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MS and NMR
Accepted Manuscript Title: A study on structural characterization of degradation products of cangrelor using LC/QTOF/MS/MS and NMR Authors: Vinodh Guvvala, Venkatesan Chidambaram Subramanian, Jaya Shree Anireddy PII: DOI: Reference:
S0731-7085(18)32181-2 https://doi.org/10.1016/j.jpba.2019.03.031 PBA 12544
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
25 September 2018 13 March 2019 14 March 2019
Please cite this article as: Guvvala V, Chidambaram Subramanian V, Anireddy JS, A study on structural characterization of degradation products of cangrelor using LC/QTOF/MS/MS and NMR, Journal of Pharmaceutical and Biomedical Analysis (2019), https://doi.org/10.1016/j.jpba.2019.03.031 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.
A study on structural characterization of degradation products of cangrelor using LC/QTOF/MS/MS and NMR
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Vinodh Guvvala a, b*, Venkatesan Chidambaram Subramanian b, Jaya Shree Anireddy a Centre for Chemical Science & Technology, Institute of Science & Technology, JNTUH, Kukatpally, Hyderabad 500 085, India b Gland Pharma Ltd, Research and Development, D.P.Pally, Hyderabad 500 043, India *Corresponding author. Email: [email protected]; [email protected]
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Graphical Abstract
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Highlights:
Cangrelor was subjected to acid, base and peroxide stress conditions. Acid and peroxide sensitive
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hydrolysis resulted in formation of six degradation products.
Structures of all the six degradation products were established by extensive characterization of LC/QTOF/MS/MS and NMR spectroscopy.
Liquid chromatography method compatible with mass spectrometry was developed.
Mass fragmentation pathway was established for the cangrelor and degradation products.
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Abstract
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A complete degradation study was performed on cangrelor drug substance as per the ICH guidelines. The study reveals that a total of six degradation products (DP-1 to DP-6) were found and out of these, three
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unknown degradation products (DP-1, DP-5 and DP-6) were not reported in the literature. Based on the degradation study, the drug substance cangrelor was found to be sensitive towards acidic, basic and
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oxidative conditions. Besides, it was stable under thermal and photolytic stress conditions. The
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degradation products were characterized by using advanced LC/QTOF and MS/MS analysis. Further, the structures were characterized by NMR studies. The identified degradation products of cangrelor are
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valuable for cangrelor manufacturing process and quality control.
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Key words: Cangrelor, stressed degradation, LC/QTOF/MS/MS, NMR, degradation products. 1. Introduction
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Cangrelor (Fig.1) is a parenteral drug that reversibly inhibits the P2Y12 receptor like ticagrelor
and does not require metabolic activation. It has rather a short half-life of 3 to 4 minutes; [1,2] and, with an infusion of 4 μg/kg per minute, peak inhibition occurs after 15 minutes. Cangrelor also has a rapid offset, with normal levels of platelet aggregation returning after 60 minutes. Inhibitors of platelet activation and aggregation are substances that are useful during percutaneous coronary intervention and other catheterization techniques in order to reduce bleeding complications and in the treatment of acute 2
coronary syndromes and clotting disorders in general. One class of antiplatelet agents includes the inhibitors of the P2Y12 receptor, a G-protein coupled purinergic receptor which is an important component of platelet activation [3]. Cangrelor (trade name kengreal in the US and kengrexal in Europe) was approved by the United States Food and Drug Administration in 2015. ICH Q3A(R2) and Q3B(R2) recommend the characterization of impurities/degradation products
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that are present at a level greater than the identification threshold in a drug substance or drug product [410]. The stability of drug substance or drug product is a critical parameter which effects on purity, potency and safety. Forced degradation studies give the impurity profile and the behavior of the drug substance under various stress conditions.
In continuation of the identification of impurities during the process development and degradation of drug substance the present work has been taken up on the degradation behavior of cangrelor. The
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present investigation deals with all the degradation studies including acid, base, thermal and photo stability on the drug substance as per the ICH guidelines. The formed degradation products were
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identified through LC/QTOF/MS/MS and NMR spectral analysis [11, 12].
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2. Experimental 2.1. Drugs and chemicals
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Sample of cangrelor API (active pharmaceutical ingredient) was obtained from synthesis by
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research and development department, Gland Pharma Limited, Hyderabad, India. Solvents and buffers used for analysis were HPLC grade Acetonitrile (Merck), Formic acid , Ammonia (Alfa Aesar) and
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hydrogen peroxide (H2O2) from Qualigens Fine Chemicals Pvt. Ltd. (Mumbai, India). Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from S.D. Fine-Chem Ltd. (Mumbai, India). Ultrapure water obtained from Millipore water purification system (Bangalore, India) was used
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throughout the studies.
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2.2 Instrumentation The forced degradation samples were analyzed on an Agilent HPLC (series 1290) equipped with
a vacuum degasser, binary pump, auto injector, diode array detector (DAD), and coupled to an Agilent QTOF 6530 (Agilent technologies Inc., CA,USA) operating in +ESI-MS studies. Chromatographic separation was achieved on Agilent AdvanceBio Peptide column, (150 x 2.1 mm, 2.7 μm). Mobile phase was degassed using Ultrasonic bath (PCI analytics, Mumbai, India). The LC/QTOF/MS/MS system
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operation was controlled using Mass hunter software. The 1H NMR studies were carried out on Bruker 400 MHz Advance-III HD (Bruker, Billerica, Massachusetts, United States).The samples were dissolved in DMSO-d6 and D2O by using tetramethylsilane (TMS) as an internal standard. Hydrolytic and thermal forced degradation studies were carried out using autoclave and hot air
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oven equipped with digital temperature control capable of controlling temperature within the range of ±1and ±2°C, respectively (Cintex precision hot air oven, Mumbai, India). Photo degradation studies were carried out in photo stability chamber ( Thermo lab Scientific equipments, Vasai, India) equipped with fluorescent lamp for 1.2 million lux hours (exposed for 144 hours) and UV light for 200 watt hour/m2 (exposed for 72 hours) and capable of controlling temperature and humidity in the range of ± 20C and ± 3% RH, respectively. The chamber was set at a temperature of 25oC and at relative humidity of 60% RH. The light system compiled with option 2 of the ICH guideline Q1B. The autoclave studies
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were carried out in Fedegari instrument (Fedegari Technologies Inc., Sellersville, USA).
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2.3. Forced degradation study
The forced degradation studies were carried out on 0.5 mg/mL of the drug stock solutions.
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Cangrelor drug substance was subjected to stress under different conditions individually as well as in combination as per ICH guidelines. All the stressed samples were diluted with mobile phase prior to the
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injection to obtain a final concentration of 0.2 mg/mL. Acidic and alkaline hydrolysis of cangrelor was
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conducted in 0.1M HCl and 0.1M NaOH, respectively. The drug substance was diluted with acidic and alkaline solutions to obtain a concentration of 0.5 mg/mL and the hydrolytic studies were carried out at
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80°C for 2 hrs. For the oxidative stress study, the sample was diluted with 0.3% peroxide solution (H2O2) to obtain a concentration of 0.5 mg/mL and subsequently kept at room temperature for 30 minutes. Photo degradation studies were carried out by exposing the drug substance in its solid state to a light energy of
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1.2 million lux hours (exposed for 144 hours) and an integrated UV energy of 200 watt hour/m2 (exposed for 72 hours). A parallel set of the drug solutions were stored in dark at the same temperature to serve as
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a control. Thermal studies were conducted on the solid drug substance by heating at 80°C for 7 days in a hot air oven. All the stresses sample solutions were diluted with the mobile phase before HPLC analysis. 2.4. High-performance Liquid Chromatography (LC-UV) The chromatographic separation of the drug from its degradation products was attained on an Agilent AdvanceBio Peptide column (150 mm × 2.1 mm, 2.7μm) using 0.2% formic acid (by adjusting 4
the pH to 8.5 with ammonia solution) as mobile phase A and ACN (100% v/v) as mobile phase B in a linear gradient program (Tmin/%ACN): 0.0/5.0, 3.0/5.0, 18.0/20, 30.0/45, 42.0/80, 45.0/5.0 , 50.0/5.0. The mobile phase flow rate, sample injection volume and auto sampler and column oven temperature were set at 0.3 mL/min, 5µL and 10oC and 35oC, respectively. Cangrelor and its degradation products
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were determined at 285 nm and 245 nm. 2.5. High Resolution Mass Spectrometry and MS/MS (HRMS and MS/MS)
The chromatographic conditions for LC-MS and MS study were the same as those for the HPLC method. The operating conditions for Q-TOF 6530 model Agilent scan of cangrelor and degradation products in +ESI mode were optimized and the source parameters are as follows; Gas Temp, 250 oC; Gas Flow 11 L/min; Nebulizer 45 psig; Sheath Gas Temp, 350 oC; Sheath Gas Flow,11 L/min and Scan
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segment Positive polarity. The Scan source parameters are as follows; VCap, 4000; Nozzle voltage, 1000 V; Fragmentor, 175; Skimmer1, 65 and Octopole RF Peak, 750. The operating conditions for
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MS/MS scan of all the degradation products of the drug were in +ESI mode and recorded in the range of
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110–1600 m/z.
