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Determination of suvorexant in human plasma using 96-well liquid–liquid extraction and HPLC with tandem mass spectrometric detection
Journal of Chromatography B, 1002 (2015) 254–259
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of suvorexant in human plasma using 96-well liquid–liquid extraction and HPLC with tandem mass spectrometric detection S.A. Breidinger ∗ , R.C. Simpson 1 , E. Mangin, E.J. Woolf Pharmacokinetics Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, WP75B-300, West Point, PA 19486, USA
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 29 July 2015 Accepted 30 July 2015 Available online 11 August 2015 Keywords: LC–MS/MS Human plasma Liquid–liquid extraction Suvorexant Bioanalytical
a b s t r a c t A method, using liquid chromatography with tandem mass spectrometric detection (LC–MS/MS), was developed for the determination of suvorexant (MK-4305, Belsomra® ), a selective dual orexin receptor antagonist for the treatment insomnia, in human plasma over the concentration range of 1–1000 ng/mL. Stable isotope labeled 13 C2 H3 -suvorexant was used as an internal standard. The sample preparation procedure utilized liquid–liquid extraction, in the 96-well format, of a 100 L plasma sample with methyl t-butyl ether. The compounds were chromatographed under isocratic conditions on a Waters dC18 (50 × 2.1 mm, 3 m) column with a mobile phase consisting of 30/70 (v/v %) 10 mM ammonium formate, pH3/acetonitrile at a flow rate of 0.3 mL/min. Multiple reaction monitoring of the precursor-toproduct ion pairs for suvorexant (m/z 451 → 186) and 13 C2 H3 -suvorexant (m/z 455 → 190) on an Applied Biosystems API 4000 tandem mass spectrometer was used for quantitation. Intraday assay precision, assessed in six different lots of control plasma, was within 10% CV at all concentrations, while assay accuracy ranged from 95.6 to 105.0% of nominal. Quality control (QC) samples in plasma were stored at −20 ◦ C. Initial within day analysis of QCs after one freeze-thaw cycle showed accuracy within 9.5% of nominal with precision (CV) of 6.7% or less. The plasma QC samples were demonstrated to be stable for up to 25 months at −20 ◦ C. The method described has been used to support clinical studies during Phase I through III of clinical development. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Insomnia is a medical condition characterized by a difficulty in falling asleep, difficulty staying asleep, poor sleep quality, and/or inadequate sleep duration despite adequate opportunity for sleep. It is estimated that about one third of the adult American population is affected by insomnia [1]. Typical pharmacologic treatments for insomnia include hypnotics, such as benzodiazepines and nonbenzodiazepine GABAA modulators, sedating antidepressants including amitriptyline and trazodone, and “over the counter” sleep aides, typically consisting of a sedating, “first generation” antihistamine (e.g., diphenhydramine, doxylamine or promethazine) sometimes in combination with an analgesic. These treatments are believed to affect neurotransmitters associated with sleep induction.
∗ Corresponding author. Tel.: +1 215 652 7509; fax: +1 215 652 8548. E-mail address: sheila [email protected] (S.A. Breidinger). 1 Current address: Quest Diagnostics, 400 Egypt Road, Norristown PA 19406, USA. http://dx.doi.org/10.1016/j.jchromb.2015.07.056 1570-0232/© 2015 Elsevier B.V. All rights reserved.
Recently, antagonism of the orexin system has emerged as a potential new mechanism for modulating sleep [2]. Neuropeptides orexin A and orexin B are produced by neurons in the lateral hypothalamus region of the brain. These neuropeptides bind to orexin receptors 1 and 2; such binding is believed to promote a feeling of wakefulness. The hypothesis regarding the function of the orexin system is supported by several experimental findings [3]. Mice, engineered to be deficient in orexin peptides demonstrate a condition similar to human narcolepsy, a sleep disorder characterized by excessive daytime sleepiness. Dogs that possess a mutation in the gene coding for the orexin receptor suffer from narcolepsy. Finally, levels of the orexin A peptide are low or undetectable in many narcoleptic humans [3]. Interest in the orexin system has led to the synthesis and testing of orexin receptor antagonists as potential treatments for insomnia. Several such compounds have entered clinical testing. Of these, suvorexant (MK-4305, Fig. 1), an antagonist of orexin receptors 1 and 2, has recently completed clinical testing and received marketing authorization in the United States as a treatment for insomnia [3,4].
S.A. Breidinger et al. / J. Chromatogr. B 1002 (2015) 254–259
In order to support the clinical development of suvorexant, an assay for its determination in human plasma was needed to enable the clinical pharmacokinetic characterization of the compound. An assay for suvorexant in human plasma, utilizing liquid–liquid extraction for sample preparation, followed by HPLC with tandem mass spectrometric detection (LC–MS/MS) is described in this publication. To our knowledge, this is the first such method to appear in the literature.
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2.3. Chromatographic conditions Chromatography was performed under isocratic conditions on a 2.1 × 50 mm Waters Atlantis dC18 (Milford, MA, USA) column (3 m) with a mobile phase consisting of 30/70 (v/v %) 10 mM ammonium formate, pH 3/acetonitrile at a flow rate of 0.3 mL/min. A 5 L partial loop injection was used. The total run time was 5 min. 2.4. Mass spectrometer conditions
2. Experimental 2.1. Materials Suvorexant (Fig. 1) was obtained from the Chemical Data Department of Merck Research Laboratories (Rahway, NJ, USA). 13 C2 H stable isotope labeled suvorexant (Fig. 1) was synthe3 sized and provided by the Merck Labeled Compound Synthesis Department (Rahway, NJ, USA). Acetonitrile (HPLC Grade), methyltert-butyl ether (MTBE), ammonium formate (99.9%), and formic acid (99+%) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). Trifluoroacetic acid (HPLC Grade) was purchased from Thermo Scientific Pierce (Rockford, IL, USA). Sodium phosphate monobasic and phosphoric acid (reagent grade) were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). K2 EDTA human control plasma was purchased from Biological Specialty Corporation (Colmar, PA, USA). A Millipore Milli-Q Plus system (Bedford, MA, USA) was used to generate deionized (18 m/cm) water.
