MS and NMR studies for characterization of its new hydrolytic and oxidative forced degradation products

Accepted Manuscript Title: Validated stability indicating assay method of olaparib: LC-ESI-Q-TOF-MS/MS and NMR studies for characterization of its new hydrolytic and oxidative forced degradation products Authors: Mohit Thummar, Bhoopendra S. Kuswah, S. Gananadhamu, Upendra Bulbake, Jitendra Gour, Wahid Khan PII: DOI: Reference:

S0731-7085(18)31042-2 https://doi.org/10.1016/j.jpba.2018.07.017 PBA 12088

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

3-5-2018 12-7-2018 14-7-2018

Please cite this article as: Mohit T, Kuswah BS, Gananadhamu S, Upendra B, Jitendra G, Wahid K, Validated stability indicating assay method of olaparib: LC-ESI-Q-TOFMS/MS and NMR studies for characterization of its new hydrolytic and oxidative forced degradation products, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.07.017 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.

Validated stability indicating assay method of olaparib: LC-ESI-QTOF-MS/MS and NMR studies for characterization of its new hydrolytic and oxidative forced degradation products

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Mohit Thummar, Bhoopendra S. Kuswah, S. Gananadhamu*, Upendra Bulbake, Jitendra

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Gour, Wahid Khan

National Institute of Pharmaceutical Education and Research (NIPER), Balanagar,

*Author

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Hyderabad, 500037, Telangana, India.

for correspondence: Dr. S. Gananadhamu,

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Graphical abstract

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Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research [NIPER], Hyderabad-500037, Telangana, India. E-mail: [email protected]

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Highlights

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Olaperib (OLA) was approved recently for the treatment of prostate, ovarian and breast cancer in the patients with hereditary BRCA1 and BRCA2 mutations.

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This is the first report on stability indicating assay method of the drug according to

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ICH guidelines. Four new degradation products (DPs) were identified and characterized by using LC-

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ESI-QTOF-MS/MS.

Major DPs (DP-1 and DP-2) were isolated and their chemical structures were

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confirmed by NMR experiments.

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Abstract

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Olaparib (OLA) is a poly ADP ribose polymerase (PARP) enzyme inhibitor used to treat

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prostate, ovarian and breast cancer. The drug substance OLA was subjected to forced degradation as per ICH prescribed guidelines. It was degraded in hydrolytic (acidic and basic)

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and oxidative stress conditions and yielded four degradation products (DPs) while it remained stable in neutral hydrolytic, dry heat and photolytic stress conditions. A stability indicating

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assay method was developed to separate OLA and its DPs using InertSustain C18 column (250 × 4.6 mm, 5 μm) with a gradient mobile phase of ammonium acetate (pH 4.5) and

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acetonitrile (ACN) at a flow rate of 1 mL min-1. The characterization of DPs was carried out by using liquid chromatography-electrospray ionization-quadrupole-time of flight tandem mass spectrometry (LC-ESI-Q-TOF-MS/MS). Major degradation products (DP-1 and DP-2) were isolated by using preparative HPLC and structures were further confirmed by using NMR spectroscopy. All the obtained DPs were new and not reported previously. The

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developed chromatographic method was validated as per ICH Q2 (R1) guideline and USP general chapter.

Keywords: Olaparib (OLA); Stability indicating assay method (SIAM); Degradation

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1. Introduction

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products (DPs); LC-ESI-QTOF-MS/MS; 1H and 13C NMR.

The BRCA1 and BRCA2 are a family of tumor suppressor genes responsible for many

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cellular functions like DNA repair, recombination, transcriptional regulation and chromatin

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remodeling [1-3]. A deficiency in these genes leads to hereditary ovarian and breast cancers;

[4].

Olaparib

(OLA)

is

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cancers

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it can also increase the risk of male breast, uterine, cervical, pancreatic, colon and prostate chemically

known

as

(cyclopropanecarbonyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one.

4-(3-(4The

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capsule formulation of drug was approved in December 2014 for the treatment of prostate, ovarian and breast cancer in the patients with hereditary BRCA1 and BRCA2 mutations [5].

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The tablet formulation of drug was approved in August 2017 for maintenance therapy [6].

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The drug acts by inhibiting poly ADP ribose polymerase 1 (PARP-1) enzyme which is involved in DNA repair work by recognizing single strand DNA break. Inhibition of this enzyme leads to accumulation of broken single strand and double strand DNA which

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ultimately leads to cell death [7]. Stability indicating assay method (SIAM) is an integral part of a pharmaceutical drug development process, as in addition to the assay it deals with the identification of degradation behavior of the drug towards various stressed conditions as well as a separation and characterization of degradation products (DPs) [8-11]. Moreover, it is also useful for the

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formulation and packaging development, shelf life determination and designing of the manufacturing process. Hence various global pharmaceutical regulatory bodies put emphasis on the development of SIAM for the assay of the DPs thereby ensuring the quality of the developed pharmaceutical products [12,13]. Hyphenated technique, LC-ESI-Q-TOF-MS/MS

for the identification and structural elucidation of DPs [14,15].