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2.6. Isolation of the degradation products (DP-2 & DP-5) by preparative HPLC A Shimadzu preparative HPLC (Shimadzu corporation, Kyoto, Japan) equipped with UV-Visible
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detector was employed for the isolation of DP-2 and DP-5 using Sunfire C18 (250 mm × 19 mm; 10µm,
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100 Å) column. The flow rate was 16 mL/min and the detection was carried out at 285 nm. The mobile phase consisted of part A and B, where part A was 5mM of ammonium carbonate (pH 6.3 adjusted with
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formic acid), where as part B was acetonitrile (ACN). A gradient elution of T/% of B: 0.01/15, 15/35, 21/65, 22/15, 26/15 was followed for linear gradient.
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3. Results and discussion
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3.1. Method development and optimization of chromatographic conditions The objective of the selective HPLC method was to separate all the degradation products from
cangrelor. Agilent AdvanceBio Peptide (2.1mm × 150 mm, 2.7μm) column showed the best results in terms of retention, peak shape and resolution compared to other columns. At first the method development was started with different MS compatible buffers (0.2% formic acid, 0.1% trifluoroacetic acid (TFA), 0.1% ammonia, ammonium formate, ammonium acetate and ammonium bicarbonate) and various mobile phase compositions were tried using buffers with pH 2.5, 4.5 6.5, 7.0, 7.5 and 8.5 with 5
organic modifiers (ACN, MeOH). Acetonitrile solvent was found to be better in terms of resolution, retention, and peak shapes. Selective separation was achieved with the mobile phase solution A: composition of 0.2% formic acid (Aq) (pH 8.5 adjusted with ammonia solution) and solution B: Acetonitrile in gradient method: Time (min) T/% of B: 0.0/5.0, 3.0/5.0, 18.0/20, 30.0/45, 42.0/80, 45.0/5.0, 50.0/5.0. The mobile phase flow rate, sample injection volume and auto sampler, and column
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oven temperature were set at 0.3 mL/min, 5µL and 10oC and 35oC, respectively. Cangrelor and its degradation products were determined at 285 nm and 245 nm. For LC/QTOF/MS/MS studies, the same method was successfully transferred to MS for the characterization of the stress degradation products. 3.2. Degradation behaviour of cangrelor
The UV absorption spectrum of cangrelor showed an absorption maximum λmax at 245 nm and
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285 nm and hence, it was chosen as detection wavelength in HPLC. Numerous variations in mobile phase compositions and columns led to the optimized chromatographic conditions that resolved cangrelor and
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all degradation products formed under different conditions in a single run. These chromatographic
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conditions were used to study the degradation behaviour of cangrelor as well as for LC/QTOF/MS/MS
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studies.
Cangrelor drug substance was degraded into six major degradants which are denoted as DP-1 to
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DP-6 (Fig. 1) in accordance with the sequence in which the peak appeared from left to right in the
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chromatogram under various stress conditions. In the acid stress conditions, a total of five degradation products (DP-1, DP-2, DP-3, DP-4 and DP-5) were observed. However, DP-3 was observed under both acid as well as basic stress. Besides, the same product (DP-3) is further converting into the corresponding
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DP-1 and DP-4 product under the acid stress. Subsequently, DP-5 is further degraded into DP-2 under the acid stress. Apart from these, one major degradant DP-6 was also observed under the peroxide stress. The
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chemical structures of the degradants (DP-1 to DP-6) were confirmed by HRMS (High Resolution Mass Spectrometry) and NMR spectral studies. The degradation products DP-3 and DP-4 are process related
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known impurities in the cangrelor intermediates [13]. There is no degradation observed under thermal and photolytic conditions and hence cangrelor drug substance is found to be stable under thermal and photolytic conditions. The HPLC chromatogram of the forced degradation products DP-1 to DP-6 of cangrelor is depicted in Fig. 2. 3.3. Mass Fragmentation of cangrelor (m/z 775.9556 [M+H] +)
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The ESI/MS/MS spectrum of cangrelor was performed to outline its mass fragmentation pattern for the characterization of the degradation products (Fig. 3). Cangrelor was detected as the parent ion [M+H]+ at m/z 775.9556 corresponding to its molecular mass of m/z 774.9543. Fragmentation of the parent ion m/z 775.9556 produced product ion of m/z 550.0801, due to cleavage of clodronic acid moiety. The product ion of m/z 550.0801 further produced a daughter ion at m/z 338.0715, attributed to the
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cleavage of ribose phosphate moiety. Similarly, the product ion m/z 338.0715 by heterolytic cleavage of ethyl methyl sulfide moiety showed a daughter ion at m/z 263.0453. Furthermore, the product ion m/z 263.0453 showed a daughter ion of m/z 194.0495 due to elimination of CF3 (m/z 68.9952) from the straight chain of the parent ion. All these and further displayed product ion m/z 167.0266 and 151.0026 fragmentations confirmed the basic structure of cangrelor and the complete fragmentation pattern is shown in Fig. 3. The elemental compositions of all these product ions have been confirmed by their
3.4. Characterization of the degradation products
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accurate mass measurements (Table 1).
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3.4.1. Characterization of DP-1(m/z 566.0751 [M+H] +)
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DP-1 with [M+H]+ at m/z 566.0751 was formed under the acid hydrolytic stress conditions. DP-1 displayed an elemental composition of C16H24F3N5O8PS2+ ([M+H]+) and showed distinctive product ions
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at m/z 354.0665 , 290.0682, 194.0495, and 151.0073 (Fig. 4). The fragmentation of the parent ion
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produced a product ion of m/z 354.0665 due to the elimination of ribose phosphate moiety (m/z 214.0237). The product ion of m/z 354.0665 produced a daughter ion at m/z 290.0682. The product ion at m/z 290.0682 further formed a daughter ion at m/z 194.0495 due to the elimination of trifluoropropyl ion.
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In addition, the product ion m/z 194.0495 produced a daughter ion at m/z 151.0073 due to elimination of ethylamine cation group. Based on the fragmentation pattern, it is suggested that the compound is having
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a sulfoxide group in the structural motif and the sulfoxide group is present in the aminoethyl methyl side chain. Thus, the MS/MS fragmentation pattern concluded the sulfoxide position and the structure of DP-
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1. Based on the fragmentation pattern and the accurate mass measurements, DP-1 was identified as ((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((2-(methylsulfinyl)ethyl)amino)-2-((3,3,3-trifluoropropyl)thio)-9Hpurin-9- yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate. The 1H NMR spectrum of DP-1 shows a down field signal for methylene protons in the region of δH 3.56-3.16 ppm due to presence of the sulfoxide group compared to DP-3 and reveals that the structure is having a sulfoxide group. Besides, the spectrum shows a singlet at δH 2.76 ppm due to methyl group 7
attached to sulfoxide. In addition, it has a multiplet at δH 4.09 ppm due to methylene group attached to NH group. Apart from these, it has a signal in the aromatic region of δH 8.32 ppm due to adenosine ring. Besides, other signals due to glucose and other side chain methylene and methane protons are also present. Moreover, the compound structure was further confirmed by
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P NMR analysis (see
3.4.2. Characterization of DP-2 (m/z 354.0665 [M+H] +)
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supplementary information).
