- Email: [email protected]
MS and 1H NMR
Accepted Manuscript Title: Forced degradation of Fingolimod: effect of co-solvent and characterization of degradation products by UHPLC-Q-TOF-MS/MS and 1 H NMR Author: Prinesh N. Patel Pradipbhai D. Kalariya S. Gananadhamu R. Srinivas PII: DOI: Reference:
S0731-7085(15)30083-2 http://dx.doi.org/doi:10.1016/j.jpba.2015.07.028 PBA 10184
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
13-3-2015 12-7-2015 22-7-2015
Please cite this article as: P.N. Patel, P.D. Kalariya, S. Gananadhamu, R. Srinivas, Forced degradation of Fingolimod: effect of co-solvent and characterization of degradation products by UHPLC-Q-TOF-MS/MS and 1 H NMR, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.07.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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*Graphical Abstract
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Forced degradation study of fingolimod was carried out as per ICH guidelines. The drug degraded in base hydrolysis to form 3 degradation products (DPs). DPs were characterized by LC-QTOF-MS/MS and NMR. Acetonitrile as co-solvent in stress studies reacted and formed acetylated DPs.
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*Revised Manuscript
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Forced degradation of Fingolimod: effect of co-solvent and characterization of degradation products by UHPLC-Q-TOF-MS/MS and 1H NMR
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Prinesh N. Patela, Pradipbhai D. Kalariyaa, S. Gananadhamu a*, R. Srinivasa, b*
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a
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad, 500037, Telangana, India
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National Centre for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology,
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Hyderabad, 500 007, Telangana, India
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a, b
*Corresponding author
: [email protected] (R. Srinivas) : [email protected]
Telephone Number
: +91-40-27193122
Fax Number
: +91-40-27193156
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Abstract:
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Fingolimod (FGL), an immunomodulator drug for treating multiple sclerosis, was subjected to
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hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per
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International Conference on Harmonization specified conditions. The drug showed extensive
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degradation under base hydrolysis, however, it was stable under all other conditions. A total of 3
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degradation products (DPs) were observed. The chromatographic separation of the drug and its
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degradation products was achieved on a Fortis C18 (100 x 2.1 mm, 1.7 µm) column with a
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mobile phase composed of 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) in gradient
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mode. All the DPs were identified and characterized by liquid chromatography-quadrupole time
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of flight-mass spectrometry (LC-Q-TOF-MS) in combination with accurate mass measurements.
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The major DP was isolated and characterized by Nuclear Magnetic resonance spectroscopy. This
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is a typical case of degradation where acetonitrile used as co-solvent in stress studies, reacts with
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FGL in base hydrolytic conditions to produce acetylated DPs. Hence, it can be suggested that
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acetonitrile is not preferable as a co-solvent for stress degradation of FGL. The developed
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UHPLC method was validated as per ICH guidelines.
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Keywords: Fingolimod; UHPLC Stability Assay, Forced Degradation; Stress Studies; LC-
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MS/MS
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1. Introduction Fingolimod hydrochloride (FGL), 2-amino-2-[2-(4-octylphenyl) ethyl] propane-1, 3-diol
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hydrochloride, a sphingosine 1-phosphate receptor modulator has been used for treating multiple
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sclerosis. On September 22, 2010, FGL became the first oral disease-modifying drug approved
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by the Food and Drug Administration to reduce relapses and delay disability progression in
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patients with relapsing forms of multiple sclerosis[1].
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Forced degradation studies involve subjecting the drug to hydrolytic, oxidative, photo
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and thermal stress conditions, liquid chromatographic (LC) method development for separation
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of the drug and its degradation products (DPs), followed by structure elucidation of DPs using
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modern hyphenated instruments [2-5]. The identification and characterization of degradation
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products (DPs) help to establish degradation pathway of the drug, which is important from the
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view point of the drug development process.
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Reported literature for analytical methods for FGL includes LC-MS/MS method for
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determination of FGL with its metabolite in human blood[6] and in murine intracellular
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compartments and human plasma[7]. Stability assay methods for FGL are also reported by
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HPLC[8-10] and UHPLC[11], however, characterization of degradation products have not been
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attempted in any of them. The main aim of the present work is to develop a validated stability-
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indicating UHPLC method for FGL and to characterize the degradation products by UHPLC-
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QTOF-MS/MS and NMR studies. In addition, this is the first report on reaction of the co-solvent
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acetonitrile with the drug to form the acetylated products during stress studies. To the best of our
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knowledge, these products are novel and have not been reported previously.
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2. Experimental
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2.1 Chemicals and reagents
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Pure fingolimod hydrochloride was purchased from Clearsynth Labs Pvt. Ltd., Mumbai,
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India. HPLC gradient grade acetonitrile (ACN) and methanol (MeOH) were purchased from
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Merck, India. HPLC grade water was prepared by filtrating through a Milli-Q- plus system
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(Millipore, Milford, MA, USA). Ammonium acetate and ammonium formate of HPLC grade
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were purchased from Finar Chemicals Pvt. Ltd. (Ahmedabad, India). All analytical grade
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reagents: formic acid, sodium hydroxide, hydrochloric acid and 30% hydrogen peroxide (H2O2)
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were purchased from Merck (Mumbai, India).
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2.2 Instrumentation
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2.2.1 Liquid chromatography
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The UPLC analysis was performed on Water’s Acquity UPLC H-Class comprised of
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quarternary solvent manager plus sample manager with a flow-through needle design and a
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Photo Diode Array (PDA) detector. The output signal was monitored and processed using
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Empower 3 software. All pH measurements were done on a pH tutor (Eutech Instruments) and
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weighing was carried out on Sartorius balance (CPA225D, Germany).
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2.2.2 Mass spectrometry
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For UHPLC-MS analysis, an Agilent 1290 series LC instrument (Agilent Technologies,
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USA) attached to a quadrupole – time of flight (Q-TOF) mass spectrometer (Q-TOF LC/MS)
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6540 series, Agilent Technologies, USA) was used. The data acquisition was under the control of
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Mass Hunter workstation software. Electrospray ionization (ESI) with positive ionization mode
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was found to be suitable. The typical operating source conditions for MS scan of PZ in positive
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ESI mode were optimized as follows: the fragmentor voltage was set at 140 V, the capillary at
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3500 V, the skimmer at 65 V, nitrogen as the drying gas (350 °C,10 L/min), and nebulising (40
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psi) gas. For collision-induced dissociation (CID) experiments, keeping MS1 static, the precursor
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ion of interest was selected using the quadrupole analyzer and the product ions were analyzed
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using a TOF analyzer. Ultra high pure nitrogen gas was used as collision gas. Photolytic studies were carried out in a photostability chamber (Osworld OPSH-G-16-
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GMP series, Osworld Scientific Equipments Pvt. Ltd. India) set at 40 ± 5°C/ 75% RH ± 3% RH
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and equipped with an illumination bank on inside top, consisting of a combination of two black
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light UV lamps and four white fluorescent lamps in accordance with of the ICH guideline Q1B.