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combined with an accurate mass measurement has been entrenched as promising technique

Comprehensive literature survey reveals this as the first report on the development of

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validated stability indicating assay method of OLA for the separation and characterization of

its forced degradation products. A few reported pharmacokinetic methods were found in the

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literature [16,17]. In the present study, we have developed SIAM and identified the

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degradation products of OLA by using LC-ESI-QTOF-MS/MS and NMR.

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2. Experimental

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2.1 Chemicals and reagents

The pure drug sample was purchased from Haoyuan Chemexpress Co., Ltd. (Shanghai,

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China). Gradient grade acetonitrile (ACN), methanol (MeOH), formic acid and trifluoroacetic

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acid (TFA) were purchased from Merck specialities Pvt. Ltd. (Mumbai, India). Buffer grade ammonium acetate and ammonium formate; analytical reagent grade (AR Grade)

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hydrochloric acid (HCl), sodium hydroxide (NaOH) and 30% w/v H2O2 were procured from S D Fine Chemicals Pvt. Ltd. (Mumbai, India). Ultra pure water was obtained by Milli-Q

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Gradient Millipore system (Bedford, MA, USA). 2.2 Instrumentation Chromatographic separation was carried out by using Waters e2695 separation module (M.A, USA) high performance liquid chromatographic (HPLC) system fitted out with an auto sampler, on-line degasser, quaternary pumps, a photo diode array detector (210-800 nm) and

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column heater. The HPLC system was controlled by Empower-3 software. The structural characterization of the DPs was accomplished by using Agilent 1290 infinity series separation modules attached with the quadrupole time-of-flight mass analyzer (Q-TOF, Agilent 6540, Agilent Technology, USA) and electrospray ionization (ESI) source. The system was controlled by mass hunter software. The proton 1H and carbon 13C NMR studies

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were carried out on a 500 MHz NMR (AVANCE III HD-500, Bruker, Switzerland) spectrometer using d6-DMSO as solvent and TMS as an internal standard. Photolytic

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degradation studies were put through Osworld OPSH S/G-16 GMP series photo stability chamber (Osworld Scientific Equipements, Pvt. Ltd. India.) fitted with UV and visible lamps

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[18]. High precision water bath and hot air oven (Osworld Scientific Equipements, Pvt. Ltd.

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India) were used for hydrolytic degradations and thermal degradation, respectively.

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2.3 Chromatography and mass spectrometric conditions

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The chromatographic peaks were selectively separated by using InertSustain C18 column

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(250 × 4.6 mm, 5 μm) with a gradient mobile phase of 10 mM ammonium acetate, pH 4.5 (solvent A) and ACN (solvent B). The gradient program was started as follows: (time in

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min/% of solvent B) 0-3/10, 3-12/75, 12-16/75, 16-17/10, and 17-18/10 at a flow rate of 1 mL min-1. All the samples were studied at an assay concentration of 200 μg mL-1 with an

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injection volume of 10 μL. The auto-sampler and column oven temperatures were controlled at 10 °C and 25 °C, respectively. All the chromatograms were monitored at λ max of 275 nm. The optimized source conditions for single MS scan of OLA in positive electrospray

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ionization (ESI) mode includes: fragmentor, capillary and skimmer voltage at 90 V, 3500 V and 70 V, respectively. Ultra high pure nitrogen was used as drying (330 °C, 10l min-1) and nebulizing (50 psi) gas. Structural characterization of DPs was carried out by collision induced dissociation (CID) experiment in which quadrupole mass analyzer was used to select precursor ion of interest and time of flight mass analyzer was used to analyze product ions of

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selected precursor ion. Mass spectra were recorded and obtained at identical mass experimental conditions with an average scan speed of 20-25 spectra/s. 2.4 Forced degradation study The forced degradation studies were carried out on 1 mg mL-1 of drug stock solutions. Acidic

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hydrolysis (1 N HCl for 4 h), basic hydrolysis (0.2 N NaOH for 10 h) and neutral hydrolysis (water for 7 days) were performed under heat at 70 °C. Oxidative degradation study was

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performed with 15 % w/v H2O2 for 120 h at room temperature. Photolytic degradation studies were carried out by irradiating solid state and solution state samples with UV light up to 200

Whrm-2 and visible light up to 1.2 million lux hr. For thermal degradation, the solid sample of

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the drug was spread on a Petri dish as a 1 mm thickness layer and kept at 85 °C for 7 days.