2.2. Instrumentation The HPLC–MS/MS system consisted of a Leap CTC Pal autosampler (Carrboro, NC, USA), a Perkin Elmer series 200 micropump (Norwalk, CT, USA), and an Applied Biosystems API 4000 (Foster City, CA, USA) triple quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface. Analyst version 1.4 software was used for data acquisition and processing. The 96-well liquid–liquid extraction procedure was accomplished by the use of a Tomtec Quadra 96, model 320 workstation (Hamden, CT, USA).
A O
Cl
N
O N
N
H3C
B Cl N
N
N
A 100 g/mL stock solution of suvorexant was prepared and serially diluted to give working standard solutions of 2, 4, 10, 20, 40, 100, 200, 400, 1000, 2000 ng/mL. A 10 g/mL stock solution of stable isotope labeled internal standard, 13 C2 H3 -suveroxant, was prepared and diluted to a 50 ng/mL working solution. All solutions were prepared in 50/50 (v/v%) acetonitrile/water. Solutions were stored at 4 ◦ C in amber glass. Plasma standards were prepared by adding 50 L of working standard stock and 50 L of working ISTD solution into 100 L of K2 EDTA human control plasma. These standards were used to quantitate clinical plasma samples over the concentration range of 1–1000 ng/mL. Clinical samples that contained more than 1000 ng/mL suvorexant were diluted with control plasma and reanalyzed. 2.6. Preparation of quality control samples A separate suvorexant stock solution at a concentration of 75 g/mL was prepared in 50/50 (v/v%) acetonitrile/water. This solution was then diluted to yield solutions at concentrations of 15 and 0.3 g/mL. The 75, 15, and 0.3 g/mL solutions were used to prepare low (3 ng/mL), mid (150 ng/mL) and high (750 ng/mL) plasma quality control (QC) samples; 0.5 mL of each of the solutions were transferred to a 50 mL volumetric flask which was then filled to the mark with human control plasma. Three hundred microliter aliquots of the plasma QC solutions were transferred to 3.6 mL polypropylene cryo tubes. The tubes were subsequently capped, and stored at −20 ◦ C. 2.7. Sample preparation
CH3
O
2.5. Preparation of standards
N
N
N
O
An atmospheric pressure chemical ionization interface (APCI) probe operated in the positive mode and set at a temperature of 500 ◦ C was used to ionize the sample. Collision induced dissociation of the protonated molecules was achieved with a CAD gas setting of 4, the optimum corona discharge needle setting was 3, and the dwell time for analyte and ISTD was 300 ms.
N
N
N CH3 13
D C D D Fig. 1. Chemical structures of suvorexant (A) and 13 C2 H3 -suvorexant (ISTD, B).
To 100 L aliquots of plasma samples contained within the 2.4 mL wells of a 96-well polypropylene plate (Sun SRI, Rockwood, TN, USA) was added 50 L of a 50 ng/mL working internal standard solution and 50 L of (50:50) v/v% ACN:H2 O; the acetonitrile/water was added to compensate for the volume of working standard solution used to prepare the calibration curve samples. A 25 L volume of a 1 M pH 2 phosphate buffer solution was then added to each well. Using a Tomtec Quadra 96 workstation, 1.2 mL of MTBE extraction solvent was added to each well. The plate was sealed with a 96-well square TFE/SIL mat (Sun SRI, Rockwood, TN, USA) and mixed on a rotator for 10 min. The extraction plate was then centrifuged for 5 min and, using the workstation, 800 L of the organic layer in each well was transferred to a well in a clean 1.2 mL 96well polypropylene autosampler plate (Arctic White, Bethlehem, PA, USA). The solvent was evaporated to dryness under N2 at 40 ◦ C for approximately 10 min. The samples were then reconstituted in
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200 L of 50/50 (v/v%) acetonitrile/water. The plate containing the samples was covered with a silicone sealing mat (Analytical Sales & Service, Pompton Plains, NJ). The resulting extracts were mixed and 5 L of each was injected into the LC–MS/MS system for analysis. 3. Results and discussion 3.1. Assay development Precursor ions for suvorexant and internal standard were determined from Q1 spectra obtained during the infusion of neat solutions in mobile phase, via the APCI source, into the mass spectrometer operating in the positive ionization mode with the collision gas off. Under these conditions, the protonated molecules at m/z 451 and 455 for suvorexant and internal standard, respectively, were predominately observed. Each of the precursor ions was subjected to collision induced dissociation (CID) in order to determine the resulting product ions. Product ion mass spectra for suvorexant and internal standard are shown in Fig. 2. Multiple reaction monitoring of the predominant precursor to product ion pairs m/z 451 → 186 for suvorexant and m/z 455 → 190 for ISTD was used for quantitation. A Waters Atlantis dC18 column (dC18, 50 × 2.1-mm; 3 m) together with an isocratic mobile phase consisting of a mixture of 30% (10 mM ammonium formate, pH 3): 70% (acetonitrile) was evaluated and found to yield symmetrical peaks for analyte and ISTD. The mobile phase was delivered at a flow rate of 0.3 mL/min. Under these conditions, both the analyte and internal standard
Table 1 Representative intraday precision and accuracy data for the determination of suvorexant in six different lots of plasma as assessed by the replicate (n = 6) analysis of standards. Nominal concentration (ng/mL)
Mean (n = 6) determined concentration (ng/mL)
Accuracya (%)
Precisionb (%)
1 2 5 10 20 50 100 200 500 1000
1.0 2.1 4.9 9.9 20.8 47.8 97.8 200.2 493.2 1047.2
100.0 105.0 98.0 99.0 104.0 95.6 97.8 100.1 98.6 104.7
10.0 4.8 8.2 6.1 6.7 4.4 4.6 5.6 4.1 5.4
a b
Expressed as [(mean observed concentration)/(nominal concentration)] × 100. Coefficient of variation.
eluted at a retention time of approximately 1.3 min. Analyte and internal standard were well resolved from the solvent front and the inactive circulating hydroxylated metabolite of suvorexant which had a retention time of approximately 0.8 min. Liquid–liquid extraction in a 96-well format at pH 2 with MTBE as extraction solvent was used to isolate the analyte from the plasma matrix. Consistent recovery was observed across the calibration curve range under these conditions. 3.2. Assay validation
Fig. 2. Product ion mass spectrum of suvorexant (A) and 13 C2 H3 -suvorexant (B).