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2.3 Sample preparation
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For all the stress studies except acid hydrolysis, 5 mg drug was dissolved in 1 mL ACN and then
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4 mL of stressor was added. Acid hydrolysis was carried out by dissolving 5 mg drug in 5 mL of
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3N HCl. Acid and base hydrolysis samples were neutralized with appropriate amount of NaOH
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and HCl solution respectively and then they were diluted to 250 µg/mL using ACN - water
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(50:50, v/v). Other stressed samples (neutral hydrolysis, oxidative, thermal, and photolytic stress)
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were diluted with ACN - water (50:50, v/v) to 250 µg/mL concentrations and filtered through
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0.22 µm membrane filter before LC and LC/MS analysis. Base hydrolysis was also carried out
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using MeOH as co-solvent instead of ACN and subjected to same stress conditions.
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2.4 Forced degradation studies
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Stress studies were carried out on the bulk drug under ICH recommended conditions of
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hydrolysis, photolysis, oxidation and dry heat. For stress studies, concentration of FGL was kept
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1.0 mg/mL. Acid hydrolysis was carried out by refluxing the drug in 3N HCl for 48 hr. Base
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hydrolysis was carried out by refluxing the drug in 0.1N NaOH for 1 hr. For neutral hydrolysis,
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drug solution was refluxed at 80 °C for 48 hr. For oxidative degradation, the drug was subjected
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to 30% H2O2 at room temperature for 7 days. Photo degradation study was done by exposing the
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drug in solution (0.1N HCl, 0.1 N NaOH and water) as well as in solid form to 1.2 × 106 lux h of
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fluorescent light and 200 W h m−2 UV light in a photostability chamber. For thermal stress study,
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the drug was kept in an oven at 90 °C in oven for 7 days.
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2.5 Chromatographic conditions
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The chromatographic conditions were optimized using Fortis C18 (100 x 2.1 mm, 1.7 µm)
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column with a mobile phase composed of 0.1 % formic acid (Solvent A) and acetonitrile
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(Solvent B) in gradient mode. The gradient programme was set as follows: (tmin/% proportion of
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solvent B):
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injection volume and detection wavelength were 1 µL and 220 nm, respectively. Column
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temperature was kept 30 °C. For LC/MS analysis, conditions such as nebulizing gas flow,
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capillary voltage, drying gas temperature, skimmer voltage, drying gas flow, and spray voltage
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were optimized to get maximum ionization of FGL and all the DPs.
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2.6 Preparative LC conditions
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A waters semi-preparative chromatograph equipped with waters 515 HPLC pump and waters
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2489 UV- visible detector was used. Waters µBondapak C18 Prep Column (125Å, 10 µm, 7.8
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mm X 300 mm) was employed for isolation. The mobile phase consisted of 0.1% formic acid:
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acetonitrile (80:20 % v/v) in isocratic mode. The flow rate was 3 mL/min.
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2.7 Isolation of degradation product F-3 by preparative HPLC
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A total of three DPs (F-1 to F-3) were formed in base hydrolysis in 0.1 N NaOH in 1 hr at reflux.
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When same base hydrolysis was extended for 3 days, F-1 and F-2 disappeared and F-3 only
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remained intact. This solution was used for isolation of F-3 by repeated injections into semi
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preparative HPLC. Fractions containing > 97 % purity of F-3 were collected and
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together; concentrated on rotavapor to remove acetonitrile and the aqueous solution was
The flow rate was 0.4 mL/min. The
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pooled
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lyophilised using a freeze dryer. The DP, F-3 was obtained as a white powder with
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chromatographic purity of 99.2%.
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2.8 NMR spectroscopy
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The 1H experiments were performed on a 500 MHz NMR (AVANCE III HD-500, Bruker,
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Switzerland) spectrometer using CDCl3 as solvent. 1H chemical shift values were reported on the
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δ scale in ppm relative to TMS (δ = 0.00 ppm) as internal standard.
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3.0 Results and discussion
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3.1 Optimization of chromatographic conditions
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In order to achieve the optimum separation of FGL and its DPs, numerous changes in the
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chromatographic conditions such as pH and composition of the mobile phase, flow rate, column,
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etc. were carried out. Various trials were done using different buffers and columns. Different
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mobile phase compositions using buffers pH 3.0, pH 4.5, pH 5.5 and acetonitrile and methanol
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were tried. Fortis C18 provided better peak symmetry than BEH C18 column. As FGL is very
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hydrophobic in nature, methanol was not suitable for elution. When using pH more than 3.0,
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FGL gave very broad peak with tailing greater than 2. To reduce tailing, 0.1% formic acid was
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tried and it gave best peak symmetry among all the trials done. Finally 0.1 % formic acid in
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water and acetonitrile using proposed gradient programme gave sufficient resolution for all the
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degradants.
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3.2 Degradation behavior of the drug
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The degradation behavior of FGL was studied using UHPLC-PDA and UHPLC-MS under
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various forced degradation conditions. FGL was found to be stable in acid and neutral hydrolytic
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conditions (Fig.S1, see supplementary material). A total of three DPs (F-1 to F-3) were formed in
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base hydrolysis (Fig.1) in 0.1 N NaOH in 1 hr (~ 45 % degradation in 1 hr). Whereas in same
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base hydrolysis conditions after 3 days only F-3 remained unchanged and F-1 and F-2
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disappeared. This was taken as an advantage for preparative isolation of F-3 for characterization
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using NMR spectroscopy. The drug was found to be stable in oxidative condition even after
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subjecting to 30% H2O2 at room temperature for 7 days (Fig. S1 (d), see supplementary
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material).
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On exposing the solid drug sample and neutral drug solution at 1.2 million lux hours and
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200W h/m2 for three days, no DPs were formed (Fig. S1, supplementary material). Drug in basic
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solution after photolysis (Fig.S1 (h), supplementary material) showed formation of the three DPs
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(F-1 to F-3) which were also observed in base hydrolysis. The drug was found to be stable in
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thermal conditions even after keeping the drug at 90 °C in oven for 7 days (Fig. S1 (I), see
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supplementary material). A total of three DPs were formed and characterized by using LC–ESI-
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QTOF-MS/MS experiments and accurate mass measurements. The proposed structures and their
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elemental compositions are given in scheme 1 and table 1, respectively. The overlay of UHPLC
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chromatograms of base hydrolysis of FGL for the effect of co-solvent, ACN and MeOH are
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given in fig. 1.
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3.3 LC/ESI/MS/MS study of FGL and its degradation products
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The developed UHPLC method was found to be suitable for UHPLC-MS analysis for
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identification of degradation products.