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2.5 Preparation of samples for chromatographic and mass spectrometric analysis

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Acidic and basic hydrolysis stress samples were neutralized and diluted with 50:50 % v/v solutions of water and ACN to make a final assay concentration of 200 μg mL-1. The final

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sample solutions were filtered through 0.22 μm membrane filter before subjected into HPLC and LC-MS/MS analysis. All the sample solutions were stored at 4 °C in refrigerator to avoid

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any degradation.

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2.6 Method validation

The proposed chromatographic method was validated with respect to selectivity, limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy, precision and robustness

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as described in ICH guideline Q2 (R1) and USP general chapter [19,20]. 2.7 Preparative HPLC conditions Preparative HPLC system from Waters (Waters, M.A, USA) equipped with 515 HPLC pumps and 2489 UV–visible detector was used for isolation of DP-1 and DP-2. The

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chromatographic conditions for isolation of above DPs includes Waters Xbridge C18 column (250 × 19 mm × 5 μm, 130 Å) with isocratic mobile phase containing a 60:40 % v/v of ammonium acetate (10 mM, 4.5 pH) and acetonitrile (ACN) at a flow rate of 12 mL min -1.

3. Results and discussion

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3.1 HPLC and LC-MS/MS method development

The purpose of method development was to obtain optimum separation of drug and its DPs

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with sufficient resolution. Initial trials on InertSustain C18 column (250 × 4.6 mm, 5 μm) were found to be appropriate for the separation. While trying with different systematic

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gradient trials of organic phase MeOH/ACN and water it showed peak tailing and merged

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peak (DP-4 and OLA). In order to have better separation, good peak shape and mass

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compatibility, the buffers like ammonium acetate at pH 4 to 6, ammonium formate at pH 3-5

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and 0.1% formic acid were tried. A gradient mobile phase of 10 mM ammonium acetate (pH 4.5) and acetonitrile (ACN) gave highly resolved peaks and acceptable peak tailing. For LC-

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MS/MS analysis, positive as well as negative ESI and APCI (atmospheric pressure chemical ionization) ionization modes were tried. The positive ESI mode showed the best ionization of

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the drug and DPs compared to other ionization modes. The optimized positive ESI tandem

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mass spectrometric conditions include: the fragmentation voltage of 90 V, the capillary voltage of 3500 V and the skimmer voltage of 70 V. Ultra pure nitrogen gas was used as drying (330°C; 10 L min-1), nebulizing (50 psi) and collision gas.

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3.2 Forced degradation behaviour of OLA The drug was found to be susceptible towards acidic hydrolysis, basic hydrolysis and oxidative stress degradation conditions while it remains stable in neutral hydrolytic and photolytic degradation conditions. A total of four DPs were observed and they are shown in overlaid chromatogram of Fig. 1.

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3.2.1 Hydrolysis In acidic hydrolysis- two DPs {(DP-1, 27.9%) and (DP-2, 4.0%)} (Fig. 1b) and in basic hydrolysis- two DPs {(DP-1, 6.0%) and (DP-2, 4.2%)} (Fig. 1c) were obtained. 3.2.2 Oxidative degradation

degradation. 3.2.3 Photolytic degradation and thermal degradation

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The drug remained stable in photolytic and thermal stress degradations.

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Two DPs {(DP-3, 1.5 %) and (DP-4, 2.4 %)} (Fig. 1d) were obtained in oxidative stress

3.3 LC–ESI-QTOF-MS/MS and NMR studies of OLA and its DPs:

The LC–ESI-Q-TOF-MS/MS experiments were carried out for characterization of all the DPs

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(DP-1 – DP-4) and most probable structures have been proposed based on the m/z values of

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their [M+H]+ions, mass fragmentation pattern and an elemental composition derived from

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accurate mass measurements (Table 1). Further, major DPs (DP-1 and DP-2) were isolated and structures were confirmed by using NMR experiments. Fig. 2 shows the structures of all

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the DPs formed during the forced degradation of OLA. 3.3.1 MS/MS and NMR of OLA

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The drug was eluted at retention time (Rt) of 11.88 min. The ESI-MS/MS spectrum (Fig. 3a) of [M+H]+ of drug showed precursor ion peak at 435.1834 Da. The precursor ion was further

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fragmented to obtain a product ions at m/z 407 (loss of CO), m/z 367 (loss of C4H4O), m/z 350 (loss of NH3 from m/z 367), m/z 324 (loss of C2H2 from m/z 350), m/z 281 (loss of C2H5N