DP-2 was formed under the acid hydrolytic stress conditions. The QTOF/MS/MS result shows that the mass was found at m/z 354.0665 for the parent ion [M+H]+ corresponding to its molecular mass of m/z 353.0592. DP-2 displayed an elemental composition of C11H15F3N5OS2+ ([M+H]+) and showed distinctive product ions at m/z 290.0682, 192.0338, and 151.0073 (Fig. 4). The degradant DP-2 is
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probably formed by the cleavage of ribose phosphate moiety from DP-1. The further fragmentation of
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DP-2 produced a daughter ion at m/z 290.0682 by the elimination of methyl sulfoxide group. Besides, the product ion at m/z 290.0682 formed a daughter ion at m/z 192.0338. Further, the product ion at m/z
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192.0338 generated a daughter ion at m/z 151.0073 by the elimination of amino alkene group which
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confirms the presence of aminoethyl group in the compound. Based on the above data, the structure of DP-2 was identified as N-(2-(methylsulfinyl) ethyl)-2-((3,3,3-trifluoropropyl)thio)-9H-purin-6-amine.
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The 1H NMR spectrum of DP-2 showed a multiplet for methylene group in the down field in the
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region of δH 2.94-3.13 ppm due to the presence of methylene group besides sulfoxide. Presence of a singlet at δH 2.69 ppm was due to methyl group. In addition, it showed a signal at δH 3.85 due to methylene group attached to NH group. Furthermore, it had two triplets at δH 2.74 and 3.25 ppm due to
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methylene groups attached to sulfur and trifluoro methyl group. In addition, the spectrum showed a
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singlet in the aromatic region due to the adenosine ring proton flanked between two nitrogen atoms. 3.4.3. Characterization of DP-3(m/z 550.0801 [M+H] +)
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The structure of DP-3 matched with that of one of the key intermediates [13] observed in
cangrelor drug synthesis. Similarly, the ESI/QTOF/MS results of DP-3 show that the mass at m/z 550.0801 for the parent ion [M+H]+ is confirming that the compound is generated by the direct cleavage of clodronic acid moiety from cangrelor. DP-3 displayed an elemental composition of C16H24F3N5O7PS2+ ([M+H]+) and showed distinctive product ions at m/z 338.0715, 263.0453, 194.0495, 167.0266 and 151.0073 (Fig.4). For further confirmation of the structure, MS/MS fragmentation study was chosen. The 8
fragmentation pattern was found to be similar to that of cangrelor. The fragmentation of the parent ion at m/z 550.0801 shows a daughter ion at m/z 338.0715 due to the heterolytic cleavage of C-N bond of ribose phosphate group, confirming that the compound is having only one phosphate moiety. Further, the product ion at m/z 338.0715 produced a daughter ion at m/z 263.0453 by the elimination of ethyl methyl sulfide moiety from the side chain. Furthermore, fragmentation of the ion at m/z 263.0453 produced a
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daughter ion at m/z 194.0495 by the elimination of trifluoromethyl group. Similarly, fragmentation of the ion m/z 194.0495 produced a daughter ion at m/z 167.0266 by the elimination of ethyl side chain. Further cleavage of the product ion at m/z 167.0266 produced a daughter ion at m/z 151.0073 confirming the structure of the compound. Based on the MS/MS fragmentation data the structure of DP-3 was identified as
((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((2-(methylthio)ethyl)amino)-2-((3,3,3-trifluoropropyl)thio)-9H-
purin-9-yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate.
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The structure of the compound was further confirmed by 1H NMR data and the results revealed
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the presence of two triplets at δH 3.77 and 2.68 ppm due to methylene groups attached to NH and sulfur group. Besides, the spectrum showed a multiplet and a broad singlet in the region of δH 2.81 and 3.30
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ppm due to methylene groups attached to sulfur and CF3 groups. Moreover, it had a signal in the aromatic
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region at δH 8.41 ppm due to CH of the adenosine and other signals due to remaining CH and CH2 groups
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supplementary information).
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of the glucose moiety. The structure of the compound was further confirmed by 31P NMR analysis (see
3.4.4. Characterization of DP-4(m/z 470.1138 [M+H] +) The structure of DP-4 matched with that of one of the key intermediates [13] observed in
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cangrelor drug synthesis. The LC-MS/MS QTOF results revealed that the parent ion mass was found at m/z 470.1138 [M+H]+ for DP-4. In order to confirm the structure further MS/MS fragmentation study
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was chosen. The fragmentation of the parent ion generated a daughter ion at m/z 338.0715 confirming the cleavage of C-N bond and is similar to the fragmentation observed in the parent molecule. Moreover, the
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fragmentation of the product ion m/z 338.0715 produced a daughter ion at m/z 263.0453 by the loss of 75 mass units due to the elimination of ethyl methyl sulfide. Further fragmentation of the product ion m/z 263.0453 produced a daughter ion at m/z 194.0495 due to the homolytic cleavage and elimination of CF3 group. The further fragmentation of the product ion m/z 194.0495 produced daughter ions at m/z 167.0266 and m/z 151.0073 confirming the structure. Based on the LC-MS/QTOF and MS/MS
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fragmentation (Fig. 4) the structure of DP-4 was identified as (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-((2(methylthio)ethyl)amino)-2-((3,3,3-trifluoropropyl)thio)-9H-purin-9-yl)tetrahydrofuran-3,4-diol. The structure of the compound was further confirmed by 1H NMR analysis. The 1H NMR spectrum of the compound is similar to that of the reported literature data [13].
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3.4.5. Characterization of DP-5(m/z 338.0715 [M+H] +)
The QTOF/MS/MS results revealed that the parent ion mass was found at m/z 338.0715 [M+H]+ for DP-5. In order to confirm the structure, further MS/MS fragmentation study was chosen. The fragmentation of the parent ion generated a daughter ion at m/z 264.0525 confirming the cleavage of ethyl methyl sulfide and is similar to the fragmentation observed in the parent drug. Moreover, the fragmentation of m/z 263.0453 produced a daughter ion at m/z 194.0495 by the loss of 69 units due to the
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elimination of CF3 group from side chain and indicated that the compound is having a trifluoro methyl
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group in the structure. Further, fragmentation of the product ion at m/z 194.0495 produced a daughter ion at m/z 167.0266 due to the heterolytic cleavage of C-S bond. Based on the fragmentation (Fig. 4) and the mass
measurements,
DP-5
identified
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N-(2-(methylthio)
ethyl)-2-((3,3,3-
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trifluoropropyl)thio)-9H-purin-6-amine.
was
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accurate
The structure of the compound was further confirmed by 1H NMR spectra. The 1H NMR
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spectrum of DP-5 showed two multiplets in the regions δH 3.63 and 2.72 ppm due to methylene groups
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attached to NH group and sulfur group. Besides, it showed a sharp singlet at δH 2.09ppm due to methyl group attached to sulfur group of adenosine side chain. In addition, there appeared two triplets at δH 2.72 and 3.23 ppm due to methylene groups attached to sulfur and trifluoro methyl group. Apart from these,
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other signals due to adenosine and glucose ring protons were also present.
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3.4. 6.Characterization of DP-6(m/z 791.9505 [M+H] +) The LC-MS/MS QTOF results revealed that the parent ion mass was found at m/z 791.9505
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[M+H]+ for DP-6. The mass of DP-6 is almost 16 mass units more than that of cangrelor mass which confirms that probable oxidation might have happened on either of the two sulfur or nitrogen atoms. In order to confirm the structure and the oxidation position, further MS/MS fragmentation study was chosen. The fragmentation of the parent ion generated a daughter ion at m/z 566.0751 confirming the cleavage of clodronic acid moiety and is similar to the fragmentation observed in the parent drug. Moreover, the fragmentation of m/z 566.0751 shows a daughter ion at m/z 354.0665 by the loss of ribose 10
phosphate moiety. Further, fragmentation of m/z 354.0665 produced a daughter ion at m/z 290.0682 due to the elimination of 63 units, which confirms the presence of methyl sulfoxide group. Based on the fragmentation, it is confirmed that the oxidation has happened on ethyl methyl sulfide instead of trifluoro ethyl methyl sulfide group. Based on the LC-MS/QTOF and MS/MS fragmentation the structure of the compound is deduced and the fragmentation pattern is shown below in Fig. 4. The 1H NMR spectrum of
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DP-6 showed a down field singlet at δH 2.73 ppm due to methyl group of side chain which confirms that the compound is sulfoxide. In addition, other multiplets at δH 3.33-3.53 and δH 3.24-3.68 ppm due to methylene groups present between NH and sulfoxide groups were observed. Besides, other signals were present due to methylene and glucose rings. In addition, the compound structure was further confirmed by 31P NMR analysis (see supplementary information).