Assay validation was performed in accordance with current regulatory guidances [5,6]. Representative chromatograms, from samples extracted and analyzed under the conditions of the assay, are shown in Fig. 3a–c for a control double blank sample, a control single blank sample containing 25 ng/mL of ISTD, and a 1 ng/mL suvorexant plasma standard (LLOQ). An assessment of intraday variability was conducted by analyzing replicate (n = 6) sets of calibration standards from 1 to 1000 ng/mL. Each set was prepared in a different lot of control human plasma. The resulting precision and accuracy data are presented in Table 1. The precision of the assay, expressed as the coefficient of variation (CV), was better than 10% at all concentrations within the standard curve range, while the assay accuracy was between 95.6 and 105.0% of nominal. Plasma quality control (QC) samples containing suvorexant were prepared at concentrations of 3.0 (Low QC), 150 (Middle QC), and 750 ng/mL (High QC). The accuracy of the initial within-day analysis of the QC samples was within 9.5% of nominal with a CV of 6.7% or below at each level. Inter-day assay performance was assessed based on the replicate (n = 31) analysis of QC samples over a 10 month span. The average QC accuracy was within 0.47% of nominal and the precision was 7.0% CV or better. Extraction recovery and the effect of the sample matrix on ionization were evaluated for suvorexant using calibration standards prepared in 6 different lots of control plasma at analyte concentrations of 1, 100, and 1000 ng/mL. Extraction recovery was determined by comparing the absolute peak areas of the standards in human plasma extracted as described, to control plasma extracted in the same manner and then spiked post extraction with the same concentration of the drug and internal standard. Matrix enhancement/suppression of ionization was evaluated by comparing the absolute peak areas of control plasma extracted and then spiked with a known amount of drug, to neat standards injected directly in the same reconstitution solvent. Results are shown in
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Fig. 3. Representative chromatograms: (A) control plasma double blank, (B) control plasma sample containing 25 ng/mL ISTD, and (C) plasma calibration standard containing 1 ng/mL suvorexant and 25 ng/mL ISTD.
Table 2. Although assay recovery was just slightly greater than 50%, it was found to be consistent across multiple lots of plasma, with the stable label internal standard able to compensate for any variability. Based on the intra-day precision results obtained using 6 different lots of control plasma, a relative matrix effect would not be expected to affect the assay. The presence of “cross-talk” between channels used for monitoring the analytes was evaluated based on the analysis of plasma samples containing ISTD at the working concentration (25 ng/mL) in the absence of suvorexant and the analysis of plasma samples containing suvorexant at the ULOQ (1000 ng/mL) in the absence of ISTD. No significant “cross-talk” was observed in either experiment. Quality control samples (n = 3 at each concentration) were subjected to replicate freeze-thaw cycles consisting of a thaw to reach room temperature (>4 h) and then refreezing (−20 ◦ C) at least overnight and repeating for a total of 3 cycles. Samples exposed to only 1 freeze-thaw cycle were used as the control samples. Samples that were subjected to additional freeze-thaw cycles analyzed within 3.6% of the controls. To assess the room temperature stability of suvorexant in human plasma, quality control samples were allowed to remain at room temperature for 6 h before analysis. These samples were analyzed and the results were compared with a set of samples that were assayed immediately after thawing. The results for the samples stored at room temperature prior to analysis were within 3.6%
Table 2 Extraction recovery and assessment of absolute matrix effects on ionization during the determination of suvorexant in six different lots of human plasma. Standard concentration in plasma (ng/mL)
Mean extraction recovery (%)a (n = 6)
Mean absolute matrix effect (%)b (n = 6)
1 100 1000 25 (ISTD)c
50.5 57.2 53.3 46.4
92.1 103.6 98.9 100.6
a Extraction recovery was calculated by dividing the mean peak area of analyte spiked before extraction by the respective mean peak area of analyte spiked into extracts of control plasma and multiplying by 100. b Matrix effect was calculated by dividing the mean peak area of analyte spiked into control plasma extracts by the mean peak area of the neat analyte standard and multiplying by 100. c (n = 18)
of the control set. These results indicate suvorexant is stable at room temperature in human plasma for up to 6 h. The ability to accurately dilute samples was assessed by preparing a set of human plasma samples containing suvorexant at a concentration 10 times greater than the ULOQ (10,000 ng/mL). These dilution samples (n = 5) were frozen at −20 ◦ C for at least 24 h, thawed, and diluted 1:20 with control plasma. The diluted samples were analyzed along with a standard curve. The average accuracy of the assayed concentration of these samples was 98.9% of nominal, with a CV of 5.4%. The concentrations of all clinical samples obtained following the administration of therapeutic doses of suvorexant were below 10,000 ng/mL. A set of calibration samples (n = 5 per concentration) was prepared and analyzed. Following initial analysis, the samples were allowed to remain on the autosampler exposed to ambient laboratory conditions for 3 days after which the samples were reinjected. Assayed standard concentrations were determined based on (1) the initial injection of the standard curve samples and (2) the standard curve samples that were reinjected. The results are presented in Table 3. The stability of suvorexant stock solutions was evaluated by comparing the instrument response of freshly prepared standard solutions from a new standard weighing to similarly prepared stock and working solutions stored for 521 days at 4 ◦ C. The results showed a difference of 3.2 and 1.0% between stock and working solutions, indicating that stock and working solutions were stable for at least 521 days. The long-term storage stability of suvorexant in human plasma was assessed based on the analysis of QC samples prepared and stored at −20 ◦ C for an extended period of time. Samples stored at −20 ◦ C for a period of 25 months were found to analyze within 10% of their initial values, indicating that suvorexant in plasma was stable for at least this period. The time period between sample collections and sample analysis did not exceed the established stability period for any of the clinical studies.