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from m/z 324), m/z 261 (loss of HF from m/z 281), m/z 253 (loss of CO from m/z 281), m/z 233 (loss of HF from m/z 253), m/z 225 (loss of C9H14N2O2 from m/z 407), m/z 205 (loss of HF from m/z 225), m/z 159 (loss of C6H2 from m/z 233), m/z 153 (loss of C16H11FN2O2),m/z 85 (loss of C4H4O from m/z 153) and m/z 69 (loss of C4H8N2 from m/z 153) (Scheme 1). The product ion peaks at m/z 281 and m/z 153 can confirm the basic skeleton of the drug. The

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elemental compositions and error ppm for all those formed ions have been confirmed by high resolution mass experiments combined with accurate mass measurements and given in Table 1. NMR signals of piperazine (atom no. 23, 24, 26 and 27) and cyclopropane carbonyl (atom no. 28, 30, 31 and 32) portions of OLA were helpful for characterization of DP-1 and DP-2 (Table 2). All the NMR spectra were given in the supplementary information (S 1).

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3.3.2 MS/MS and NMR of degradation products

DP-1 was eluted at Rt of 9.71 min. The ESI-MS/MS spectrum (Fig. 3b) of [M+H]+ of

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hydrolytic degradation product (DP-1) showed precursor ion peak at 299.0827 Da. Further it

was fragmented to obtain product ions at m/z 281 (loss of H2O), m/z 153 (loss of C8H6N2O),

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m/z 261 (loss of HF from m/z 281), m/z 253 (loss of CO from m/z 281), m/z 233 (loss of HF

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from m/z 253), m/z 225 (loss of CO from m/z 253), m/z 205 (loss of HF from m/z 225), m/z

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159 (loss of C6H2 from m/z 233) and m/z 133 (loss of HF from m/z 153) (Scheme 2). The

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positive ESI-MS/MS spectrum of DP-1 showed the common product ions at m/z 281, m/z 261, m/z 253, m/z 233, m/z 225, m/z 205 and m/z 159 compared to drug and additional

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product ion peaks at m/z 153 and m/z 133 which proves the DP-1 having free carboxylic group and formed by hydrolysis of amide bond between fluorobenzyl and piperazine rings of

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drug. Further proposed structure was confirmed by NMR study which showed absence of peaks related to piperazine and cyclopropane carbonyl portions in both proton and carbon

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NMR spectra compared to drug (Table 2). Based on these data DP-1 was identified as 2fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid, possessing an elemental

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composition of C16H12FN2O3+ and -0.33 ppm mass error. DP-2 was eluted at Rt of 10.20 min. The ESI-MS/MS spectrum of [M+H]+ (Fig. 3c) of DP-2 showed precursor ion peak at 367.1568 Da. Further DP-2 fragmented to give product ions at m/z 350 (loss of NH3), m/z 85 (loss of C16H11FN2O2), m/z 324 (loss of C2H2 from m/z 350), m/z 281 (loss of C2H5N from m/z 324), m/z 261 (loss of HF from m/z 281) m/z 253 (loss of

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CO from m/z 281), m/z 233 (loss of HF from m/z 253), m/z 225 (loss of CO from m/z 253), m/z 205 (loss of HF from m/z 225) and m/z 159 (loss of C6H2 from m/z 233) (Scheme 1). The common product ions at m/z 350, m/z 324, m/z 281, m/z 261, m/z 253, m/z 233, m/z 225, m/z 205, m/z 159, m/z 85 and absence of product ion peaks at m/z 153, m/z 69 compared to drug proves the degradation product formed by hydrolysis of amide bond connecting the

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piperazine and cyclopropane rings. The proton NMR spectrum of DP-2 shows absence of cyclopropane carbonyl peaks and presence of two –NH peaks (at 12.61 and 2.08 ppm) as

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compared to drug which is having only one –NH peak (at 12.61 ppm). While carbon NMR spectrum shows absence of cyclopropane carbonyl peaks compared to drug spectrum (Table

2). Based on these MS/MS and NMR data DP-2 was identified as 4-(4-fluoro-3-(piperazine-

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1-carbonyl)benzyl)phthalazin-1(2H)-one, having an elemental composition of C20H20FN4O2+

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with -0.82 ppm mass error.