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3.3.1 MS/MS of FGL
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To understand the degradation behaviour of FGL, the MS/MS spectrum (Fig. 2(a)) of protonated
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FGL ([M+H]+, m/z 308; retention time (Rt) = 3.40 min) was studied. The spectrum shows
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abundant product ions at m/z 273 (loss of -H2O and NH3), m/z 255 (loss of -2H2O and NH3), m/z
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243 (loss of -CHOH from m/z 273), m/z 185 (loss of C5H10 from m/z 255). A series of product
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ions were formed from the octyl side chain of FGL. It can be noted that the fragment ion at m/z
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255 is characteristic for (4-octylphenyl)but-3-en-1-ylium skeleton of FGL. The elemental
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compositions of all these ions have been confirmed by accurate mass measurements (Table 1).
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3.3.2 MS/MS of degradation products
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On line LC/ESI/MS/MS experiments were performed to characterize all the DPs ((F-1 to F-
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3) formed under stress conditions. Most plausible structures have been proposed for all the DPs
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of FGL based on the m/z values of their [M+H]+ ions and the MS/MS data in combination with
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elemental compositions derived from accurate mass measurements as discussed below.
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The MS/MS spectra of both F-1(Rt = 3.52 min., Fig 2(b)) and F-3 (Rt = 4.94 min., Fig. 3(b))
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show [M+H]+ ion at m/z 350 with an elemental composition of C21H36NO3. The increase of 42
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Da in molecular weight as compared to that of the drug can be attributed to the addition of an
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acetyl group to the drug. To elucidate the structure of F-1 and F-3, the MS/MS spectra were
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examined. As shown in scheme 1, the MS/MS of [M+H]+ of F-1 gives the product ions at m/z
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290 (loss of –CH3COOH), m/z 272 (loss of -H2O from m/z 290), m/z 255 (loss of NH3 from m/z
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272). Protonated F-3 gives the product ions at m/z 332 (loss of -H2O), m/z 308 (loss of –COCH2),
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m/z 290 (loss of –COCH2 from m/z 332), m/z 272 (loss of -H2O from m/z 290) and m/z 60
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(protonated acetamide ion) (scheme 1). The formation of an abundant product ion at m/z 60
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suggests acetylation of free amine group of FGL in F-3 whereas absence of m/z 60 and m/z 332
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confirms acetylation at –OH group of FGL in F-1. The structure of F-3 was also confirmed by
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NMR. 1H NMR values (Fig. 4) in CDCl3, δ (ppm) 7.10 (s, 4 H), 5.77 (s, 1 H), 3.88-3.86 (d, J =
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11.2 Hz, 2 H), 3.71 (s, 2 H), 3.63-3.61 (d, J = 11.1 Hz, 2 H), 2.64 (t, J = 8.0 Hz, 2 H), 2.57 (t, J =
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7.6 Hz, 2 H), 1.96-1.91 (m, 5 H), 1.29-1.27 (m, 10 H), 0.89 (t, J = 6.8 Hz, 3 H). The NMR
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spectra of FGL (Fig. S3, supplementary material) and F-3 were compared for characterization.
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The most characteristic peak at δ 5.77 (s, 1 H) of amine proton and δ 1.95 (s, 3H) of CH3 of –
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NHCOCH3 confirmed the N-acetylation on free –NH2 group of FGL in F-3. The above spectral
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data supports the assigned structure, 2-amino-2-(hydroxy methyl)-4-(4-octyl phenyl) butyl
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acetate for F-1 and N-(1-hydroxy-2-(hydroxy methyl)-4-(4-octyl phenyl) butan-2-yl) acetamide
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for F-3.
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The DP, F-2 (Rt = 4.41 min) exhibited the [M+H]+ at m/z 332 with an elemental composition
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of C21H34NO2. Its MS/MS spectrum (Fig. 3(a)) shows characteristic product ions at m/z 314 (loss
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of -H2O), m/z 290 (loss of COCH2), m/z 272 (loss of H2O from m/z 290) and m/z 60 (protonated
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acetamide) (scheme 1). The elemental compositions of product ions have been confirmed by
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accurate mass measurements (Table 1). All these data are consistent with the proposed structure,
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1-(2-(hydroxy methyl)-2-(4-octyl phenethyl) aziridin-1-yl) ethanone for F-2.
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3.4 Plausible mechanisms of formation DPs
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As shown in the fig. 1, all the DPs were formed only when ACN was used as co-solvent in base
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hydrolysis. However, they were not formed when exposed to same stress conditions using
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MeOH as co-solvent. This suggested that they were formed due to presence of ACN. As
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explained in the scheme 2, ACN may get hydrolyzed to form sodium acetate in presence of
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NaOH [12] which reacts with FGL to form acetylated DPs, F-1 and F-3. F-2 may be formed by
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dehydration of F-3.
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3.5 Method Validation
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The stability indicating UHPLC-PDA method for FGL was validated for specificity, linearity,
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accuracy and precision as per ICH guideline Q2 (R1). The specificity of the method was
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established by determining peak purity for FGL in a mixture of stressed samples using a
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photodiode array (PDA) detector and evaluation of the resolution factor. UHPLC-MS showed an
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excellent purity for FGL and every degradation product for all the degradation samples, which
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unambiguously confirms the specificity of the method. To establish linearity and range, solutions
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in the concentration range of 5 – 200 µg/mL of FGL were prepared. The linearity test solutions
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were prepared and analyzed in triplicate. The response for the drug was linear in the investigated
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concentration (r2= 0.999) and the % RSD for each investigated concentration was < 0.14%.
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(Table S1, supplementary material)). Table S2 (supplementary material) shows accuracy data at
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three different concentrations 20, 50 and 100 µg/mL of FGL in triplicate analysis. The recoveries
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of the added drug were calculated from the difference between peak areas of fortified and
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unfortified degraded samples. The recovery of FGL in the presence of degradation products
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ranged from 99.07 to 99.89 %. The intra- and inter-day precisions were determined at three
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different concentrations 20, 50 and 100 µg/mL, on the same day (n= 3) and consecutive days (n
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= 3). Intermediate precision was determined on different days and also by different analysts.
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Table S4 (supplementary material) shows that the %RSD for intra-day and intermediate
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precision was < 0.62 % and 0.88 % respectively, indicating that the method was sufficiently
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precise. Robustness of proposed method was determined by purposely changing the flow rate
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(0.35 – 0.45 mL/min), column temperature (30 ± 5 °C) and change in the % of formic acid of
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mobile phase (0.1 ± 0.02 %) at three different concentrations (20, 50, 100 µg/mL). Each sample
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was injected in triplicate (n = 3), and peak areas obtained were used to calculate means and %
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RSD values. The % RSD was < 1%. Lack of significant change in assay value observed by
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changing these chromatographic conditions confirmed the robustness of the method.