The chemical composition and the HRMS data of all the degradation products (DP-1 to DP-6)
3.5. Postulated degradation pathway mechanism
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were obtained and the MS/MS fragmentation results are tabulated in Table 1.
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The most probable mechanistic explanation for the formation of the degradation products DP-1 to
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DP-6 from cangrelor is depicted in Scheme1 and Scheme 2 (see supplementary information). The degradation product DP-6 was formed by the oxidation of cangrelor under peroxide stress. Similarly, the
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degradation product DP-3 was observed under both acid and base hydrolysis stress of cangrelor.
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However, further degradation of DP-3 occurred under acid stress to form DP-4 and DP-1. Apart from these, DP-5 and DP-2 were formed in acid hydrolysis of DP-3. The postulated degradation pathway and
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deglycosylation mechanism are shown in Scheme 3 (see supplementary information).
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4. Conclusion
Degradation behavior of cangrelor was explored by exposing it to ICH defined stressed
conditions. The drug was found to degrade under acidic, basic and oxidative conditions, while it was
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stable under thermal and photolytic stress conditions. The hyphenated LC-MS method with high resolution mass determination facilitated the qualitative analysis of the unknown degradation products unlike the conventional HPLC-UV. A total of six degradation products of cangrelor (DP-1 to DP-6) were characterized by the LC/QTOF/MS/MS and NMR spectral studies.
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Acknowledgements The authors are grateful to Gland Pharma Limited, Hyderabad for providing facilities to carry out the work and also thanks to Prof. D. B. Ramachary and Dr. Anebouselvy, School of Chemistry, University of
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Hyderabad, Hyderabad for their valuable suggestions and helpful discussions in the preparation of manuscript.
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of modern sophisticated hyphenated tools in the characterization of impurities and degradation products, J. Pharm. Biomed. Anal. 69 (2012) 148-173. [8] P.D. Kalariya, B. Raju, R.M. Borkar, D. Namdev, S. Gananadhamu, P.P. Nandekar, A.T. Sangamwar, R. Srinivas, Characterization of forced degradation products of ketorolac tromethamine using LC/ESI/Q/TOF/MS/MS and in silico toxicity prediction, J. Mass Spectrom. 49 (2014) 380-391.
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[9] B. Raju, M. Ramesh, R. Srinivas, S. Satyanarayana Raju, Y. Venkateswarlu, Identification and characterization of stressed degradation products of prulifloxacin using LC-ESI-MS/Q-TOF, MSn experiments: development of a validated specific stability-indicating LC-MS method, J.Pharm. Biomed. Anal. 56 (2011) 560-568. [10] M.V.N. Kumar Talluri, N.R. Kandimalla, R. Bandu, D. Chundi, R. Marupaka, R. Srinivas,
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Selective separation, detection of zotepine and mass spectral characterization of degradants by LCMS/MS/QTOF, J. Pharma. Anal. 4 (2014) 107-116.
[11] V. Guvvala, V.C. Subramanian, J. Anireddy, M. Konda, Study on the Isolation and Chemical Investigation of Potential Impurities in Dexrazoxane Using 2D-NMR and LC-PDA-MS, Org. Process Res. Dev., 21 (2016) 11-17.
[12] V. Guvvala, V.C. Subramanian, J. Anireddy, M. Konda, Novel degradation products of
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argatroban: Isolation, synthesis and extensive characterization using NMR and LC-PDA-MS/Q-TOF,
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J. Pharm. Anal. 8 (2018) 86-95.
[13] A.H. Ingall, J. Dixon, A. Bailey, M.E. Coombs, D. Cox,
J.I. McInally, S.F. Hunt, N.D.
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Kindon, B. J. Teobald, P.A. Willis, R.G. Humphries, P. Leff, J.A. Clegg, J.A. Smith, W. Tomlinson,
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Antagonists of the Platelet P2T Receptor: A Novel Approach to Antithrombotic Therapy, J. Med.
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Chem., 42 (1999) 213-220.
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Fig. 1 Cangrelor and degradation products (DP-1 to DP-6)
Fig. 2 HPLC chromatograms of (a) Blank, (b) Acid, (c) Base, (d) Peroxide, (e) Cangrelor API 14
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Fig. 3 Proposed mass fragmentation pattern of cangrelor
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(a)
15
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(b)
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(c)
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Fig. 4 (a) Fragmentation patterns of (DP-1& DP-2), (b) Fragmentation patterns of (DP-3, DP-4 & DP-5), (c) Fragmentation patterns of (DP-6).
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Table 1 LC/QTOF/MS/MS data of (DP-1 to DP-6) along with their possible molecular formulae and major
Degradation Experimenta impurities l Mass 755.9556
Best Possible molecular formula C17H26Cl2F3N5O12P3S2+
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fragments.
Theoretical mass 775.9561
550.0801 Cangrelor
566.0769
-1.8 354.0668 (-0.3, C11H15F3N5OS2+) 336.0599 (8.7, C11H15F2N5OS2) 290.0683 (-0.1, C10H11F3N5S+) 194.0489 (0.6, C7H8N5S+) 151.0066 (0.7, C3H7S+)
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354.0665 336.0686
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DP-1 290.0682
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C16H24F3N5O8PS2+
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75.0263 566.0751
194.0495
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151.0073 354.0665
C11H15F3N5OS2+
354.0683
-1.8 336.0604 (6.1, C11H15F2N5OS2) 290.0731 (-11.1, C10H11F3N5S+) 194.0496 (-0.1, C7H6N5S+) 151.0075 (-0.2, C3H7S+)
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336.0686
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DP-2
DP-3
290.0682 194.0495 151.0073 550.0801 338.0715 194.0495
Major fragments (error in mmu, chemical formula)
550.0790 (1.1, C16H24F3N5O7PS2+) 338.0714 (0.1, C11H15F3N5S2+) 75.0275 (-1.2, C3H7S+)
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338.715
Error in mmu -0.5
C16H24F3N5O7PS2+
550.0838
-3.7 338.0724 (-0.9, C11H15F3N5S2+) 194.0489 (0.6, C7H8N5S+) 17
75.0263 470.1138
75.0270 (-0.7, C3H7S+) C16H23F3N5O4S2+
470.1154
-1.6 338.0732 (-1.7, C11H15F3N5S2+) 264.0532 (-0.7, C8H9F3N5S+) 194.0501(-0.6, C7H8N5S+) 151.0082 (-0.9, C5H3N4S+) 75.0277 (-1.4, C3H7S+)
338.0715 264.0525
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DP-4 194.0495 151.0073 75.0263 338.0715
C11H15F3N5S2+
338.0749
194.0495
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DP-5
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151.0073
C17H26Cl2F3N5O13P3S2+
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75.0263 791.9506
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DP-6
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566.0751 354.0665 290.0682
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194.0514
791.9515
-0.9 566.0757 (-0.6, C16H24F3N5O8PS2+) 354.0670 (-0.5, C11H15F3N5OS2+) 290.0692 (-0.8, C10H11F3N5S+) 194.0503 (1.5, C7H8N5S+) 151.0094 (0.8, C5H3N4S+)
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151.0073
264.0550 (-2.5, C8H9F3N5S+) 194.0500(-0.5, C7H8N5S+) 151.0077 (-0.4, C5H3N4S+) 75.0273 (-1.0, C3H7S+)
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264.0525
-3.4
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S0731-7085(18)32181-2 https://doi.org/10.1016/j.jpba.2019.03.031 PBA 12544
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
25 September 2018 13 March 2019 14 March 2019
Please cite this article as: Guvvala V, Chidambaram Subramanian V, Anireddy JS, A study on structural characterization of degradation products of cangrelor using LC/QTOF/MS/MS and NMR, Journal of Pharmaceutical and Biomedical Analysis (2019), https://doi.org/10.1016/j.jpba.2019.03.031 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.