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DP-3 was eluted at Rt of 10.74 min. The ESI-MS/MS spectrum of [M+H]+ (Fig. 4a) of oxidative degradation product showed 16 Da more molecular mass compare to drug and

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having precursor ion peak at mass of 451.1759 Da. Further DP-3 fragmented to obtain product ions at m/z 422 (loss of CHO), m/z 383 (loss of C4H4O), m/z 155 (loss of

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C16H9FN2O3), m/z 297 (loss of C4H10N2 from m/z 383), m/z 269 (loss of CO from m/z 297),

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m/z 241 (loss of C9H13N2O2 from m/z 422), m/z 159 (loss of C6H3FO from m/z 269), m/z 87 (loss of C4H4O from m/z 155) and m/z 69 (loss of C4H10N2 from m/z 155) (Scheme 3). Based on the Structure indicative product ions at m/z 383, m/z 297 and m/z 269 (addition of one

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more oxygen which are corresponding to the drug product ions at m/z 367, m/z 281 and m/z 253, respectively) and intact product ions at m/z 159 and m/z 155 proves hydroxylation site should be on the fluoro-benzyl ring system. Based on these MS/MS data DP-3 was identified as

4-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-4-fluoro-5-hydroxybenzyl)

phthalazin-1(2H)-one, containing formula of C24H24FN4O4+ with 0.22 ppm mass error.

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DP-4 was eluted at Rt of 11.35 min. The ESI-MS/MS spectrum (Fig. 4b) of [M+H]+ of oxidative degradation product showed 2 Da less molecular weight compared to the drug and showed precursor ion peak at mass of 433.1661 Da. Further DP-4 fragmented to obtain a product ions at m/z 365 (loss of C4H4O), m/z 152 (loss of C16H10FN2O2), m/z 345 (loss of HF from m/z 365), m/z 281 (loss of C4H8N2 from m/z 365), m/z 261 (loss of HF from m/z 281),

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m/z 253 (loss of CO from m/z 281), m/z 233 (loss of HF from m/z 253), m/z 225 (loss of CO from m/z 253), m/z 205 (loss of HF from m/z 225), m/z 84 (loss of C4H4O from m/z 152) and

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m/z 69 (loss of C4H9N2 from m/z 152) (Scheme 2). The product ion at m/z 365 is comparable to the drug product ion at m/z 367 which proves DP formed by dehydrogenation of OLA.

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Based on common product ions at m/z 281, m/z 261, m/z 253, m/z 233, m/z 225, m/z 205 and

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m/z 69 compare to drug proves 4-(4-fluorobenzyl)phthalazin-1(2H)-one ring system should

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be intact in DP-4 structure. Further, the product ions at m/z 152 and m/z 84 proved DP-4

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formed by dehydrogenation of the piperazine ring of OLA. Based on these mass data DP-4 was identified as 4-(3-(4-(cyclopropanecarbonyl)-1,2,3,4-tetrahydropyrazine-1-carbonyl)-4-

2.08 ppm mass error.

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fluorobenzyl)phthalazin-1(2H)-one, having an elemental composition of C24H22FN4O3+ with

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3.4 Degradation pathway of OLA

The chemical structure of OLA contains cyclopropane, piperazine, fluorobenzene and

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phthalazin-1(2H)-one moieties and two amide bonds. Due to presence of amide bonds, two major degradation products (DP-1 and DP-2) were obtained in acidic and basic hydrolysis.

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Two minor degradation products (DP-3 and DP-4) were observed in oxidative stress degradation. DP-1 was formed by hydrolysis of amide bond between fluorobenzyl and piperazine rings of drug while DP-2 was formed by hydrolysis of amide bond between piperazine and cyclopropane rings. Minor degradation products, DP-3 and DP-4 were formed by hydroxylation of the 4-fluorobenzyl ring and dehydrogenation of the piperazine ring of the

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drug, respectively in the presence of hydrogen peroxide. The degradation mechanism of the drug is given in Scheme 4.

4. Method validation The proposed stability indicating assay method for OLA was validated as described in ICH Q2 (R1) guideline and US Pharmacopeia (USP) general chapter on method validation

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[19,20]. 4.1 Selectivity

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The selectivity of the developed chromatographic method was proved by measuring a peak purity of all chromatographic peaks by using PDA detector and mass detector. All peaks were

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well separated from each other with sufficient resolution and purity angle values were found

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to be within purity threshold values. Hence, the developed HPLC method was selectively

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stability indicating.

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4.2 Limit of detection (LOD) and Limit of quantification (LOQ) The different concentrations of drug standard solutions (0.1-2 μg mL-1) were injected to find

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out the signal-to-noise ratio (S/N) values to determine the LOD and LOQ concentrations of OLA. The approximate (S/N) ratios of 3 and 10 were obtained at 0.79 μg mL-1 (LOD) and

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2.48 μg mL-1 (LOQ) concentrations of drug standard solutions, respectively. The obtained

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LOD and LOQ values further confirmed by analyzing replicate injections of same drug concentrations (n=5). 4.3 Linearity

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The linearity of the proposed HPLC method was performed on seven different concentrations of working standard solutions (10-280 μg mL-1) in the triplicate analysis. The standard calibration curve plotted between the obtained mean peak area on y-axis and concentration of OLA on x-axis shows an excellent relationship between concentrations and respective peak areas. The determination coefficient (r2) value was found to be 0.999 with a linear regression

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equation of 15906 x – 14224. Hence, the method has linear response over performed concentration range. 4.4 Accuracy The accuracy of HPLC method was assessed by determining the recovery of olaperib from drug spiked stressed samples compared to un-spiked stressed samples in triplicate. Recovery

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was found to be 100.03 to 100.13 % (Table. 3). Hence, developed method is sufficiently accurate.