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4. Conclusion
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Stress degradation studies on fingolimod, carried out according to ICH guidelines, provided
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information on the degradation behavior of the drug under conditions of hydrolysis, oxidation,
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photolysis and thermal stress. This is a typical case of degradation where co-solvent acetonitrile
248
reacts with FGL in base hydrolytic stress conditions to produce 3 acetylated degradation
249
products, all of which were hitherto unknown. The degradation products were characterized
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using LC–Q-TOF-MS/MS and 1H NMR studies. The structures were justified by mechanisms of
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their formation. The UHPLC method was validated as per ICH guidelines and can be used for
252
routine analysis and stability studies.
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Acknowledgements
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The authors thank Dr. Ahmed Kamal, Project Director, NIPER, Hyderabad and Dr. S.
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Chandrasekhar, Director, IICT, Hyderabad for facilities (AARF) and their cooperation. P.P and
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P.K are thankful to Department of Pharmaceuticals, Ministry of chemicals and fertilizers, Govt.
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of India, New Delhi for providing Research Fellowship.
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Sci Pharm, 83 (2015) 85-93.
291
[11] N. Rajan, K.A. Basha, Rapid determination of fingolimod hydrochloride-related Substances
292
and degradation products in API and pharmaceutical dosage forms by use of a stability-
293
indicating UPLC method, Chromatographia, 77 (2014) 1545-1552.
294
[12] S.W. Baertschi, K.M. Alsante, R.A. Reed, Pharmaceutical stress testing: predicting drug
295
degradation, CRC Press, 2011.
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296 297 298
Scheme 1: Proposed fragmentation pathway of protonated fingolimod (FGL) and its DPs (F-1 to F-3)
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Scheme 2: Probable mechanism of formation of DPs
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Figure Captions:
303
Fig. 1.Overlay of base hydrolysis chromatograms for effect of co-solvent [a] ACN, [b] MeOH
304
Fig. 2 ESI/MS/MS spectrum of (a) FGL (m/z 308) at 20 eV, (b) F-1 (m/z 350) at 20eV
305
Fig. 3 ESI/MS/MS spectrum of (a) F-2 (m/z 332) at 20 eV, (b) F-3 (m/z 350) at 20 eV
306
Fig. 4 1H NMR spectrum of F-3
307
List of tables:
308
Table 1: High resolution mass spectrometry (HRMS) data of FGL and degradation products
309
along with their Elemental composition and major fragments
310
Supplementary material:
311
Fig. S1. [a] Acid degradation [b] Base degradation [c] Neutral degradation [d] Oxidative
312
degradation [e] Photo degradation of Solid sample [f] Photo degradation of neutral solution,
313
[g] Photo degradation sample in 0.1N HCl, [h] Photo degradation sample in 0.1N NaOH [i]
314
Photo degradation of Solid sample in florescent light
315
Fig. S2 Base degradation showing the separation of F-1 at 0.1% level of FGL
316
Fig. S3 NMR spectrum of fingolimod hydrochloride
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317 318
Table S1. Parameters for linear regression equation
319
Table S2: Accuracy and Precision data
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table 1.doc
Table 1: High resolution - mass spectrometry (HR-MS) data of fingolimod and degradation products along with their elemental composition and major fragments
F-3
131.0859 117.0699 105.0700 91.0542 71.0857 57.0700
131.0855 117.0699 105.0699 91.0542 71.0855 57.0699
C21H36NO3+ C21H32NO+ C19H30N+ C19H27+ C21H34NO2+ C21H32NO+ C21H32NO+
350.2693 290.2478 272.2366 255.2097 332.2575 314.2470 290.2467
-4.52 -4.80 -1.48 0.54 0.00 -0.70
M
an
-3.05 0.00 -0.95 0.00 -2.81 -1.75
350.2690 290.2478 272.2373 255.2107 332.2584 314.2478 290.2478
-0.86 0.00 2.57 3.92 2.71 2.55 3.79
C19H30N+ C19H27+ C2H5NO+ C21H36NO3+ C21H34NO2+ C19H34NO2+
272.2369 255.2104 60.0442 350.2685 332.2567 308.2570
272.2373 255.2107 60.0444 350.2690 332.2584 308.2584
1.47 1.18 3.33 1.43 5.12 4.54
C19H30N+
272.2372 255.2097 60.0448
272.2373 255.2107 60.0444
0.37 3.92 -6.66
C19H27
+
C2H5NO+
ip t
C10H11+ C9H9+ C8H9+ C7H7+ C5H11+ C4H9+
-3.24 -1.10 -5.09
cr
243.2107 229.1951 203.1794 185.1325 157.1012 143.0855
Error
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243.2118 229.1962 203.1797 185.1324 157.1012 143.0856
d
F-2
C18H27+ C17H25+ C15H23+ C14H17+ C12H13+ C11H11+
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F-1
308.2594 273.2216 255.2120
Calculated m/z 308.2584 273.2213 255.2107
Observed m/z
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Molecular Formula C19H34NO2+ C19H29O+ C19H27+
Page 19 of 23
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Fig. 1
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Fig. 2
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Fig. 3
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S0731-7085(15)30083-2 http://dx.doi.org/doi:10.1016/j.jpba.2015.07.028 PBA 10184
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
13-3-2015 12-7-2015 22-7-2015
Please cite this article as: P.N. Patel, P.D. Kalariya, S. Gananadhamu, R. Srinivas, Forced degradation of Fingolimod: effect of co-solvent and characterization of degradation products by UHPLC-Q-TOF-MS/MS and 1 H NMR, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.07.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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*Graphical Abstract
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highlights_.doc
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Forced degradation study of fingolimod was carried out as per ICH guidelines. The drug degraded in base hydrolysis to form 3 degradation products (DPs). DPs were characterized by LC-QTOF-MS/MS and NMR. Acetonitrile as co-solvent in stress studies reacted and formed acetylated DPs.
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*Revised Manuscript
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Forced degradation of Fingolimod: effect of co-solvent and characterization of degradation products by UHPLC-Q-TOF-MS/MS and 1H NMR
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Prinesh N. Patela, Pradipbhai D. Kalariyaa, S. Gananadhamu a*, R. Srinivasa, b*
4
a
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad, 500037, Telangana, India
7 b
National Centre for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology,
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Hyderabad, 500 007, Telangana, India
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a, b
*Corresponding author
: [email protected] (R. Srinivas) : [email protected]
Telephone Number
: +91-40-27193122
Fax Number
: +91-40-27193156
21
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Abstract:
23
Fingolimod (FGL), an immunomodulator drug for treating multiple sclerosis, was subjected to
24
hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per
25
International Conference on Harmonization specified conditions. The drug showed extensive
26
degradation under base hydrolysis, however, it was stable under all other conditions. A total of 3
27
degradation products (DPs) were observed. The chromatographic separation of the drug and its
28
degradation products was achieved on a Fortis C18 (100 x 2.1 mm, 1.7 µm) column with a
29
mobile phase composed of 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) in gradient
30
mode. All the DPs were identified and characterized by liquid chromatography-quadrupole time
31
of flight-mass spectrometry (LC-Q-TOF-MS) in combination with accurate mass measurements.