3.3. Clinical sample analysis Human plasma samples were analyzed following the administration of oral doses of suvorexant. A representative post-dose chromatogram from a sample collected 2 h after administration of
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Table 3 Processed sample stability and re-injection accuracy and precision after 3 days. Nominal conc. (ng/mL)
1 2 5 10 20 50 100 200 500 1000
Reinjection reproducibilitya
Initial injection (Control) d
Processed sample stabilityb
Mean assayed conc.c (ng/mL)
Accuracy (%)
Mean assayed conc.c (ng/mL)
Difference from controle (%)
Mean assayed conc.c (ng/mL)
Difference from controle (%)
1.0 (8.6) 2.1 (5.3) 5.0 (9.0) 10.1 (6.4) 21.3 (7.7) 48.7 (5.0) 100.0 (5.0) 206.9 (4.8) 504.4 (4.4) 1074.4 (5.6)
100.0 105.0 100.0 101.0 106.5 97.4 100.0 103.5 100.9 107.4
1.0 (11.2) 2.1 (10.1) 4.9 (8.3) 10.0 (6.2) 21.6 (7.7) 49.1 (5.6) 100.2 (4.8) 206.9 (4.1) 496.4 (3.1) 1078.0 (4.0)
0.0 0.0 −2.0 −1.0 1.4 0.8 0.2 0.0 −1.6 0.3
1.0 (8.6) 2.1 (10.8) 5.0 (8.4) 10.0 (6.2) 21.6 (7.6) 48.9 (5.5) 99.6 (4.8) 205.8 (4.1) 493.4 (3.1) 1071.5 (4.0)
0.0 0.0 0.0 −1.0 1.4 0.4 −0.4 −0.5 −2.2 −0.3
a
Reinjected standard extracts calculated using reinjected standard curve. 3 Day old standard extracts calculated from initial injection of standard curve. c n = 5. d Expressed as [(mean observed concentration)/(nominal concentration)] × 100. e Calculated as [(mean assayed concentration) − (mean assayed control concentration)]/(mean assayed control concentration) × 100. Numbers in parentheses are coefficients of variation (%CV). b
300
Concentration (ng/mL)
a 10 mg suvorexant dose is shown in Fig. 4. Plasma concentration vs. time profiles of suvorexant after oral administration of a single 10 mg dose to 6 subjects are shown in Fig. 5; 10 mg is the recommended starting dose of suvorexant in patients being treated for insomnia [7]. Ten percent of the plasma samples from a clinical study were reanalyzed to determine the reproducibility and accuracy of the determined concentration of incurred samples. All of the reassayed samples analyzed within ±20% of the original value, demonstrating the reproducibility of the method.
250 200 150 100 50 0 0
5
10
15
20
25
30
35
40
45
50
Time (Hours) Fig. 5. Mean (n = 6 subjects) plasma concentration vs. time profile following a single 10 mg oral dose of suvorexant.
4. Conclusions A bioanalytical method for the determination of suvorexant in human plasma has been validated and successfully applied to the analysis of human clinical studies. The sensitivity of the assay is sufficient to permit the pharmacokinetics of clinically relevant doses of suvorexant to be characterized. The analysis, at multiple laboratories around the world, of samples from over thirty clinical studies has been supported using this method, thus demonstrating that the described procedure is rugged and reliable. References
Fig. 4. Chromatogram from a plasma sample collected 2 h after administration of 10 mg suvorexant (analyzed suvorexant concentration = 311.1 ng/mL).
[1] T. Roth, Prevalence, associated risks, and treatment patterns of Insomnia, J. Clin. Psychiatry 66 (Suppl. 9) (2005) 10–13. [2] C. Hopkins, ACS chemical neuroscience molecule spotlight on suvorexant, ACS Chem. Neurosci. 3 (2012) 647–648. [3] C.D. Cox, M.J. Breslin, D.B. Whitman, J.D. Schreier, G.B. McGaughey, M.J. Bogusky, A.J. Roecker, S.P. Mercer, R.A. Bednar, W. Lemaire, J.G. Bruno, D.R. Reiss, C.M. Harrell, K.L. Murphy, S.L. Garson, S.M. Doran, T. Prueksaritanont, W.B. Anderson, C. Tang, S. Roller, T.D. Cabalu, D. Cui, G.D. Hartman, S.D. Young, K.S. Koblan, C.J. Winrow, J.J. Renger, P.J. Coleman, Discovery of the dual orexin receptor antagonist [(7R)-4-(5-chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan1-yl][5-methyl-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (MK-4305) for the treatment of insomnia, J. Med. Chem. 53 (2010) 5320–5332.