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4.5 Precision

The intraday (repeatability) and inter-day (reproducibility) precisions were measured at three

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different concentrations on the same day (n=3) and three consecutive days (n=3),

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respectively. The value of % RSD was found to be below 1% (Table. 3). Hence, the

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developed method was sufficiently precise.

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4.6 Robustness

Robustness of the developed method was proved by analyzing deliberate changes in method

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variables (Table 4). The study was performed by changing in column oven temperature (25 ± 5.0 °C), flow rate (1 ± 0.1 mL min-1), mobile phase pH (4.5 ± 0.2 units) and % of organic

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solvent composition (± 2 %) at an assay concentration of 200 μg mL-1 in triplicate. Obtained peak areas were used for calculation of % RSD and it was found to be below 1 %. The other

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assay components like the resolution between the critical pair of DP-4 and OLA, USP plate counts and tailing factor of drug peak is within the limits which proved the robustness of the

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developed method (Table 4).

5. Conclusion Forced degradation of the drug was performed according to the ICH prescribed guidelines. The drug was found to be susceptible towards acidic and basic hydrolytic and oxidative stressed conditions, while it remained stable in neutral hydrolytic and photolytic stress

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conditions. The drug and DPs (two hydrolytic and two oxidative DPs) were selectively separated by proposed chromatographic method and well characterized by LC-ESI-Q-TOFMS/MS experiments. Major degradation products (DP-1 and DP-2) were isolated by preparative HPLC and subjected for NMR studies. All DPs were new and not reported previously. The mechanistic drug degradation pathway was elaborated and explained. The

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fragmentation pathway of drug was outlined and can be used in future appraisal for the

characterization of drug-drug/drug-excipient/drug-packaging interaction products, process

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related impurities and metabolites of OLA. The developed HPLC method was validated as

per ICH guideline Q2 (R1) and USP general chapter. As the proposed method is able to

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selectively separate all the DPs from the OLA, it can be used in quality control laboratories

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for stability studies and routine analysis of OLA.

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Acknowledgements

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The authors would like to show appreciation towards the National Institute of Pharmaceutical Education & Research [NIPER], Hyderabad, and the Ministry of Chemicals and Fertilizers,

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New Delhi, India, for providing a facilities and research fellowship.

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J.K. Litton, Cancers associated with BRCA1 and BRCA2 mutations other than breast and ovarian, Cancer, 121 (2015) 269–75. [5] E.D. Deeks, Olaparib: First Global Approval, Drugs, 75 (2015) 231-240.

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[6] FDA approves olaparib tablets for maintenance treatment in ovarian cancer. https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm572143.htm/,

2017

(accessed 17 August 2017). [7] R.S. Meehan, A.P. Chen, New treatment option for ovarian cancer: PARP inhibitors, Gynecologic Oncology Research and Practice, 3 (2016) 3.

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[8] M. Thummar, P.N. Patel, G. Samanthula, S. Ragampeta, Stability indicating assay method for acotiamide: separation, identification and characterization of its hydroxylated and hydrolytic degradation products along with a process related impurity by UHPLC-ESIQTOF-MS/MS, Rapid communication in mass spectrometry, 31 (2017) 1813-1824. [9] S. Singh, M. Junwal, G. Modhe, H. Tiwari, M. Kurmi, N. Parashar, P. Sidduri, Forced degradation studies to assess the stability of drugs and products, Trends Anal. Chem., 49

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(2013) 71–88. [10] S. Görög, Drug safety, drug quality, drug analysis, J. Pharm. Biomed. Anal. 48 (2008),

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247-253.

[11] S. Klick, P.G. Muijselaar, J. Waterval, T. Eichinger, C. Korn, T.K. Gerding, A.J. Debets, C.S. Griend, C. Beld, G.W. Somsen, G.J. De Jong, Stress Testing of Drug Substances and

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Drug Products, Pharm. Technol., 29 (2005) 48-66.

[12] ICH, Harmonised Tripartite Guideline, Stability Testing of New Drug Substances and

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Products, Q1A (R2), Current Step, 4 (2003).