32
The major DP was isolated and characterized by Nuclear Magnetic resonance spectroscopy. This
33
is a typical case of degradation where acetonitrile used as co-solvent in stress studies, reacts with
34
FGL in base hydrolytic conditions to produce acetylated DPs. Hence, it can be suggested that
35
acetonitrile is not preferable as a co-solvent for stress degradation of FGL. The developed
36
UHPLC method was validated as per ICH guidelines.
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Keywords: Fingolimod; UHPLC Stability Assay, Forced Degradation; Stress Studies; LC-
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MS/MS
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1. Introduction Fingolimod hydrochloride (FGL), 2-amino-2-[2-(4-octylphenyl) ethyl] propane-1, 3-diol
42
hydrochloride, a sphingosine 1-phosphate receptor modulator has been used for treating multiple
43
sclerosis. On September 22, 2010, FGL became the first oral disease-modifying drug approved
44
by the Food and Drug Administration to reduce relapses and delay disability progression in
45
patients with relapsing forms of multiple sclerosis[1].
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Forced degradation studies involve subjecting the drug to hydrolytic, oxidative, photo
47
and thermal stress conditions, liquid chromatographic (LC) method development for separation
48
of the drug and its degradation products (DPs), followed by structure elucidation of DPs using
49
modern hyphenated instruments [2-5]. The identification and characterization of degradation
50
products (DPs) help to establish degradation pathway of the drug, which is important from the
51
view point of the drug development process.
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Reported literature for analytical methods for FGL includes LC-MS/MS method for
53
determination of FGL with its metabolite in human blood[6] and in murine intracellular
54
compartments and human plasma[7]. Stability assay methods for FGL are also reported by
55
HPLC[8-10] and UHPLC[11], however, characterization of degradation products have not been
56
attempted in any of them. The main aim of the present work is to develop a validated stability-
57
indicating UHPLC method for FGL and to characterize the degradation products by UHPLC-
58
QTOF-MS/MS and NMR studies. In addition, this is the first report on reaction of the co-solvent
59
acetonitrile with the drug to form the acetylated products during stress studies. To the best of our
60
knowledge, these products are novel and have not been reported previously.
61
2. Experimental
62
2.1 Chemicals and reagents
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Pure fingolimod hydrochloride was purchased from Clearsynth Labs Pvt. Ltd., Mumbai,
64
India. HPLC gradient grade acetonitrile (ACN) and methanol (MeOH) were purchased from
65
Merck, India. HPLC grade water was prepared by filtrating through a Milli-Q- plus system
66
(Millipore, Milford, MA, USA). Ammonium acetate and ammonium formate of HPLC grade
67
were purchased from Finar Chemicals Pvt. Ltd. (Ahmedabad, India). All analytical grade
68
reagents: formic acid, sodium hydroxide, hydrochloric acid and 30% hydrogen peroxide (H2O2)
69
were purchased from Merck (Mumbai, India).
70
2.2 Instrumentation
71
2.2.1 Liquid chromatography
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The UPLC analysis was performed on Water’s Acquity UPLC H-Class comprised of
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quarternary solvent manager plus sample manager with a flow-through needle design and a
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Photo Diode Array (PDA) detector. The output signal was monitored and processed using
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Empower 3 software. All pH measurements were done on a pH tutor (Eutech Instruments) and
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weighing was carried out on Sartorius balance (CPA225D, Germany).
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2.2.2 Mass spectrometry
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For UHPLC-MS analysis, an Agilent 1290 series LC instrument (Agilent Technologies,
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USA) attached to a quadrupole – time of flight (Q-TOF) mass spectrometer (Q-TOF LC/MS)
80
6540 series, Agilent Technologies, USA) was used. The data acquisition was under the control of
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Mass Hunter workstation software. Electrospray ionization (ESI) with positive ionization mode
82
was found to be suitable. The typical operating source conditions for MS scan of PZ in positive
83
ESI mode were optimized as follows: the fragmentor voltage was set at 140 V, the capillary at
84
3500 V, the skimmer at 65 V, nitrogen as the drying gas (350 °C,10 L/min), and nebulising (40
85
psi) gas. For collision-induced dissociation (CID) experiments, keeping MS1 static, the precursor
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ion of interest was selected using the quadrupole analyzer and the product ions were analyzed
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using a TOF analyzer. Ultra high pure nitrogen gas was used as collision gas. Photolytic studies were carried out in a photostability chamber (Osworld OPSH-G-16-
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GMP series, Osworld Scientific Equipments Pvt. Ltd. India) set at 40 ± 5°C/ 75% RH ± 3% RH
90
and equipped with an illumination bank on inside top, consisting of a combination of two black
91
light UV lamps and four white fluorescent lamps in accordance with of the ICH guideline Q1B.
92
2.3 Sample preparation
93
For all the stress studies except acid hydrolysis, 5 mg drug was dissolved in 1 mL ACN and then
94
4 mL of stressor was added. Acid hydrolysis was carried out by dissolving 5 mg drug in 5 mL of
95
3N HCl. Acid and base hydrolysis samples were neutralized with appropriate amount of NaOH
96
and HCl solution respectively and then they were diluted to 250 µg/mL using ACN - water
97
(50:50, v/v). Other stressed samples (neutral hydrolysis, oxidative, thermal, and photolytic stress)
98
were diluted with ACN - water (50:50, v/v) to 250 µg/mL concentrations and filtered through
99
0.22 µm membrane filter before LC and LC/MS analysis. Base hydrolysis was also carried out
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using MeOH as co-solvent instead of ACN and subjected to same stress conditions.
101
2.4 Forced degradation studies
102
Stress studies were carried out on the bulk drug under ICH recommended conditions of
103
hydrolysis, photolysis, oxidation and dry heat. For stress studies, concentration of FGL was kept
104
1.0 mg/mL. Acid hydrolysis was carried out by refluxing the drug in 3N HCl for 48 hr. Base
105
hydrolysis was carried out by refluxing the drug in 0.1N NaOH for 1 hr. For neutral hydrolysis,
106
drug solution was refluxed at 80 °C for 48 hr. For oxidative degradation, the drug was subjected
107
to 30% H2O2 at room temperature for 7 days. Photo degradation study was done by exposing the
108
drug in solution (0.1N HCl, 0.1 N NaOH and water) as well as in solid form to 1.2 × 106 lux h of
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fluorescent light and 200 W h m−2 UV light in a photostability chamber. For thermal stress study,
110
the drug was kept in an oven at 90 °C in oven for 7 days.