S.A. Breidinger et al. / J. Chromatogr. B 1002 (2015) 254–259 [4] C.J. Winrow, A.L. Gotter, C.D. Cox, S.M. Doran, P.L. Tannenbaum, M.J. Breslin, S.L. Garson, S.V. Fox, C.M. Harrell, J. Stevens, D.R. Reiss, D. Cui, P.J. Coleman, J.J. Renger, Promotion of sleep by suvorexant—a novel dual orexin receptor antagonist, J. Neurogenet. 25 (2011) 52–61. [5] United States Food and Drug Administration, Guidance for Industry – Bioanalytical Method Validation, 2001, May, http://www.fda.gov/downloads/Drugs/ Guidances/ucm070107.pdf
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[6] European Medicines Agency, Guideline on Bioanalytical Method Validation, 2011, July, http://www.ema.europa.eu/docs/en GB/document library/ Scientific guideline/2011/08/WC500109686.pdf [7] Merck & Co., Suvorexant Prescribing Information, 2014, http://www.merck.com/ product/usa/pi circulars/b/belsomra/belsomra pi.pdf
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of suvorexant in human plasma using 96-well liquid–liquid extraction and HPLC with tandem mass spectrometric detection S.A. Breidinger ∗ , R.C. Simpson 1 , E. Mangin, E.J. Woolf Pharmacokinetics Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, WP75B-300, West Point, PA 19486, USA
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 29 July 2015 Accepted 30 July 2015 Available online 11 August 2015 Keywords: LC–MS/MS Human plasma Liquid–liquid extraction Suvorexant Bioanalytical
a b s t r a c t A method, using liquid chromatography with tandem mass spectrometric detection (LC–MS/MS), was developed for the determination of suvorexant (MK-4305, Belsomra® ), a selective dual orexin receptor antagonist for the treatment insomnia, in human plasma over the concentration range of 1–1000 ng/mL. Stable isotope labeled 13 C2 H3 -suvorexant was used as an internal standard. The sample preparation procedure utilized liquid–liquid extraction, in the 96-well format, of a 100 L plasma sample with methyl t-butyl ether. The compounds were chromatographed under isocratic conditions on a Waters dC18 (50 × 2.1 mm, 3 m) column with a mobile phase consisting of 30/70 (v/v %) 10 mM ammonium formate, pH3/acetonitrile at a flow rate of 0.3 mL/min. Multiple reaction monitoring of the precursor-toproduct ion pairs for suvorexant (m/z 451 → 186) and 13 C2 H3 -suvorexant (m/z 455 → 190) on an Applied Biosystems API 4000 tandem mass spectrometer was used for quantitation. Intraday assay precision, assessed in six different lots of control plasma, was within 10% CV at all concentrations, while assay accuracy ranged from 95.6 to 105.0% of nominal. Quality control (QC) samples in plasma were stored at −20 ◦ C. Initial within day analysis of QCs after one freeze-thaw cycle showed accuracy within 9.5% of nominal with precision (CV) of 6.7% or less. The plasma QC samples were demonstrated to be stable for up to 25 months at −20 ◦ C. The method described has been used to support clinical studies during Phase I through III of clinical development. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Insomnia is a medical condition characterized by a difficulty in falling asleep, difficulty staying asleep, poor sleep quality, and/or inadequate sleep duration despite adequate opportunity for sleep. It is estimated that about one third of the adult American population is affected by insomnia [1]. Typical pharmacologic treatments for insomnia include hypnotics, such as benzodiazepines and nonbenzodiazepine GABAA modulators, sedating antidepressants including amitriptyline and trazodone, and “over the counter” sleep aides, typically consisting of a sedating, “first generation” antihistamine (e.g., diphenhydramine, doxylamine or promethazine) sometimes in combination with an analgesic. These treatments are believed to affect neurotransmitters associated with sleep induction.
∗ Corresponding author. Tel.: +1 215 652 7509; fax: +1 215 652 8548. E-mail address: sheila [email protected] (S.A. Breidinger). 1 Current address: Quest Diagnostics, 400 Egypt Road, Norristown PA 19406, USA. http://dx.doi.org/10.1016/j.jchromb.2015.07.056 1570-0232/© 2015 Elsevier B.V. All rights reserved.
Recently, antagonism of the orexin system has emerged as a potential new mechanism for modulating sleep [2]. Neuropeptides orexin A and orexin B are produced by neurons in the lateral hypothalamus region of the brain. These neuropeptides bind to orexin receptors 1 and 2; such binding is believed to promote a feeling of wakefulness. The hypothesis regarding the function of the orexin system is supported by several experimental findings [3]. Mice, engineered to be deficient in orexin peptides demonstrate a condition similar to human narcolepsy, a sleep disorder characterized by excessive daytime sleepiness. Dogs that possess a mutation in the gene coding for the orexin receptor suffer from narcolepsy. Finally, levels of the orexin A peptide are low or undetectable in many narcoleptic humans [3]. Interest in the orexin system has led to the synthesis and testing of orexin receptor antagonists as potential treatments for insomnia. Several such compounds have entered clinical testing. Of these, suvorexant (MK-4305, Fig. 1), an antagonist of orexin receptors 1 and 2, has recently completed clinical testing and received marketing authorization in the United States as a treatment for insomnia [3,4].
S.A. Breidinger et al. / J. Chromatogr. B 1002 (2015) 254–259
In order to support the clinical development of suvorexant, an assay for its determination in human plasma was needed to enable the clinical pharmacokinetic characterization of the compound. An assay for suvorexant in human plasma, utilizing liquid–liquid extraction for sample preparation, followed by HPLC with tandem mass spectrometric detection (LC–MS/MS) is described in this publication. To our knowledge, this is the first such method to appear in the literature.
255
2.3. Chromatographic conditions Chromatography was performed under isocratic conditions on a 2.1 × 50 mm Waters Atlantis dC18 (Milford, MA, USA) column (3 m) with a mobile phase consisting of 30/70 (v/v %) 10 mM ammonium formate, pH 3/acetonitrile at a flow rate of 0.3 mL/min. A 5 L partial loop injection was used. The total run time was 5 min. 2.4. Mass spectrometer conditions
2. Experimental 2.1. Materials Suvorexant (Fig. 1) was obtained from the Chemical Data Department of Merck Research Laboratories (Rahway, NJ, USA). 13 C2 H stable isotope labeled suvorexant (Fig. 1) was synthe3 sized and provided by the Merck Labeled Compound Synthesis Department (Rahway, NJ, USA). Acetonitrile (HPLC Grade), methyltert-butyl ether (MTBE), ammonium formate (99.9%), and formic acid (99+%) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). Trifluoroacetic acid (HPLC Grade) was purchased from Thermo Scientific Pierce (Rockford, IL, USA). Sodium phosphate monobasic and phosphoric acid (reagent grade) were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). K2 EDTA human control plasma was purchased from Biological Specialty Corporation (Colmar, PA, USA). A Millipore Milli-Q Plus system (Bedford, MA, USA) was used to generate deionized (18 m/cm) water.