A

[13] WHO, Draft Stability Testing of Active Pharmaceutical Ingredients and Pharmaceutical

M

Products, World Health Organization, Geneva, 2007.

[14] M. Thummar, P.N. Patel, A.L. Petkar, D. Swain, R. Srinivas, S. Gananadhamu,

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Identification of degradation products of saquinavir mesylate by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass

(2017) 771-781.

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spectrometry and its application to quality control, Rapid Commun. Mass Spectrometry, 31

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[15] S. Singh, T. Handa, M. Narayanam, A. Sahu, M. Junwal, R.P. Shah, A critical review on the use of modern sophisticated hyphenated tools in the characterization of impurities and degradation products, J. Pharm. Biomed. Anal. 69 (2012) 148–173.

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[16] R.W. Sparidans, I. Martens, L.B.J. Valkengurg-van-Iersel, J.D. Hartigh, J.H.M. Schellens, J.H. Beijnen, Liquid chromatography–tandem mass spectrometric assay for the PARP-1 inhibitor olaparib in combination with the nitrogen mustard melphalan in human plasma, J. Chromatogr. B, 879 (2011) 1851-1856.

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[17] C.M. Nijenhuis, L.Lucas, H. Rosing, J.H.M. Schellens, J.H. Beijnen, Development and validation of a high-performance liquid chromatography–tandem mass spectrometry assay quantifying olaparib in human plasma, J. Chromatogr. B, 940 (2013) 121-125. [18] ICH Guideline, Photostability Testing of New Drug Substances and Products, Q1B, IFPMA, Geneva, Switzerland, (1996). [19] ICH guideline, Q2 (R1): Validation of Analytical Procedures: Text and Methodology,

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International Conference on Harmonization IFPMA, Geneva, Switzerland, (2005).

[20] US Pharmacopoeia, Validation of compendia methods, section <1223>, United States

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Pharmacopeal Convention, Rockville, MD, (2013) 979.

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A Fig 1

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Figure

18

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Fig 2

19

A ED

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Fig 3

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A ED

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Fig 4

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Scheme

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ED

M

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Scheme 1

Scheme 2

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Scheme 3

A

Scheme 4

23

Table

Table 1. High resolution mass data corresponding to elemental composition of OLA and DPs

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PT

ED

A

DP-2

DP-3

Error (ppm) -1.61 2.70 -1.63 4.28 0.62 -1.42 0.38 1.19 3.00 6.22 1.95 3.14 7.18 3.53 -4.35 -0.33 6.05 6.51 2.37 4.29 1.33 3.41 -2.51 5.23 2.26 -0.82 -3.43 -0.31 -1.42 2.68 -2.37 6.01 4.44 4.39 -4.40 1.18 0.22

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Calculated m/z 435.1827 407.1878 367.1565 350.1299 324.1143 281.0721 261.0659 253.0772 233.0709 225.0823 205.0760 159.0553 153.1022 85.0760 69.0335 299.0826 281.0721 261.0659 253.0772 233.0709 225.0823 205.0760 159.0553 153.0346 133.0284 367.1565 350.1299 324.1143 281.0721 261.0659 253.0772 233.0709 225.0823 205.0760 159.0553 85.0760 451.1776

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Observed m/z 435.1834 407.1867 367.1571 350.1284 324.1141 281.0725 261.0658 253.0769 233.0702 225.0809 205.0756 159.0548 153.1011 85.0757 69.0338 299.0827 281.0704 261.0642 253.0766 233.0699 225.0820 205.0753 159.0557 153.0338 133.0281 367.1568 350.1311 324.1144 281.0725 261.0652 253.0778 233.0695 225.0813 205.0751 159.0560 85.0759 451.1775

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M

DP-1

Molecular Formula [M+H]+ C24H24FN4O3+ C23H24FN4O2+ C20H20FN4O2+ C20H17FN3O2+ C18H15FN3O2+ C16H10FN2O2+ C16H9N2O2+ C15H10FN2O+ C15H9N2O+ C14H10FN2+ C14H9N2+ C9H7N2O+ C8H15N2O+ C4H11N2+ C4H5O+ C16H12FN2O3+ C16H10FN2O2+ C16H9N2O2+ C15H10FN2O+ C15H9N2O+ C14H10FN2+ C14H9N2+ C9H7N2O+ C8H6FO2+ C8H5O2+ C20H20FN4O2+ C20H17FN3O2+ C18H15FN3O2+ C16H10FN2O2+ C16H9N2O2+ C15H10FN2O+ C15H9N2O+ C14H10FN2+ C14H9N2+ C9H7N2O+ C4H11N2+ C24H24FN4O4+