A study on structural characterization of degradation products of cangrelor using LC/QTOF/MS/MS and NMR
a
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Vinodh Guvvala a, b*, Venkatesan Chidambaram Subramanian b, Jaya Shree Anireddy a Centre for Chemical Science & Technology, Institute of Science & Technology, JNTUH, Kukatpally, Hyderabad 500 085, India b Gland Pharma Ltd, Research and Development, D.P.Pally, Hyderabad 500 043, India *Corresponding author. Email: [email protected]; [email protected]
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Graphical Abstract
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Highlights:
Cangrelor was subjected to acid, base and peroxide stress conditions. Acid and peroxide sensitive
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hydrolysis resulted in formation of six degradation products.
Structures of all the six degradation products were established by extensive characterization of LC/QTOF/MS/MS and NMR spectroscopy.
Liquid chromatography method compatible with mass spectrometry was developed.
Mass fragmentation pathway was established for the cangrelor and degradation products.
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Abstract
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A complete degradation study was performed on cangrelor drug substance as per the ICH guidelines. The study reveals that a total of six degradation products (DP-1 to DP-6) were found and out of these, three
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unknown degradation products (DP-1, DP-5 and DP-6) were not reported in the literature. Based on the degradation study, the drug substance cangrelor was found to be sensitive towards acidic, basic and
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oxidative conditions. Besides, it was stable under thermal and photolytic stress conditions. The
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degradation products were characterized by using advanced LC/QTOF and MS/MS analysis. Further, the structures were characterized by NMR studies. The identified degradation products of cangrelor are
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valuable for cangrelor manufacturing process and quality control.
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Key words: Cangrelor, stressed degradation, LC/QTOF/MS/MS, NMR, degradation products. 1. Introduction
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Cangrelor (Fig.1) is a parenteral drug that reversibly inhibits the P2Y12 receptor like ticagrelor
and does not require metabolic activation. It has rather a short half-life of 3 to 4 minutes; [1,2] and, with an infusion of 4 μg/kg per minute, peak inhibition occurs after 15 minutes. Cangrelor also has a rapid offset, with normal levels of platelet aggregation returning after 60 minutes. Inhibitors of platelet activation and aggregation are substances that are useful during percutaneous coronary intervention and other catheterization techniques in order to reduce bleeding complications and in the treatment of acute 2
coronary syndromes and clotting disorders in general. One class of antiplatelet agents includes the inhibitors of the P2Y12 receptor, a G-protein coupled purinergic receptor which is an important component of platelet activation [3]. Cangrelor (trade name kengreal in the US and kengrexal in Europe) was approved by the United States Food and Drug Administration in 2015. ICH Q3A(R2) and Q3B(R2) recommend the characterization of impurities/degradation products
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that are present at a level greater than the identification threshold in a drug substance or drug product [410]. The stability of drug substance or drug product is a critical parameter which effects on purity, potency and safety. Forced degradation studies give the impurity profile and the behavior of the drug substance under various stress conditions.
In continuation of the identification of impurities during the process development and degradation of drug substance the present work has been taken up on the degradation behavior of cangrelor. The
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present investigation deals with all the degradation studies including acid, base, thermal and photo stability on the drug substance as per the ICH guidelines. The formed degradation products were
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identified through LC/QTOF/MS/MS and NMR spectral analysis [11, 12].
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2. Experimental 2.1. Drugs and chemicals
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Sample of cangrelor API (active pharmaceutical ingredient) was obtained from synthesis by
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research and development department, Gland Pharma Limited, Hyderabad, India. Solvents and buffers used for analysis were HPLC grade Acetonitrile (Merck), Formic acid , Ammonia (Alfa Aesar) and
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hydrogen peroxide (H2O2) from Qualigens Fine Chemicals Pvt. Ltd. (Mumbai, India). Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from S.D. Fine-Chem Ltd. (Mumbai, India). Ultrapure water obtained from Millipore water purification system (Bangalore, India) was used
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throughout the studies.
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2.2 Instrumentation The forced degradation samples were analyzed on an Agilent HPLC (series 1290) equipped with
a vacuum degasser, binary pump, auto injector, diode array detector (DAD), and coupled to an Agilent QTOF 6530 (Agilent technologies Inc., CA,USA) operating in +ESI-MS studies. Chromatographic separation was achieved on Agilent AdvanceBio Peptide column, (150 x 2.1 mm, 2.7 μm). Mobile phase was degassed using Ultrasonic bath (PCI analytics, Mumbai, India). The LC/QTOF/MS/MS system
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operation was controlled using Mass hunter software. The 1H NMR studies were carried out on Bruker 400 MHz Advance-III HD (Bruker, Billerica, Massachusetts, United States).The samples were dissolved in DMSO-d6 and D2O by using tetramethylsilane (TMS) as an internal standard. Hydrolytic and thermal forced degradation studies were carried out using autoclave and hot air
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oven equipped with digital temperature control capable of controlling temperature within the range of ±1and ±2°C, respectively (Cintex precision hot air oven, Mumbai, India). Photo degradation studies were carried out in photo stability chamber ( Thermo lab Scientific equipments, Vasai, India) equipped with fluorescent lamp for 1.2 million lux hours (exposed for 144 hours) and UV light for 200 watt hour/m2 (exposed for 72 hours) and capable of controlling temperature and humidity in the range of ± 20C and ± 3% RH, respectively. The chamber was set at a temperature of 25oC and at relative humidity of 60% RH. The light system compiled with option 2 of the ICH guideline Q1B. The autoclave studies
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were carried out in Fedegari instrument (Fedegari Technologies Inc., Sellersville, USA).
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2.3. Forced degradation study
The forced degradation studies were carried out on 0.5 mg/mL of the drug stock solutions.
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Cangrelor drug substance was subjected to stress under different conditions individually as well as in combination as per ICH guidelines. All the stressed samples were diluted with mobile phase prior to the
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injection to obtain a final concentration of 0.2 mg/mL. Acidic and alkaline hydrolysis of cangrelor was
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conducted in 0.1M HCl and 0.1M NaOH, respectively. The drug substance was diluted with acidic and alkaline solutions to obtain a concentration of 0.5 mg/mL and the hydrolytic studies were carried out at
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80°C for 2 hrs. For the oxidative stress study, the sample was diluted with 0.3% peroxide solution (H2O2) to obtain a concentration of 0.5 mg/mL and subsequently kept at room temperature for 30 minutes. Photo degradation studies were carried out by exposing the drug substance in its solid state to a light energy of
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1.2 million lux hours (exposed for 144 hours) and an integrated UV energy of 200 watt hour/m2 (exposed for 72 hours). A parallel set of the drug solutions were stored in dark at the same temperature to serve as
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a control. Thermal studies were conducted on the solid drug substance by heating at 80°C for 7 days in a hot air oven. All the stresses sample solutions were diluted with the mobile phase before HPLC analysis. 2.4. High-performance Liquid Chromatography (LC-UV) The chromatographic separation of the drug from its degradation products was attained on an Agilent AdvanceBio Peptide column (150 mm × 2.1 mm, 2.7μm) using 0.2% formic acid (by adjusting 4
the pH to 8.5 with ammonia solution) as mobile phase A and ACN (100% v/v) as mobile phase B in a linear gradient program (Tmin/%ACN): 0.0/5.0, 3.0/5.0, 18.0/20, 30.0/45, 42.0/80, 45.0/5.0 , 50.0/5.0. The mobile phase flow rate, sample injection volume and auto sampler and column oven temperature were set at 0.3 mL/min, 5µL and 10oC and 35oC, respectively. Cangrelor and its degradation products
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were determined at 285 nm and 245 nm. 2.5. High Resolution Mass Spectrometry and MS/MS (HRMS and MS/MS)
The chromatographic conditions for LC-MS and MS study were the same as those for the HPLC method. The operating conditions for Q-TOF 6530 model Agilent scan of cangrelor and degradation products in +ESI mode were optimized and the source parameters are as follows; Gas Temp, 250 oC; Gas Flow 11 L/min; Nebulizer 45 psig; Sheath Gas Temp, 350 oC; Sheath Gas Flow,11 L/min and Scan
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segment Positive polarity. The Scan source parameters are as follows; VCap, 4000; Nozzle voltage, 1000 V; Fragmentor, 175; Skimmer1, 65 and Octopole RF Peak, 750. The operating conditions for
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MS/MS scan of all the degradation products of the drug were in +ESI mode and recorded in the range of
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110–1600 m/z.