2.2. Instrumentation The HPLC–MS/MS system consisted of a Leap CTC Pal autosampler (Carrboro, NC, USA), a Perkin Elmer series 200 micropump (Norwalk, CT, USA), and an Applied Biosystems API 4000 (Foster City, CA, USA) triple quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface. Analyst version 1.4 software was used for data acquisition and processing. The 96-well liquid–liquid extraction procedure was accomplished by the use of a Tomtec Quadra 96, model 320 workstation (Hamden, CT, USA).
A O
Cl
N
O N
N
H3C
B Cl N
N
N
A 100 g/mL stock solution of suvorexant was prepared and serially diluted to give working standard solutions of 2, 4, 10, 20, 40, 100, 200, 400, 1000, 2000 ng/mL. A 10 g/mL stock solution of stable isotope labeled internal standard, 13 C2 H3 -suveroxant, was prepared and diluted to a 50 ng/mL working solution. All solutions were prepared in 50/50 (v/v%) acetonitrile/water. Solutions were stored at 4 ◦ C in amber glass. Plasma standards were prepared by adding 50 L of working standard stock and 50 L of working ISTD solution into 100 L of K2 EDTA human control plasma. These standards were used to quantitate clinical plasma samples over the concentration range of 1–1000 ng/mL. Clinical samples that contained more than 1000 ng/mL suvorexant were diluted with control plasma and reanalyzed. 2.6. Preparation of quality control samples A separate suvorexant stock solution at a concentration of 75 g/mL was prepared in 50/50 (v/v%) acetonitrile/water. This solution was then diluted to yield solutions at concentrations of 15 and 0.3 g/mL. The 75, 15, and 0.3 g/mL solutions were used to prepare low (3 ng/mL), mid (150 ng/mL) and high (750 ng/mL) plasma quality control (QC) samples; 0.5 mL of each of the solutions were transferred to a 50 mL volumetric flask which was then filled to the mark with human control plasma. Three hundred microliter aliquots of the plasma QC solutions were transferred to 3.6 mL polypropylene cryo tubes. The tubes were subsequently capped, and stored at −20 ◦ C. 2.7. Sample preparation
CH3
O
2.5. Preparation of standards
N
N
N
O
An atmospheric pressure chemical ionization interface (APCI) probe operated in the positive mode and set at a temperature of 500 ◦ C was used to ionize the sample. Collision induced dissociation of the protonated molecules was achieved with a CAD gas setting of 4, the optimum corona discharge needle setting was 3, and the dwell time for analyte and ISTD was 300 ms.
N
N
N CH3 13
D C D D Fig. 1. Chemical structures of suvorexant (A) and 13 C2 H3 -suvorexant (ISTD, B).
To 100 L aliquots of plasma samples contained within the 2.4 mL wells of a 96-well polypropylene plate (Sun SRI, Rockwood, TN, USA) was added 50 L of a 50 ng/mL working internal standard solution and 50 L of (50:50) v/v% ACN:H2 O; the acetonitrile/water was added to compensate for the volume of working standard solution used to prepare the calibration curve samples. A 25 L volume of a 1 M pH 2 phosphate buffer solution was then added to each well. Using a Tomtec Quadra 96 workstation, 1.2 mL of MTBE extraction solvent was added to each well. The plate was sealed with a 96-well square TFE/SIL mat (Sun SRI, Rockwood, TN, USA) and mixed on a rotator for 10 min. The extraction plate was then centrifuged for 5 min and, using the workstation, 800 L of the organic layer in each well was transferred to a well in a clean 1.2 mL 96well polypropylene autosampler plate (Arctic White, Bethlehem, PA, USA). The solvent was evaporated to dryness under N2 at 40 ◦ C for approximately 10 min. The samples were then reconstituted in
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200 L of 50/50 (v/v%) acetonitrile/water. The plate containing the samples was covered with a silicone sealing mat (Analytical Sales & Service, Pompton Plains, NJ). The resulting extracts were mixed and 5 L of each was injected into the LC–MS/MS system for analysis. 3. Results and discussion 3.1. Assay development Precursor ions for suvorexant and internal standard were determined from Q1 spectra obtained during the infusion of neat solutions in mobile phase, via the APCI source, into the mass spectrometer operating in the positive ionization mode with the collision gas off. Under these conditions, the protonated molecules at m/z 451 and 455 for suvorexant and internal standard, respectively, were predominately observed. Each of the precursor ions was subjected to collision induced dissociation (CID) in order to determine the resulting product ions. Product ion mass spectra for suvorexant and internal standard are shown in Fig. 2. Multiple reaction monitoring of the predominant precursor to product ion pairs m/z 451 → 186 for suvorexant and m/z 455 → 190 for ISTD was used for quantitation. A Waters Atlantis dC18 column (dC18, 50 × 2.1-mm; 3 m) together with an isocratic mobile phase consisting of a mixture of 30% (10 mM ammonium formate, pH 3): 70% (acetonitrile) was evaluated and found to yield symmetrical peaks for analyte and ISTD. The mobile phase was delivered at a flow rate of 0.3 mL/min. Under these conditions, both the analyte and internal standard
Table 1 Representative intraday precision and accuracy data for the determination of suvorexant in six different lots of plasma as assessed by the replicate (n = 6) analysis of standards. Nominal concentration (ng/mL)
Mean (n = 6) determined concentration (ng/mL)
Accuracya (%)
Precisionb (%)
1 2 5 10 20 50 100 200 500 1000
1.0 2.1 4.9 9.9 20.8 47.8 97.8 200.2 493.2 1047.2
100.0 105.0 98.0 99.0 104.0 95.6 97.8 100.1 98.6 104.7
10.0 4.8 8.2 6.1 6.7 4.4 4.6 5.6 4.1 5.4
a b
Expressed as [(mean observed concentration)/(nominal concentration)] × 100. Coefficient of variation.
eluted at a retention time of approximately 1.3 min. Analyte and internal standard were well resolved from the solvent front and the inactive circulating hydroxylated metabolite of suvorexant which had a retention time of approximately 0.8 min. Liquid–liquid extraction in a 96-well format at pH 2 with MTBE as extraction solvent was used to isolate the analyte from the plasma matrix. Consistent recovery was observed across the calibration curve range under these conditions. 3.2. Assay validation
Fig. 2. Product ion mass spectrum of suvorexant (A) and 13 C2 H3 -suvorexant (B).