111
2.5 Chromatographic conditions
112
The chromatographic conditions were optimized using Fortis C18 (100 x 2.1 mm, 1.7 µm)
113
column with a mobile phase composed of 0.1 % formic acid (Solvent A) and acetonitrile
114
(Solvent B) in gradient mode. The gradient programme was set as follows: (tmin/% proportion of
115
solvent B):
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injection volume and detection wavelength were 1 µL and 220 nm, respectively. Column
117
temperature was kept 30 °C. For LC/MS analysis, conditions such as nebulizing gas flow,
118
capillary voltage, drying gas temperature, skimmer voltage, drying gas flow, and spray voltage
119
were optimized to get maximum ionization of FGL and all the DPs.
120
2.6 Preparative LC conditions
121
A waters semi-preparative chromatograph equipped with waters 515 HPLC pump and waters
122
2489 UV- visible detector was used. Waters µBondapak C18 Prep Column (125Å, 10 µm, 7.8
123
mm X 300 mm) was employed for isolation. The mobile phase consisted of 0.1% formic acid:
124
acetonitrile (80:20 % v/v) in isocratic mode. The flow rate was 3 mL/min.
125
2.7 Isolation of degradation product F-3 by preparative HPLC
126
A total of three DPs (F-1 to F-3) were formed in base hydrolysis in 0.1 N NaOH in 1 hr at reflux.
127
When same base hydrolysis was extended for 3 days, F-1 and F-2 disappeared and F-3 only
128
remained intact. This solution was used for isolation of F-3 by repeated injections into semi
129
preparative HPLC. Fractions containing > 97 % purity of F-3 were collected and
130
together; concentrated on rotavapor to remove acetonitrile and the aqueous solution was
The flow rate was 0.4 mL/min. The
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lyophilised using a freeze dryer. The DP, F-3 was obtained as a white powder with
132
chromatographic purity of 99.2%.
133
2.8 NMR spectroscopy
134
The 1H experiments were performed on a 500 MHz NMR (AVANCE III HD-500, Bruker,
135
Switzerland) spectrometer using CDCl3 as solvent. 1H chemical shift values were reported on the
136
δ scale in ppm relative to TMS (δ = 0.00 ppm) as internal standard.
137
3.0 Results and discussion
138
3.1 Optimization of chromatographic conditions
139
In order to achieve the optimum separation of FGL and its DPs, numerous changes in the
140
chromatographic conditions such as pH and composition of the mobile phase, flow rate, column,
141
etc. were carried out. Various trials were done using different buffers and columns. Different
142
mobile phase compositions using buffers pH 3.0, pH 4.5, pH 5.5 and acetonitrile and methanol
143
were tried. Fortis C18 provided better peak symmetry than BEH C18 column. As FGL is very
144
hydrophobic in nature, methanol was not suitable for elution. When using pH more than 3.0,
145
FGL gave very broad peak with tailing greater than 2. To reduce tailing, 0.1% formic acid was
146
tried and it gave best peak symmetry among all the trials done. Finally 0.1 % formic acid in
147
water and acetonitrile using proposed gradient programme gave sufficient resolution for all the
148
degradants.
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3.2 Degradation behavior of the drug
151
The degradation behavior of FGL was studied using UHPLC-PDA and UHPLC-MS under
152
various forced degradation conditions. FGL was found to be stable in acid and neutral hydrolytic
153
conditions (Fig.S1, see supplementary material). A total of three DPs (F-1 to F-3) were formed in
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base hydrolysis (Fig.1) in 0.1 N NaOH in 1 hr (~ 45 % degradation in 1 hr). Whereas in same
155
base hydrolysis conditions after 3 days only F-3 remained unchanged and F-1 and F-2
156
disappeared. This was taken as an advantage for preparative isolation of F-3 for characterization
157
using NMR spectroscopy. The drug was found to be stable in oxidative condition even after
158
subjecting to 30% H2O2 at room temperature for 7 days (Fig. S1 (d), see supplementary
159
material).
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On exposing the solid drug sample and neutral drug solution at 1.2 million lux hours and
161
200W h/m2 for three days, no DPs were formed (Fig. S1, supplementary material). Drug in basic
162
solution after photolysis (Fig.S1 (h), supplementary material) showed formation of the three DPs
163
(F-1 to F-3) which were also observed in base hydrolysis. The drug was found to be stable in
164
thermal conditions even after keeping the drug at 90 °C in oven for 7 days (Fig. S1 (I), see
165
supplementary material). A total of three DPs were formed and characterized by using LC–ESI-
166
QTOF-MS/MS experiments and accurate mass measurements. The proposed structures and their
167
elemental compositions are given in scheme 1 and table 1, respectively. The overlay of UHPLC
168
chromatograms of base hydrolysis of FGL for the effect of co-solvent, ACN and MeOH are
169
given in fig. 1.
170
3.3 LC/ESI/MS/MS study of FGL and its degradation products
171
The developed UHPLC method was found to be suitable for UHPLC-MS analysis for
172
identification of degradation products.
173
3.3.1 MS/MS of FGL
174
To understand the degradation behaviour of FGL, the MS/MS spectrum (Fig. 2(a)) of protonated
175
FGL ([M+H]+, m/z 308; retention time (Rt) = 3.40 min) was studied. The spectrum shows
176
abundant product ions at m/z 273 (loss of -H2O and NH3), m/z 255 (loss of -2H2O and NH3), m/z
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243 (loss of -CHOH from m/z 273), m/z 185 (loss of C5H10 from m/z 255). A series of product
178
ions were formed from the octyl side chain of FGL. It can be noted that the fragment ion at m/z
179
255 is characteristic for (4-octylphenyl)but-3-en-1-ylium skeleton of FGL. The elemental
180
compositions of all these ions have been confirmed by accurate mass measurements (Table 1).
181
3.3.2 MS/MS of degradation products
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On line LC/ESI/MS/MS experiments were performed to characterize all the DPs ((F-1 to F-
183
3) formed under stress conditions. Most plausible structures have been proposed for all the DPs
184
of FGL based on the m/z values of their [M+H]+ ions and the MS/MS data in combination with
185
elemental compositions derived from accurate mass measurements as discussed below.
