078823 0.077335 0.077946 0.079354
0.98 0.96 0.98 0.97 1.01 0.97 0.98 1.66
1 2 3 4 5 6
4.751258 4.872861 4.838011 4.742248 4.794225 4.704802
4.837975 4.764476 4.809272 4.925102 4.823232 4.819331 4.829898
0.98 1.01 1.00 0.98 0.99 0.97 0.99 1.32
1 2 3 4 5 6
8.833995 8.719925 8.773085 8.779580 8.652599 8.807475
8.703801 8.712858 8.745092 8.763914 8.773859 8.814204 8.752288
1.01 1.00 1.00 1.00 0.99 1.01 1.00 0.75
Mean CV (%) MQC 50 ng/mL
Mean CV (%) HQC 80 ng/mL
Matrix factor
Matrix present (post-extract)
Mean CV (%) Overall mean matrix factor
0.99
CV = (SD/mean) × 100. Normalized matrix factor = (peak area ratio in presence of matrix)/(mean peak area ratio in absence of matrix).
factor was calculated as the PAR (analyte/IS) for the analyte in each individual sample with matrix ions present to the mean PAR (analyte/IS) for the analyte in samples (n = 6 at each concentration) with matrix ions absent. The IS normalized matrix factor results are listed in Table 4. The %CV at each concentration level was ≤15%, indicating that there is acceptable variability in matrix effects among six lots of control human plasma. In addition, the matrix effect was also evaluated by comparing the peak area of cobimetinib from the post-extract and neat spike. The results (not shown) show the matrix factor is close to 1.0. 3.8. Stability The stock and working solution stability, freeze–thaw stability, bench-top stability, room temperature stability, long-term storage stability (−70 ◦ C), reinjection reproducibility and whole blood stability were assessed in this validation and the results are summarized in Table 5. Cobimetinib was shown to be stable in human plasma after five freeze–thaw cycles and stable on the bench-top at room temperature for 24 h prior to extraction. Cobimetinib was also shown to be stable in human plasma after storage between −60 ◦ C and −80 ◦ C for 400 days. The reinjection reproducibility test showed an acceptable run could be repeated after 103 h storage at room temperature. The whole blood stability test showed that cobimetinib was stable for at least 1 h at room temperature and in an ice bath. Both stock and working solutions of cobimetinib were stable at room temperature for at least 24 h when compared to solutions stored at 4 ◦ C. 3.9. Dilution capability In order to measure the concentration of cobimetinib above the calibration curve range, a 25-fold dilution was evaluated in this validation. Dilution QC was made at the 2000 ng/mL level.
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Table 5 Stability assessments for cobimetinib. Nominal conc. (ng/mL)
Cobimetinib Stability after 5 freeze–thaw cycles (at −70 ◦ C)
0.6 80
Mean observed Conc. Accuracy (%RE)
CV (%)
(ng/mL, n = 4)
(n = 4)
(n = 4)
0.575 77.1
−4.3 −3.7
1.4 0.4
Bench-top stability at room temperature for 24 h
0.6 80
Mean observed Conc. Accuracy (%)
CV (%)
(ng/mL, n = 4)
(n = 4)
(n = 4)
0.583 71.6
−2.9 −10.6
3 0.9
Storage at −60 to −80 ◦ C for 400 days
0.6 80
Mean observed Conc. Accuracy (%RE)
CV (%)
(ng/mL, n = 6)
(n = 6)
(n = 6)
0.531 72
−11.6 −10.1
2.3 1.3
Injection repordocibility at room temperature for 103 h
0.6 50 80
Mean observed Conc. Accuracy (%)
CV (%)
(ng/mL, n = 6)
(n = 6)
(n = 6)
0.531 49.2 79.4
−11.5 −1.6 −0.7
4.7 1.5 4.2
Fig. 4. Selected reaction monitoring chromatograms of cobimetinib (A) and its internal standard, 13 C6 -cobimetinib (B) from one of the patient samples in human K2 EDTA plasma.
Whole blood stability (ng/mL, n = 6)
Mean CV (%) %Dev from baseline
Baseline
1 h (room temp) 1 h (ice bath)
54.2 7.9
49.2 3.7 −9.19
55.3 1.7 2
Stock solution (1 mg/mL) at RT for 24 h (PAR (analyte/IS), n = 6) Stored at 4 ◦ C Mean 0.923 CV (%) 1.67 %Dev from stored at 4 ◦ C
Stored at RT 0.974 0.73 5.6
Working solution (10 g/mL) at RT for 24 h (PAR (analyte/IS), n = 6) Stored at RT Stored at 4 ◦ C Mean 8.476 5.5 CV (%) ◦ %Dev from stored at 4 C
7.96 2.1 −6.1 Fig. 5. Mean ± SD cobimetinib plasma concentration versus time profiles after a single 20 mg oral dose and 2 mg IV dose.
The accuracy (%RE) of the dilution QCs was within ±13.3% for cobimetinib (Table 3).
t1/2 = 59.5 (32.5) h. The 2 mg iv infusion results of this study and the metabolism information were reported previously [8,9].
3.10. Pharmacokinetics Validation of the LC–MS/MS assay was performed over the calibration curve range 0.20–100 ng/mL, which was sufficient to detect cobimetinib human plasma concentration throughout the course of the 8-day study in healthy human volunteers (n = 12). Fig. 4 showed the selected reaction monitoring chromatograms from one of the patient samples and Fig. 5 showed the mean cobimetinib human plasma concentrations versus time profile from the 12 subjects that were administered a single 20 mg oral dose. Cobimetinib plasma concentration of the 20 mg oral dose ranged from 0.253 to 24.4 ng/mL, with Cmax ranging from 6.5 to 24.4 ng/mL. Mean (SD) pharmacokinetic parameters for the twelve subjects were: Cmax (%CV) = 13.8 (35.6) ng/mL, AUC0-inf = 716 (38.0) ng/mL h, and
4. Conclusions A LC–MS/MS assay for the analysis of cobimetinib in human plasma has been developed and validated in the present study. This validated assay was proven to be rugged, accurate and sensitive and it has been utilized for the analysis of human plasma samples generated from multiple clinical studies. Acknowledgement The authors would like to thank the Medicinal Chemistry Groups in Genentech, Inc. for providing compounds for the validation.
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