Assay validation was performed in accordance with current regulatory guidances [5,6]. Representative chromatograms, from samples extracted and analyzed under the conditions of the assay, are shown in Fig. 3a–c for a control double blank sample, a control single blank sample containing 25 ng/mL of ISTD, and a 1 ng/mL suvorexant plasma standard (LLOQ). An assessment of intraday variability was conducted by analyzing replicate (n = 6) sets of calibration standards from 1 to 1000 ng/mL. Each set was prepared in a different lot of control human plasma. The resulting precision and accuracy data are presented in Table 1. The precision of the assay, expressed as the coefficient of variation (CV), was better than 10% at all concentrations within the standard curve range, while the assay accuracy was between 95.6 and 105.0% of nominal. Plasma quality control (QC) samples containing suvorexant were prepared at concentrations of 3.0 (Low QC), 150 (Middle QC), and 750 ng/mL (High QC). The accuracy of the initial within-day analysis of the QC samples was within 9.5% of nominal with a CV of 6.7% or below at each level. Inter-day assay performance was assessed based on the replicate (n = 31) analysis of QC samples over a 10 month span. The average QC accuracy was within 0.47% of nominal and the precision was 7.0% CV or better. Extraction recovery and the effect of the sample matrix on ionization were evaluated for suvorexant using calibration standards prepared in 6 different lots of control plasma at analyte concentrations of 1, 100, and 1000 ng/mL. Extraction recovery was determined by comparing the absolute peak areas of the standards in human plasma extracted as described, to control plasma extracted in the same manner and then spiked post extraction with the same concentration of the drug and internal standard. Matrix enhancement/suppression of ionization was evaluated by comparing the absolute peak areas of control plasma extracted and then spiked with a known amount of drug, to neat standards injected directly in the same reconstitution solvent. Results are shown in
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Fig. 3. Representative chromatograms: (A) control plasma double blank, (B) control plasma sample containing 25 ng/mL ISTD, and (C) plasma calibration standard containing 1 ng/mL suvorexant and 25 ng/mL ISTD.
Table 2. Although assay recovery was just slightly greater than 50%, it was found to be consistent across multiple lots of plasma, with the stable label internal standard able to compensate for any variability. Based on the intra-day precision results obtained using 6 different lots of control plasma, a relative matrix effect would not be expected to affect the assay. The presence of “cross-talk” between channels used for monitoring the analytes was evaluated based on the analysis of plasma samples containing ISTD at the working concentration (25 ng/mL) in the absence of suvorexant and the analysis of plasma samples containing suvorexant at the ULOQ (1000 ng/mL) in the absence of ISTD. No significant “cross-talk” was observed in either experiment. Quality control samples (n = 3 at each concentration) were subjected to replicate freeze-thaw cycles consisting of a thaw to reach room temperature (>4 h) and then refreezing (−20 ◦ C) at least overnight and repeating for a total of 3 cycles. Samples exposed to only 1 freeze-thaw cycle were used as the control samples. Samples that were subjected to additional freeze-thaw cycles analyzed within 3.6% of the controls. To assess the room temperature stability of suvorexant in human plasma, quality control samples were allowed to remain at room temperature for 6 h before analysis. These samples were analyzed and the results were compared with a set of samples that were assayed immediately after thawing. The results for the samples stored at room temperature prior to analysis were within 3.6%
Table 2 Extraction recovery and assessment of absolute matrix effects on ionization during the determination of suvorexant in six different lots of human plasma. Standard concentration in plasma (ng/mL)
Mean extraction recovery (%)a (n = 6)
Mean absolute matrix effect (%)b (n = 6)
1 100 1000 25 (ISTD)c
50.5 57.2 53.3 46.4
92.1 103.6 98.9 100.6
a Extraction recovery was calculated by dividing the mean peak area of analyte spiked before extraction by the respective mean peak area of analyte spiked into extracts of control plasma and multiplying by 100. b Matrix effect was calculated by dividing the mean peak area of analyte spiked into control plasma extracts by the mean peak area of the neat analyte standard and multiplying by 100. c (n = 18)
of the control set. These results indicate suvorexant is stable at room temperature in human plasma for up to 6 h. The ability to accurately dilute samples was assessed by preparing a set of human plasma samples containing suvorexant at a concentration 10 times greater than the ULOQ (10,000 ng/mL). These dilution samples (n = 5) were frozen at −20 ◦ C for at least 24 h, thawed, and diluted 1:20 with control plasma. The diluted samples were analyzed along with a standard curve. The average accuracy of the assayed concentration of these samples was 98.9% of nominal, with a CV of 5.4%. The concentrations of all clinical samples obtained following the administration of therapeutic doses of suvorexant were below 10,000 ng/mL. A set of calibration samples (n = 5 per concentration) was prepared and analyzed. Following initial analysis, the samples were allowed to remain on the autosampler exposed to ambient laboratory conditions for 3 days after which the samples were reinjected. Assayed standard concentrations were determined based on (1) the initial injection of the standard curve samples and (2) the standard curve samples that were reinjected. The results are presented in Table 3. The stability of suvorexant stock solutions was evaluated by comparing the instrument response of freshly prepared standard solutions from a new standard weighing to similarly prepared stock and working solutions stored for 521 days at 4 ◦ C. The results showed a difference of 3.2 and 1.0% between stock and working solutions, indicating that stock and working solutions were stable for at least 521 days. The long-term storage stability of suvorexant in human plasma was assessed based on the analysis of QC samples prepared and stored at −20 ◦ C for an extended period of time. Samples stored at −20 ◦ C for a period of 25 months were found to analyze within 10% of their initial values, indicating that suvorexant in plasma was stable for at least this period. The time period between sample collections and sample analysis did not exceed the established stability period for any of the clinical studies.