A

Description OLA

24

422.1749 383.1514 297.0670 269.0721 241.0772 159.0553 155.1179 87.0917 69.0335 433.1670 365.1408

C20H17N4O2+ C16H10FN2O2+

345.1336 281.0718

345.1346 281.0721

C16H9N2O2+

261.0665 +

253.0762 233.0699 225.0818 205.0748 152.0949 84.0686 69.0337

2.90 1.07

261.0659

-2.30

253.0772 233.0709 225.0823 205.0760 152.0944 84.0682 69.0335

3.95 4.29 2.22 5.85 -3.29 -4.76 -2.90

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C15H10FN2O C15H9N2O+ C14H10FN2+ C14H9N2+ C8H12N2O•+ C4H4N2•+ C4H5O+

-7.34 1.04 1.01 -1.49 4.98 5.03 -1.29 6.89 -2.90 2.08 0.27

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422.1780 383.1510 297.0667 269.0725 241.0760 159.0545 155.1181 87.0911 69.0337 433.1661 365.1407

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DP-4

C23H23FN4O3•+ C20H20FN4O3+ C16H10FN2O3+ C15H10FN2O3+ C14H10FN2O+ C9H7N2O+ C8H15N2O+ C4H11N2+ C4H5O+ C24H22FN4O3+ C20H18FN4O2+

25

Table 2. NMR chemical shift assignments for OLA, DP-1 and DP-2 OLA

DP-1

DP-2

Atom position

13

H ppm

C ppm

1

H ppm

13

C ppm

1

H ppm

12.63 (NH, s)

164.53 -

12.63 (NH, s)

165.21 -

4 5 6 7 8 9 10 12 13 14 15 16 17 18 20 23, 24, 26 and 27

7.98 (d) 7.90 (t) 7.84 (m) 8.27 (dd) 4.35 (s) 7.39 (s) 7.25 (t) 7.48 (m) 3.25, 3.53 (m)

7.95 (d) 7.88 (dd) 7.82 (t) 8.26 (d) 4.26 (s) 7.33 (m) 7.05 (t) 7.61 (d) -

157.77 124.39 126.21 129.43 131.86 123.94 143.57 34.99 131.68 (d) 129.87 (d) 116.44 157.41 (d) 114.45 (d) 127.44 170.51 -

25 28 30 31 and 32

-

159.82 126.54 128.36 132.03 133.96 125.92 145.32 36.90 135.30 (d) 129.43 (m) 124.05 (d) 156.85 (d) 116.40 (d) 132.23 (d) 177.77 41.71, 42.17, 45.19 and 46.97 177.77 10.83 7.60

12.61 (NH, s) 7.97 (d) 7.89 (m) 7.83 (dd) 8.27 (m) 4.33 (s) 7.34 (dd) 7.23 (t) 7.43 (m) 2.6-3.6 (m)

-

2.08 (s) -

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M

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1.95 (m) 0.75 (m)

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1 2

-

13

C ppm

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1

162.19 -

157.78 124.44 126.23 127.42 131.87 123.83 143.30 34.79 133.16 129.61 122.14 154.68 114.24 127.14 170.52 39.99, 43.20, 45.31 and 46.95 -

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s: singlet; d: doublet; m: multiplet; t: triplet; dd: double doublet

26

Table 3. Accuracy and precision data for OLA Concentration of OLA added (μg mL-1)

Concentration of OLA obtained

Intra-day precision (n=3) % Recover y

(µg mL-1) ± SD; %RSD

Inter-day precision (n=3)

Mean concentration of OLA (µg mL-1) ± SD; %RSD

160.05 ± 0.61; 0.38

100.03

160.17± 0.59; 0.37

160.26± 0.63; 0.39

200

200.26± 0.43; 0.21

100.13

200.12 ± 0.50; 0.25

200.13 ± 0.57; 0.28

240

240.18 ± 0.37; 0.14

100.07

240.15± 0.43; 0.17

240.18± 0.48; 0.20

Changed conditions

Tailing factor (n=3)

Theoretical plate count (n=3)

Column oven temperature Flow rate

20 °C 30 °C 0.9 mL min−1 1.1 mL min−1 +2% - 2% 4.7 pH 4.3 pH

1.30 1.18 1.25 1.23 1.24 1.27 1.28 1.24

37632 43454 36328 41462 41012 38423 35965 38663

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M

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% RSD for assay of OLA (n=3) 0.25 0.24 0.27 0.36 0.21 0.28 0.28 0.21

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Percentage of organic solvent pH of mobile phase

Resolution b/w OLA and DP-4 (n=3) 2.44 2.62 2.41 2.51 2.50 2.42 2.40 2.44

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Method variables

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Table 4. Robustness data for OLA

IP T

160

27