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2.6. Isolation of the degradation products (DP-2 & DP-5) by preparative HPLC A Shimadzu preparative HPLC (Shimadzu corporation, Kyoto, Japan) equipped with UV-Visible
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detector was employed for the isolation of DP-2 and DP-5 using Sunfire C18 (250 mm × 19 mm; 10µm,
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100 Å) column. The flow rate was 16 mL/min and the detection was carried out at 285 nm. The mobile phase consisted of part A and B, where part A was 5mM of ammonium carbonate (pH 6.3 adjusted with
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formic acid), where as part B was acetonitrile (ACN). A gradient elution of T/% of B: 0.01/15, 15/35, 21/65, 22/15, 26/15 was followed for linear gradient.
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3. Results and discussion
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3.1. Method development and optimization of chromatographic conditions The objective of the selective HPLC method was to separate all the degradation products from
cangrelor. Agilent AdvanceBio Peptide (2.1mm × 150 mm, 2.7μm) column showed the best results in terms of retention, peak shape and resolution compared to other columns. At first the method development was started with different MS compatible buffers (0.2% formic acid, 0.1% trifluoroacetic acid (TFA), 0.1% ammonia, ammonium formate, ammonium acetate and ammonium bicarbonate) and various mobile phase compositions were tried using buffers with pH 2.5, 4.5 6.5, 7.0, 7.5 and 8.5 with 5
organic modifiers (ACN, MeOH). Acetonitrile solvent was found to be better in terms of resolution, retention, and peak shapes. Selective separation was achieved with the mobile phase solution A: composition of 0.2% formic acid (Aq) (pH 8.5 adjusted with ammonia solution) and solution B: Acetonitrile in gradient method: Time (min) T/% of B: 0.0/5.0, 3.0/5.0, 18.0/20, 30.0/45, 42.0/80, 45.0/5.0, 50.0/5.0. The mobile phase flow rate, sample injection volume and auto sampler, and column
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oven temperature were set at 0.3 mL/min, 5µL and 10oC and 35oC, respectively. Cangrelor and its degradation products were determined at 285 nm and 245 nm. For LC/QTOF/MS/MS studies, the same method was successfully transferred to MS for the characterization of the stress degradation products. 3.2. Degradation behaviour of cangrelor
The UV absorption spectrum of cangrelor showed an absorption maximum λmax at 245 nm and
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285 nm and hence, it was chosen as detection wavelength in HPLC. Numerous variations in mobile phase compositions and columns led to the optimized chromatographic conditions that resolved cangrelor and
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all degradation products formed under different conditions in a single run. These chromatographic
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conditions were used to study the degradation behaviour of cangrelor as well as for LC/QTOF/MS/MS
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studies.
Cangrelor drug substance was degraded into six major degradants which are denoted as DP-1 to
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DP-6 (Fig. 1) in accordance with the sequence in which the peak appeared from left to right in the
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chromatogram under various stress conditions. In the acid stress conditions, a total of five degradation products (DP-1, DP-2, DP-3, DP-4 and DP-5) were observed. However, DP-3 was observed under both acid as well as basic stress. Besides, the same product (DP-3) is further converting into the corresponding
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DP-1 and DP-4 product under the acid stress. Subsequently, DP-5 is further degraded into DP-2 under the acid stress. Apart from these, one major degradant DP-6 was also observed under the peroxide stress. The
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chemical structures of the degradants (DP-1 to DP-6) were confirmed by HRMS (High Resolution Mass Spectrometry) and NMR spectral studies. The degradation products DP-3 and DP-4 are process related
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known impurities in the cangrelor intermediates [13]. There is no degradation observed under thermal and photolytic conditions and hence cangrelor drug substance is found to be stable under thermal and photolytic conditions. The HPLC chromatogram of the forced degradation products DP-1 to DP-6 of cangrelor is depicted in Fig. 2. 3.3. Mass Fragmentation of cangrelor (m/z 775.9556 [M+H] +)
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The ESI/MS/MS spectrum of cangrelor was performed to outline its mass fragmentation pattern for the characterization of the degradation products (Fig. 3). Cangrelor was detected as the parent ion [M+H]+ at m/z 775.9556 corresponding to its molecular mass of m/z 774.9543. Fragmentation of the parent ion m/z 775.9556 produced product ion of m/z 550.0801, due to cleavage of clodronic acid moiety. The product ion of m/z 550.0801 further produced a daughter ion at m/z 338.0715, attributed to the
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cleavage of ribose phosphate moiety. Similarly, the product ion m/z 338.0715 by heterolytic cleavage of ethyl methyl sulfide moiety showed a daughter ion at m/z 263.0453. Furthermore, the product ion m/z 263.0453 showed a daughter ion of m/z 194.0495 due to elimination of CF3 (m/z 68.9952) from the straight chain of the parent ion. All these and further displayed product ion m/z 167.0266 and 151.0026 fragmentations confirmed the basic structure of cangrelor and the complete fragmentation pattern is shown in Fig. 3. The elemental compositions of all these product ions have been confirmed by their
3.4. Characterization of the degradation products
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accurate mass measurements (Table 1).
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3.4.1. Characterization of DP-1(m/z 566.0751 [M+H] +)
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DP-1 with [M+H]+ at m/z 566.0751 was formed under the acid hydrolytic stress conditions. DP-1 displayed an elemental composition of C16H24F3N5O8PS2+ ([M+H]+) and showed distinctive product ions
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at m/z 354.0665 , 290.0682, 194.0495, and 151.0073 (Fig. 4). The fragmentation of the parent ion
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produced a product ion of m/z 354.0665 due to the elimination of ribose phosphate moiety (m/z 214.0237). The product ion of m/z 354.0665 produced a daughter ion at m/z 290.0682. The product ion at m/z 290.0682 further formed a daughter ion at m/z 194.0495 due to the elimination of trifluoropropyl ion.
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In addition, the product ion m/z 194.0495 produced a daughter ion at m/z 151.0073 due to elimination of ethylamine cation group. Based on the fragmentation pattern, it is suggested that the compound is having
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a sulfoxide group in the structural motif and the sulfoxide group is present in the aminoethyl methyl side chain. Thus, the MS/MS fragmentation pattern concluded the sulfoxide position and the structure of DP-
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1. Based on the fragmentation pattern and the accurate mass measurements, DP-1 was identified as ((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((2-(methylsulfinyl)ethyl)amino)-2-((3,3,3-trifluoropropyl)thio)-9Hpurin-9- yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate. The 1H NMR spectrum of DP-1 shows a down field signal for methylene protons in the region of δH 3.56-3.16 ppm due to presence of the sulfoxide group compared to DP-3 and reveals that the structure is having a sulfoxide group. Besides, the spectrum shows a singlet at δH 2.76 ppm due to methyl group 7
attached to sulfoxide. In addition, it has a multiplet at δH 4.09 ppm due to methylene group attached to NH group. Apart from these, it has a signal in the aromatic region of δH 8.32 ppm due to adenosine ring. Besides, other signals due to glucose and other side chain methylene and methane protons are also present. Moreover, the compound structure was further confirmed by
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P NMR analysis (see
3.4.2. Characterization of DP-2 (m/z 354.0665 [M+H] +)
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supplementary information).
DP-2 was formed under the acid hydrolytic stress conditions. The QTOF/MS/MS result shows that the mass was found at m/z 354.0665 for the parent ion [M+H]+ corresponding to its molecular mass of m/z 353.0592. DP-2 displayed an elemental composition of C11H15F3N5OS2+ ([M+H]+) and showed distinctive product ions at m/z 290.0682, 192.0338, and 151.0073 (Fig. 4). The degradant DP-2 is
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probably formed by the cleavage of ribose phosphate moiety from DP-1. The further fragmentation of
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DP-2 produced a daughter ion at m/z 290.0682 by the elimination of methyl sulfoxide group. Besides, the product ion at m/z 290.0682 formed a daughter ion at m/z 192.0338. Further, the product ion at m/z
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192.0338 generated a daughter ion at m/z 151.0073 by the elimination of amino alkene group which
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confirms the presence of aminoethyl group in the compound. Based on the above data, the structure of DP-2 was identified as N-(2-(methylsulfinyl) ethyl)-2-((3,3,3-trifluoropropyl)thio)-9H-purin-6-amine.