3.3. Clinical sample analysis Human plasma samples were analyzed following the administration of oral doses of suvorexant. A representative post-dose chromatogram from a sample collected 2 h after administration of
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Table 3 Processed sample stability and re-injection accuracy and precision after 3 days. Nominal conc. (ng/mL)
1 2 5 10 20 50 100 200 500 1000
Reinjection reproducibilitya
Initial injection (Control) d
Processed sample stabilityb
Mean assayed conc.c (ng/mL)
Accuracy (%)
Mean assayed conc.c (ng/mL)
Difference from controle (%)
Mean assayed conc.c (ng/mL)
Difference from controle (%)
1.0 (8.6) 2.1 (5.3) 5.0 (9.0) 10.1 (6.4) 21.3 (7.7) 48.7 (5.0) 100.0 (5.0) 206.9 (4.8) 504.4 (4.4) 1074.4 (5.6)
100.0 105.0 100.0 101.0 106.5 97.4 100.0 103.5 100.9 107.4
1.0 (11.2) 2.1 (10.1) 4.9 (8.3) 10.0 (6.2) 21.6 (7.7) 49.1 (5.6) 100.2 (4.8) 206.9 (4.1) 496.4 (3.1) 1078.0 (4.0)
0.0 0.0 −2.0 −1.0 1.4 0.8 0.2 0.0 −1.6 0.3
1.0 (8.6) 2.1 (10.8) 5.0 (8.4) 10.0 (6.2) 21.6 (7.6) 48.9 (5.5) 99.6 (4.8) 205.8 (4.1) 493.4 (3.1) 1071.5 (4.0)
0.0 0.0 0.0 −1.0 1.4 0.4 −0.4 −0.5 −2.2 −0.3
a
Reinjected standard extracts calculated using reinjected standard curve. 3 Day old standard extracts calculated from initial injection of standard curve. c n = 5. d Expressed as [(mean observed concentration)/(nominal concentration)] × 100. e Calculated as [(mean assayed concentration) − (mean assayed control concentration)]/(mean assayed control concentration) × 100. Numbers in parentheses are coefficients of variation (%CV). b
300
Concentration (ng/mL)
a 10 mg suvorexant dose is shown in Fig. 4. Plasma concentration vs. time profiles of suvorexant after oral administration of a single 10 mg dose to 6 subjects are shown in Fig. 5; 10 mg is the recommended starting dose of suvorexant in patients being treated for insomnia [7]. Ten percent of the plasma samples from a clinical study were reanalyzed to determine the reproducibility and accuracy of the determined concentration of incurred samples. All of the reassayed samples analyzed within ±20% of the original value, demonstrating the reproducibility of the method.
250 200 150 100 50 0 0
5
10
15
20
25
30
35
40
45
50
Time (Hours) Fig. 5. Mean (n = 6 subjects) plasma concentration vs. time profile following a single 10 mg oral dose of suvorexant.
4. Conclusions A bioanalytical method for the determination of suvorexant in human plasma has been validated and successfully applied to the analysis of human clinical studies. The sensitivity of the assay is sufficient to permit the pharmacokinetics of clinically relevant doses of suvorexant to be characterized. The analysis, at multiple laboratories around the world, of samples from over thirty clinical studies has been supported using this method, thus demonstrating that the described procedure is rugged and reliable. References
Fig. 4. Chromatogram from a plasma sample collected 2 h after administration of 10 mg suvorexant (analyzed suvorexant concentration = 311.1 ng/mL).
[1] T. Roth, Prevalence, associated risks, and treatment patterns of Insomnia, J. Clin. Psychiatry 66 (Suppl. 9) (2005) 10–13. [2] C. Hopkins, ACS chemical neuroscience molecule spotlight on suvorexant, ACS Chem. Neurosci. 3 (2012) 647–648. [3] C.D. Cox, M.J. Breslin, D.B. Whitman, J.D. Schreier, G.B. McGaughey, M.J. Bogusky, A.J. Roecker, S.P. Mercer, R.A. Bednar, W. Lemaire, J.G. Bruno, D.R. Reiss, C.M. Harrell, K.L. Murphy, S.L. Garson, S.M. Doran, T. Prueksaritanont, W.B. Anderson, C. Tang, S. Roller, T.D. Cabalu, D. Cui, G.D. Hartman, S.D. Young, K.S. Koblan, C.J. Winrow, J.J. Renger, P.J. Coleman, Discovery of the dual orexin receptor antagonist [(7R)-4-(5-chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan1-yl][5-methyl-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (MK-4305) for the treatment of insomnia, J. Med. Chem. 53 (2010) 5320–5332.
S.A. Breidinger et al. / J. Chromatogr. B 1002 (2015) 254–259 [4] C.J. Winrow, A.L. Gotter, C.D. Cox, S.M. Doran, P.L. Tannenbaum, M.J. Breslin, S.L. Garson, S.V. Fox, C.M. Harrell, J. Stevens, D.R. Reiss, D. Cui, P.J. Coleman, J.J. Renger, Promotion of sleep by suvorexant—a novel dual orexin receptor antagonist, J. Neurogenet. 25 (2011) 52–61. [5] United States Food and Drug Administration, Guidance for Industry – Bioanalytical Method Validation, 2001, May, http://www.fda.gov/downloads/Drugs/ Guidances/ucm070107.pdf
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[6] European Medicines Agency, Guideline on Bioanalytical Method Validation, 2011, July, http://www.ema.europa.eu/docs/en GB/document library/ Scientific guideline/2011/08/WC500109686.pdf [7] Merck & Co., Suvorexant Prescribing Information, 2014, http://www.merck.com/ product/usa/pi circulars/b/belsomra/belsomra pi.pdf