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The MS/MS spectra of both F-1(Rt = 3.52 min., Fig 2(b)) and F-3 (Rt = 4.94 min., Fig. 3(b))
187
show [M+H]+ ion at m/z 350 with an elemental composition of C21H36NO3. The increase of 42
188
Da in molecular weight as compared to that of the drug can be attributed to the addition of an
189
acetyl group to the drug. To elucidate the structure of F-1 and F-3, the MS/MS spectra were
190
examined. As shown in scheme 1, the MS/MS of [M+H]+ of F-1 gives the product ions at m/z
191
290 (loss of –CH3COOH), m/z 272 (loss of -H2O from m/z 290), m/z 255 (loss of NH3 from m/z
192
272). Protonated F-3 gives the product ions at m/z 332 (loss of -H2O), m/z 308 (loss of –COCH2),
193
m/z 290 (loss of –COCH2 from m/z 332), m/z 272 (loss of -H2O from m/z 290) and m/z 60
194
(protonated acetamide ion) (scheme 1). The formation of an abundant product ion at m/z 60
195
suggests acetylation of free amine group of FGL in F-3 whereas absence of m/z 60 and m/z 332
196
confirms acetylation at –OH group of FGL in F-1. The structure of F-3 was also confirmed by
197
NMR. 1H NMR values (Fig. 4) in CDCl3, δ (ppm) 7.10 (s, 4 H), 5.77 (s, 1 H), 3.88-3.86 (d, J =
198
11.2 Hz, 2 H), 3.71 (s, 2 H), 3.63-3.61 (d, J = 11.1 Hz, 2 H), 2.64 (t, J = 8.0 Hz, 2 H), 2.57 (t, J =
199
7.6 Hz, 2 H), 1.96-1.91 (m, 5 H), 1.29-1.27 (m, 10 H), 0.89 (t, J = 6.8 Hz, 3 H). The NMR
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spectra of FGL (Fig. S3, supplementary material) and F-3 were compared for characterization.
201
The most characteristic peak at δ 5.77 (s, 1 H) of amine proton and δ 1.95 (s, 3H) of CH3 of –
202
NHCOCH3 confirmed the N-acetylation on free –NH2 group of FGL in F-3. The above spectral
203
data supports the assigned structure, 2-amino-2-(hydroxy methyl)-4-(4-octyl phenyl) butyl
204
acetate for F-1 and N-(1-hydroxy-2-(hydroxy methyl)-4-(4-octyl phenyl) butan-2-yl) acetamide
205
for F-3.
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The DP, F-2 (Rt = 4.41 min) exhibited the [M+H]+ at m/z 332 with an elemental composition
207
of C21H34NO2. Its MS/MS spectrum (Fig. 3(a)) shows characteristic product ions at m/z 314 (loss
208
of -H2O), m/z 290 (loss of COCH2), m/z 272 (loss of H2O from m/z 290) and m/z 60 (protonated
209
acetamide) (scheme 1). The elemental compositions of product ions have been confirmed by
210
accurate mass measurements (Table 1). All these data are consistent with the proposed structure,
211
1-(2-(hydroxy methyl)-2-(4-octyl phenethyl) aziridin-1-yl) ethanone for F-2.
212
3.4 Plausible mechanisms of formation DPs
213
As shown in the fig. 1, all the DPs were formed only when ACN was used as co-solvent in base
214
hydrolysis. However, they were not formed when exposed to same stress conditions using
215
MeOH as co-solvent. This suggested that they were formed due to presence of ACN. As
216
explained in the scheme 2, ACN may get hydrolyzed to form sodium acetate in presence of
217
NaOH [12] which reacts with FGL to form acetylated DPs, F-1 and F-3. F-2 may be formed by
218
dehydration of F-3.
219
3.5 Method Validation
220
The stability indicating UHPLC-PDA method for FGL was validated for specificity, linearity,
221
accuracy and precision as per ICH guideline Q2 (R1). The specificity of the method was
222
established by determining peak purity for FGL in a mixture of stressed samples using a
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photodiode array (PDA) detector and evaluation of the resolution factor. UHPLC-MS showed an
224
excellent purity for FGL and every degradation product for all the degradation samples, which
225
unambiguously confirms the specificity of the method. To establish linearity and range, solutions
226
in the concentration range of 5 – 200 µg/mL of FGL were prepared. The linearity test solutions
227
were prepared and analyzed in triplicate. The response for the drug was linear in the investigated
228
concentration (r2= 0.999) and the % RSD for each investigated concentration was < 0.14%.
229
(Table S1, supplementary material)). Table S2 (supplementary material) shows accuracy data at
230
three different concentrations 20, 50 and 100 µg/mL of FGL in triplicate analysis. The recoveries
231
of the added drug were calculated from the difference between peak areas of fortified and
232
unfortified degraded samples. The recovery of FGL in the presence of degradation products
233
ranged from 99.07 to 99.89 %. The intra- and inter-day precisions were determined at three
234
different concentrations 20, 50 and 100 µg/mL, on the same day (n= 3) and consecutive days (n
235
= 3). Intermediate precision was determined on different days and also by different analysts.
236
Table S4 (supplementary material) shows that the %RSD for intra-day and intermediate
237
precision was < 0.62 % and 0.88 % respectively, indicating that the method was sufficiently
238
precise. Robustness of proposed method was determined by purposely changing the flow rate
239
(0.35 – 0.45 mL/min), column temperature (30 ± 5 °C) and change in the % of formic acid of
240
mobile phase (0.1 ± 0.02 %) at three different concentrations (20, 50, 100 µg/mL). Each sample
241
was injected in triplicate (n = 3), and peak areas obtained were used to calculate means and %
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RSD values. The % RSD was < 1%. Lack of significant change in assay value observed by
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changing these chromatographic conditions confirmed the robustness of the method.
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4. Conclusion
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Stress degradation studies on fingolimod, carried out according to ICH guidelines, provided
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information on the degradation behavior of the drug under conditions of hydrolysis, oxidation,
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photolysis and thermal stress. This is a typical case of degradation where co-solvent acetonitrile
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reacts with FGL in base hydrolytic stress conditions to produce 3 acetylated degradation
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products, all of which were hitherto unknown. The degradation products were characterized
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using LC–Q-TOF-MS/MS and 1H NMR studies. The structures were justified by mechanisms of
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their formation. The UHPLC method was validated as per ICH guidelines and can be used for
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routine analysis and stability studies.
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Acknowledgements
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The authors thank Dr. Ahmed Kamal, Project Director, NIPER, Hyderabad and Dr. S.
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Chandrasekhar, Director, IICT, Hyderabad for facilities (AARF) and their cooperation. P.P and
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P.K are thankful to Department of Pharmaceuticals, Ministry of chemicals and fertilizers, Govt.
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of India, New Delhi for providing Research Fellowship.
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References:
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[1] L. Kappos, E.-W. Radue, P. O'Connor, C. Polman, R. Hohlfeld, P. Calabresi, K. Selmaj, C.