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The 1H NMR spectrum of DP-2 showed a multiplet for methylene group in the down field in the
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region of δH 2.94-3.13 ppm due to the presence of methylene group besides sulfoxide. Presence of a singlet at δH 2.69 ppm was due to methyl group. In addition, it showed a signal at δH 3.85 due to methylene group attached to NH group. Furthermore, it had two triplets at δH 2.74 and 3.25 ppm due to
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methylene groups attached to sulfur and trifluoro methyl group. In addition, the spectrum showed a
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singlet in the aromatic region due to the adenosine ring proton flanked between two nitrogen atoms. 3.4.3. Characterization of DP-3(m/z 550.0801 [M+H] +)
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The structure of DP-3 matched with that of one of the key intermediates [13] observed in
cangrelor drug synthesis. Similarly, the ESI/QTOF/MS results of DP-3 show that the mass at m/z 550.0801 for the parent ion [M+H]+ is confirming that the compound is generated by the direct cleavage of clodronic acid moiety from cangrelor. DP-3 displayed an elemental composition of C16H24F3N5O7PS2+ ([M+H]+) and showed distinctive product ions at m/z 338.0715, 263.0453, 194.0495, 167.0266 and 151.0073 (Fig.4). For further confirmation of the structure, MS/MS fragmentation study was chosen. The 8
fragmentation pattern was found to be similar to that of cangrelor. The fragmentation of the parent ion at m/z 550.0801 shows a daughter ion at m/z 338.0715 due to the heterolytic cleavage of C-N bond of ribose phosphate group, confirming that the compound is having only one phosphate moiety. Further, the product ion at m/z 338.0715 produced a daughter ion at m/z 263.0453 by the elimination of ethyl methyl sulfide moiety from the side chain. Furthermore, fragmentation of the ion at m/z 263.0453 produced a
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daughter ion at m/z 194.0495 by the elimination of trifluoromethyl group. Similarly, fragmentation of the ion m/z 194.0495 produced a daughter ion at m/z 167.0266 by the elimination of ethyl side chain. Further cleavage of the product ion at m/z 167.0266 produced a daughter ion at m/z 151.0073 confirming the structure of the compound. Based on the MS/MS fragmentation data the structure of DP-3 was identified as
((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((2-(methylthio)ethyl)amino)-2-((3,3,3-trifluoropropyl)thio)-9H-
purin-9-yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate.
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The structure of the compound was further confirmed by 1H NMR data and the results revealed
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the presence of two triplets at δH 3.77 and 2.68 ppm due to methylene groups attached to NH and sulfur group. Besides, the spectrum showed a multiplet and a broad singlet in the region of δH 2.81 and 3.30
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ppm due to methylene groups attached to sulfur and CF3 groups. Moreover, it had a signal in the aromatic
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region at δH 8.41 ppm due to CH of the adenosine and other signals due to remaining CH and CH2 groups
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supplementary information).
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of the glucose moiety. The structure of the compound was further confirmed by 31P NMR analysis (see
3.4.4. Characterization of DP-4(m/z 470.1138 [M+H] +) The structure of DP-4 matched with that of one of the key intermediates [13] observed in
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cangrelor drug synthesis. The LC-MS/MS QTOF results revealed that the parent ion mass was found at m/z 470.1138 [M+H]+ for DP-4. In order to confirm the structure further MS/MS fragmentation study
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was chosen. The fragmentation of the parent ion generated a daughter ion at m/z 338.0715 confirming the cleavage of C-N bond and is similar to the fragmentation observed in the parent molecule. Moreover, the
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fragmentation of the product ion m/z 338.0715 produced a daughter ion at m/z 263.0453 by the loss of 75 mass units due to the elimination of ethyl methyl sulfide. Further fragmentation of the product ion m/z 263.0453 produced a daughter ion at m/z 194.0495 due to the homolytic cleavage and elimination of CF3 group. The further fragmentation of the product ion m/z 194.0495 produced daughter ions at m/z 167.0266 and m/z 151.0073 confirming the structure. Based on the LC-MS/QTOF and MS/MS
9
fragmentation (Fig. 4) the structure of DP-4 was identified as (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-((2(methylthio)ethyl)amino)-2-((3,3,3-trifluoropropyl)thio)-9H-purin-9-yl)tetrahydrofuran-3,4-diol. The structure of the compound was further confirmed by 1H NMR analysis. The 1H NMR spectrum of the compound is similar to that of the reported literature data [13].
SC RI PT
3.4.5. Characterization of DP-5(m/z 338.0715 [M+H] +)
The QTOF/MS/MS results revealed that the parent ion mass was found at m/z 338.0715 [M+H]+ for DP-5. In order to confirm the structure, further MS/MS fragmentation study was chosen. The fragmentation of the parent ion generated a daughter ion at m/z 264.0525 confirming the cleavage of ethyl methyl sulfide and is similar to the fragmentation observed in the parent drug. Moreover, the fragmentation of m/z 263.0453 produced a daughter ion at m/z 194.0495 by the loss of 69 units due to the
U
elimination of CF3 group from side chain and indicated that the compound is having a trifluoro methyl
N
group in the structure. Further, fragmentation of the product ion at m/z 194.0495 produced a daughter ion at m/z 167.0266 due to the heterolytic cleavage of C-S bond. Based on the fragmentation (Fig. 4) and the mass
measurements,
DP-5
identified
as
N-(2-(methylthio)
ethyl)-2-((3,3,3-
M
trifluoropropyl)thio)-9H-purin-6-amine.
was
A
accurate
The structure of the compound was further confirmed by 1H NMR spectra. The 1H NMR
D
spectrum of DP-5 showed two multiplets in the regions δH 3.63 and 2.72 ppm due to methylene groups
TE
attached to NH group and sulfur group. Besides, it showed a sharp singlet at δH 2.09ppm due to methyl group attached to sulfur group of adenosine side chain. In addition, there appeared two triplets at δH 2.72 and 3.23 ppm due to methylene groups attached to sulfur and trifluoro methyl group. Apart from these,
EP
other signals due to adenosine and glucose ring protons were also present.
CC
3.4. 6.Characterization of DP-6(m/z 791.9505 [M+H] +) The LC-MS/MS QTOF results revealed that the parent ion mass was found at m/z 791.9505
A
[M+H]+ for DP-6. The mass of DP-6 is almost 16 mass units more than that of cangrelor mass which confirms that probable oxidation might have happened on either of the two sulfur or nitrogen atoms. In order to confirm the structure and the oxidation position, further MS/MS fragmentation study was chosen. The fragmentation of the parent ion generated a daughter ion at m/z 566.0751 confirming the cleavage of clodronic acid moiety and is similar to the fragmentation observed in the parent drug. Moreover, the fragmentation of m/z 566.0751 shows a daughter ion at m/z 354.0665 by the loss of ribose 10
phosphate moiety. Further, fragmentation of m/z 354.0665 produced a daughter ion at m/z 290.0682 due to the elimination of 63 units, which confirms the presence of methyl sulfoxide group. Based on the fragmentation, it is confirmed that the oxidation has happened on ethyl methyl sulfide instead of trifluoro ethyl methyl sulfide group. Based on the LC-MS/QTOF and MS/MS fragmentation the structure of the compound is deduced and the fragmentation pattern is shown below in Fig. 4. The 1H NMR spectrum of
SC RI PT
DP-6 showed a down field singlet at δH 2.73 ppm due to methyl group of side chain which confirms that the compound is sulfoxide. In addition, other multiplets at δH 3.33-3.53 and δH 3.24-3.68 ppm due to methylene groups present between NH and sulfoxide groups were observed. Besides, other signals were present due to methylene and glucose rings. In addition, the compound structure was further confirmed by 31P NMR analysis (see supplementary information).
The chemical composition and the HRMS data of all the degradation products (DP-1 to DP-6)
3.5. Postulated degradation pathway mechanism
N
U
were obtained and the MS/MS fragmentation results are tabulated in Table 1.
A
The most probable mechanistic explanation for the formation of the degradation products DP-1 to
M
DP-6 from cangrelor is depicted in Scheme1 and Scheme 2 (see supplementary information). The degradation product DP-6 was formed by the oxidation of cangrelor under peroxide stress. Similarly, the
D
degradation product DP-3 was observed under both acid and base hydrolysis stress of cangrelor.
TE
However, further degradation of DP-3 occurred under acid stress to form DP-4 and DP-1. Apart from these, DP-5 and DP-2 were formed in acid hydrolysis of DP-3. The postulated degradation pathway and
EP
deglycosylation mechanism are shown in Scheme 3 (see supplementary information).
CC
4. Conclusion
Degradation behavior of cangrelor was explored by exposing it to ICH defined stressed
conditions. The drug was found to degrade under acidic, basic and oxidative conditions, while it was
A
stable under thermal and photolytic stress conditions. The hyphenated LC-MS method with high resolution mass determination facilitated the qualitative analysis of the unknown degradation products unlike the conventional HPLC-UV. A total of six degradation products of cangrelor (DP-1 to DP-6) were characterized by the LC/QTOF/MS/MS and NMR spectral studies.
11
Acknowledgements The authors are grateful to Gland Pharma Limited, Hyderabad for providing facilities to carry out the work and also thanks to Prof. D. B. Ramachary and Dr. Anebouselvy, School of Chemistry, University of
SC RI PT
Hyderabad, Hyderabad for their valuable suggestions and helpful discussions in the preparation of manuscript.
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[2] D.L. Bhatt, G.W. Stone, K.W. Mahaffey, C.M. Gibson, P.G. Steg, C.W. Hamm, M.J. Price, S.
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Leonardi, D. Gallup, E. Bramucci, P.W. Radke, P. Widimský, F. Tousek, J. Tauth, D. Spriggs, B.T. McLaurin, D.J. Angiolillo, P. Généreux, T. Liu, J. Prats, M. Todd, S. Skerjanec, H.D. White, R.A.
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Med. 368 (2013) 1303-1313.
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Proceeding of International Conference on Harmonization, IFPMA, Geneva, 1996. [6] ICH, Guidelines on impurities in new drug substances Q3A(R2), in: International Conference on
CC
Harmonization, IFPMA, Geneva, 2006. [7] S. Singh, T. Handa, M. Narayanam, A. Sahu, M. Junwal, R.P. Shah, A critical review on the use
A
of modern sophisticated hyphenated tools in the characterization of impurities and degradation products, J. Pharm. Biomed. Anal. 69 (2012) 148-173. [8] P.D. Kalariya, B. Raju, R.M. Borkar, D. Namdev, S. Gananadhamu, P.P. Nandekar, A.T. Sangamwar, R. Srinivas, Characterization of forced degradation products of ketorolac tromethamine using LC/ESI/Q/TOF/MS/MS and in silico toxicity prediction, J. Mass Spectrom. 49 (2014) 380-391.
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[9] B. Raju, M. Ramesh, R. Srinivas, S. Satyanarayana Raju, Y. Venkateswarlu, Identification and characterization of stressed degradation products of prulifloxacin using LC-ESI-MS/Q-TOF, MSn experiments: development of a validated specific stability-indicating LC-MS method, J.Pharm. Biomed. Anal. 56 (2011) 560-568. [10] M.V.N. Kumar Talluri, N.R. Kandimalla, R. Bandu, D. Chundi, R. Marupaka, R. Srinivas,
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Selective separation, detection of zotepine and mass spectral characterization of degradants by LCMS/MS/QTOF, J. Pharma. Anal. 4 (2014) 107-116.
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[12] V. Guvvala, V.C. Subramanian, J. Anireddy, M. Konda, Novel degradation products of
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argatroban: Isolation, synthesis and extensive characterization using NMR and LC-PDA-MS/Q-TOF,
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[13] A.H. Ingall, J. Dixon, A. Bailey, M.E. Coombs, D. Cox,
J.I. McInally, S.F. Hunt, N.D.
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Kindon, B. J. Teobald, P.A. Willis, R.G. Humphries, P. Leff, J.A. Clegg, J.A. Smith, W. Tomlinson,
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A
CC
EP
TE
D
Chem., 42 (1999) 213-220.
13
A
CC
EP
TE
D
M
A
N
U
SC RI PT
Fig. 1 Cangrelor and degradation products (DP-1 to DP-6)
Fig. 2 HPLC chromatograms of (a) Blank, (b) Acid, (c) Base, (d) Peroxide, (e) Cangrelor API 14
SC RI PT U N
M
A
Fig. 3 Proposed mass fragmentation pattern of cangrelor
A
CC
EP
TE
D
(a)
15
A
N
U
SC RI PT
(b)
CC
EP
TE
D
M
(c)
A
Fig. 4 (a) Fragmentation patterns of (DP-1& DP-2), (b) Fragmentation patterns of (DP-3, DP-4 & DP-5), (c) Fragmentation patterns of (DP-6).
16
Table 1 LC/QTOF/MS/MS data of (DP-1 to DP-6) along with their possible molecular formulae and major
Degradation Experimenta impurities l Mass 755.9556
Best Possible molecular formula C17H26Cl2F3N5O12P3S2+
SC RI PT
fragments.
Theoretical mass 775.9561
550.0801 Cangrelor
566.0769
-1.8 354.0668 (-0.3, C11H15F3N5OS2+) 336.0599 (8.7, C11H15F2N5OS2) 290.0683 (-0.1, C10H11F3N5S+) 194.0489 (0.6, C7H8N5S+) 151.0066 (0.7, C3H7S+)
M
354.0665 336.0686
TE
D
DP-1 290.0682
N
C16H24F3N5O8PS2+
A
75.0263 566.0751
194.0495
EP
151.0073 354.0665
C11H15F3N5OS2+
354.0683
-1.8 336.0604 (6.1, C11H15F2N5OS2) 290.0731 (-11.1, C10H11F3N5S+) 194.0496 (-0.1, C7H6N5S+) 151.0075 (-0.2, C3H7S+)
CC
336.0686
A
DP-2
DP-3
290.0682 194.0495 151.0073 550.0801 338.0715 194.0495
Major fragments (error in mmu, chemical formula)
550.0790 (1.1, C16H24F3N5O7PS2+) 338.0714 (0.1, C11H15F3N5S2+) 75.0275 (-1.2, C3H7S+)
U
338.715
Error in mmu -0.5
C16H24F3N5O7PS2+
550.0838
-3.7 338.0724 (-0.9, C11H15F3N5S2+) 194.0489 (0.6, C7H8N5S+) 17
75.0263 470.1138
75.0270 (-0.7, C3H7S+) C16H23F3N5O4S2+
470.1154
-1.6 338.0732 (-1.7, C11H15F3N5S2+) 264.0532 (-0.7, C8H9F3N5S+) 194.0501(-0.6, C7H8N5S+) 151.0082 (-0.9, C5H3N4S+) 75.0277 (-1.4, C3H7S+)
338.0715 264.0525
SC RI PT
DP-4 194.0495 151.0073 75.0263 338.0715
C11H15F3N5S2+
338.0749
194.0495
N
DP-5
A
151.0073
C17H26Cl2F3N5O13P3S2+
M
75.0263 791.9506
TE
DP-6
D
566.0751 354.0665 290.0682
EP
194.0514
791.9515
-0.9 566.0757 (-0.6, C16H24F3N5O8PS2+) 354.0670 (-0.5, C11H15F3N5OS2+) 290.0692 (-0.8, C10H11F3N5S+) 194.0503 (1.5, C7H8N5S+) 151.0094 (0.8, C5H3N4S+)
A
CC
151.0073
264.0550 (-2.5, C8H9F3N5S+) 194.0500(-0.5, C7H8N5S+) 151.0077 (-0.4, C5H3N4S+) 75.0273 (-1.0, C3H7S+)
U
264.0525
-3.4
18