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Agoropoulou, M. Leyk, L. Zhang-Auberson, A placebo-controlled trial of oral fingolimod in
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relapsing multiple sclerosis, N. Engl. J. Med., 362 (2010) 387-401.
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[2] S. Singh, T. Handa, M. Narayanam, A. Sahu, M. Junwal, R.P. Shah, A critical review on the
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use of modern sophisticated hyphenated tools in the characterization of impurities and
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degradation products, J. Pharm. Biomed. Anal., 69 (2012) 148-173.
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[3] P.N. Patel, R.M. Borkar, P.D. Kalariya, R.P. Gangwal, A.T. Sangamwar, G. Samanthula, S.
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Ragampeta, Characterization of degradation products of Ivabradine by LC‐HR‐MS/MS: a typical
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case of exhibition of different degradation behaviour in HCl and H2SO4 acid hydrolysis, J. Mass
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Spectrom., 50 (2015) 344-353.
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[4] P.D. Kalariya, M.K. Talluri, P.N. Patel, R. Srinivas, Identification of hydrolytic and isomeric
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N-oxide degradants of vilazodone by on line LC–ESI–MS/MS and APCI–MS, J. Pharm.
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Biomed. Anal., 102 (2015) 353-365.
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[5] P.N. Patel, R.K. Dornala, G. Samanthula, S. Ragampeta, Characterization of stress
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degradation products of tolvaptan by UHPLC-Q-TOF-MS/MS, RSC Adv. 5 (2015) 21142-
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21152.
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[6] C. Emotte, F. Deglave, O. Heudi, F. Picard, O. Kretz, Fast simultaneous quantitative analysis
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of FTY720 and its metabolite FTY720-P in human blood by on-line solid phase extraction
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coupled with liquid chromatography-tandem mass spectrometry, J. Pharm. Biomed. Anal., 58
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(2012) 102-112.
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[7] N. Ferreirós, S. Labocha, M. Schröder, H. Radeke, G. Geisslinger, LC–MS/MS determination
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of FTY720 and FTY720-phosphate in murine intracellular compartments and human plasma, J.
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Chromatogr. B, 887 (2012) 122-127.
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[8] N.R. Kotla, S. Rajan, S. Eshwaraiah, M. Rakesh, M. Kishore, I. Chakravarthy, Stability
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indicating HPLC method for the quantification of fingolimog hydrochloride and its related
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substances, Der pharma chem., 6 (2014) 335-342.
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[9] P.J. Chhabda, M. Balaji, V. Srinivasarao., Development and validation of a stability
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indicating RP-HPLC method for quantification of fingolimod in bulk and pharmaceutical dosage
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form, Pharmanest, 4 (2013) 1206-1218.
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[10] E. Souri, M. Zargarpoor, S. Mottaghi, R. Ahmadkhaniha, A. Kebriaeezadeh, A stability-
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indicating HPLC method for the determination of fingolimod in pharmaceutical dosage forms,
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Sci Pharm, 83 (2015) 85-93.
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[11] N. Rajan, K.A. Basha, Rapid determination of fingolimod hydrochloride-related Substances
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and degradation products in API and pharmaceutical dosage forms by use of a stability-
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indicating UPLC method, Chromatographia, 77 (2014) 1545-1552.
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[12] S.W. Baertschi, K.M. Alsante, R.A. Reed, Pharmaceutical stress testing: predicting drug
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degradation, CRC Press, 2011.
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296 297 298
Scheme 1: Proposed fragmentation pathway of protonated fingolimod (FGL) and its DPs (F-1 to F-3)
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Scheme 2: Probable mechanism of formation of DPs
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Figure Captions:
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Fig. 1.Overlay of base hydrolysis chromatograms for effect of co-solvent [a] ACN, [b] MeOH
304
Fig. 2 ESI/MS/MS spectrum of (a) FGL (m/z 308) at 20 eV, (b) F-1 (m/z 350) at 20eV
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Fig. 3 ESI/MS/MS spectrum of (a) F-2 (m/z 332) at 20 eV, (b) F-3 (m/z 350) at 20 eV
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Fig. 4 1H NMR spectrum of F-3
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List of tables:
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Table 1: High resolution mass spectrometry (HRMS) data of FGL and degradation products
309
along with their Elemental composition and major fragments
310
Supplementary material:
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Fig. S1. [a] Acid degradation [b] Base degradation [c] Neutral degradation [d] Oxidative
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degradation [e] Photo degradation of Solid sample [f] Photo degradation of neutral solution,
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[g] Photo degradation sample in 0.1N HCl, [h] Photo degradation sample in 0.1N NaOH [i]
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Photo degradation of Solid sample in florescent light
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Fig. S2 Base degradation showing the separation of F-1 at 0.1% level of FGL
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Fig. S3 NMR spectrum of fingolimod hydrochloride
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317 318
Table S1. Parameters for linear regression equation
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Table S2: Accuracy and Precision data
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table 1.doc
Table 1: High resolution - mass spectrometry (HR-MS) data of fingolimod and degradation products along with their elemental composition and major fragments
F-3
131.0859 117.0699 105.0700 91.0542 71.0857 57.0700
131.0855 117.0699 105.0699 91.0542 71.0855 57.0699
C21H36NO3+ C21H32NO+ C19H30N+ C19H27+ C21H34NO2+ C21H32NO+ C21H32NO+
350.2693 290.2478 272.2366 255.2097 332.2575 314.2470 290.2467
-4.52 -4.80 -1.48 0.54 0.00 -0.70
M
an
-3.05 0.00 -0.95 0.00 -2.81 -1.75
350.2690 290.2478 272.2373 255.2107 332.2584 314.2478 290.2478
-0.86 0.00 2.57 3.92 2.71 2.55 3.79
C19H30N+ C19H27+ C2H5NO+ C21H36NO3+ C21H34NO2+ C19H34NO2+
272.2369 255.2104 60.0442 350.2685 332.2567 308.2570
272.2373 255.2107 60.0444 350.2690 332.2584 308.2584
1.47 1.18 3.33 1.43 5.12 4.54
C19H30N+
272.2372 255.2097 60.0448
272.2373 255.2107 60.0444
0.37 3.92 -6.66
C19H27
+
C2H5NO+
ip t
C10H11+ C9H9+ C8H9+ C7H7+ C5H11+ C4H9+
-3.24 -1.10 -5.09
cr
243.2107 229.1951 203.1794 185.1325 157.1012 143.0855
Error
us
243.2118 229.1962 203.1797 185.1324 157.1012 143.0856
d
F-2
C18H27+ C17H25+ C15H23+ C14H17+ C12H13+ C11H11+
te
F-1
308.2594 273.2216 255.2120
Calculated m/z 308.2584 273.2213 255.2107
Observed m/z
Ac ce p
FGL
Molecular Formula C19H34NO2+ C19H29O+ C19H27+
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