Identification and characterization of forced degradation products of pralatrexate injection by LC-PDA and LC–MS

Accepted Manuscript Title: Identification and characterization of forced degradation products of pralatrexate injection by LC-PDA and LC-MS Author: R.V.R Prabhakara Sastry C.S. Venkatesan B.S. Sastry K. Mahesh PII: DOI: Reference:

S0731-7085(16)30468-X http://dx.doi.org/doi:10.1016/j.jpba.2016.08.023 PBA 10819

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

25-3-2016 13-8-2016 27-8-2016

Please cite this article as: R.V.R Prabhakara Sastry, C.S.Venkatesan, B.S.Sastry, K.Mahesh, Identification and characterization of forced degradation products of pralatrexate injection by LC-PDA and LC-MS, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2016.08.023 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.

Identification and characterization of forced degradation products of pralatrexate injection by LC-PDA and LC-MS

R.V.R Prabhakara Sastrya, b, *, C.S.Venkatesanb , B.S.Sastryb, K.Maheshb

a Centre

for Chemical Sciences & Technology, Institute of Science & Technology department of Chemistry, JNTU,

Hyderabad 500 085 b Gland

Pharma Ltd., Hyderabad 500 043

*Corresponding author. Tel.: +91 040 3051099; E-mail address:[email protected]

1

Graphical abstract

O O

13' 8'

6'

OH

5'

2'

12' 7' N 4' 3' 5 H 15' 4 10 N 6 11' 9' 3N 14' 10' NH2

H2 N 2 N 9 N 7 8 1

16'

1' OH O

O

OH

H2N

18'

PTXT

O

OH

N H

N

O

N

N H2N

N

Product I

N

N

Product II

O

O

NH2 N H2N

O

COOH

N

O N H

N

OH

N

OH

NH2

OH

N

N

17'

O

O

OH

O

OH

NH2

OH O

N H2N

NH2

NH2

N

N

N

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H 2N

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N H

NH2

OH O

N H2N

N

OH

N H

O N

O

N

O NH2

H 2N

O

NH2

N

N N

N

Product III Product VIII

Product IV

Product V

Product VI

Product VII

O

NH2 N

N

H2N

O

N

N

OH

N H O

Product

IX

2

Highlight

  

Liquid chromatography method compatible with mass spectrometry was developed. Nine degradation products of Pralatrexate injection were identified and characterized. The drug product was found to be sensitive for acid, alkali, peroxide, thermal and light.

Abstract Pralatrexate (PTXT) is an antineoplastic folate analog and the chemical name is (2S)-2-[[4-[(1RS)-1[(2,4-diaminopteridin-6-yl)methyl]but-3-ynyl] benzoyl] amino] pentanedioic acid. Degradation products of PTXT drug product (DP) under different forced degradation conditions have been studied using LC-PDA and LC-MS techniques. PTXT DP was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, and heat in accordance with ICH guidelines. The LC-MS compatible HPLC method was developed and stressed solutions were chromatographed on reversed phase HPLC. The degradation products were monitored at a wavelength of 242 nm. Stress study reveals that PTXT is sensitive towards acid, alkali, peroxide, light and heat. The degradation impurities (I-IX) were identified and characterized using LC-PDA and mass spectral data.

Keywords: Pralatrexate, Degradation products, forced degradation, LC-MS, LC-PDA.

3

1. Introduction:

Pralatrexate (PTXT) is chemically designated as (2S)-2-[[4-[(1RS)-1-[(2,4-diaminopteridin-6yl)methyl]but-3-ynyl]benzoyl]amino] pentanedioic acid (Fig.1). Pralatrexate injection is a preservativefree, sterile, isotonic, non-pyrogenic clear yellow aqueous solution for intravenous administration. PTXT drug product (DP) is an antineoplastic folate analogue. Folates are members of the B vitamins. These are involved in the synthesis of purines, pyrimidines, serine and methionine, and hence are important in deoxyribonucleic acid (DNA) replication mechanisms. PTXT contains both acidic and basic functionality (pKa values are 3.25, 4.76, and 6.17). It is a 1:1 racemic mixture of S- and Rdiastereomers at the C10 chiral centre. Both the diastereomers of PTXT are expected to show same cytoxic activity. PTXT is more efficiently internalized and retained in the cancer cells than methotrexate (similar antifolate drug) [1]. A comparative study of PTXT plus gemcitabine and the standard combination of methotrexate plus ara-C in in vitro and in vivo models showed that the activity of the former drug combination was superior to the latter [2]. The stability of drug substance or drug product is a critical parameter which effects on purity, potency and safety. Forced degradation studies give impurity profile and behavior of drug product under various stress conditions. Stress studies provide information about degradation mechanisms and potential degradation products. The purpose of stability testing is to provide evidence on how the quality of a drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a shelf life for the drug product and recommended storage conditions [3]. ICH Q3A (R2) and Q3B (R2) recommends the characterization of impurities/degradation products that are present at a level greater than the identification threshold in a drug substance or drug product [4,5]. The objective of the current study was to identify the degradation products of PTXT (Fig. 2) obtained in stress conditions using LC-PDA and LC-MS techniques. Firstly PTXT was subjected to stress conditions such as acid, base, peroxide, heat and light. The degradation products formed were identified through possible degradation mechanisms based on polarity and mass fragmentation pattern. To the best our knowledge, there is no published literature exists on PTXT injection degradation products.

LC-MS compatible HPLC method was

developed for the identification and characterization of PTXT injection degradation products.

2. Experimental 2.1. Drugs and chemicals Sample of PTXT active pharmaceutical ingredient was obtained from Synthesis research and development department Gland Pharma Limited, Hyderabad, India. Methanol (HPLC grade), 4

Hydrochloric acid (Emplura) and Sodium hydroxide (Emparta, AR/GR grade) were purchased from Merck (Mumbai, India). Ammonium formate buffer (Analytical grade) was purchased from Acros organics (New Jersey, USA). Sodium Chloride (HPLC grade) and hydrogen peroxide (ExcelaR) were purchased from Qualigens (Mumbai, India). Formic acid (ULC/MS grade) was purchased from Biosolve Chemie (Valkenswaard, The Netherlands). Ultra pure water was obtained from MilliQ water system (Millipore integral water purification system, Darmstadt, Germany). PTXT formulation sample was prepared in house and subjected to stress to obtain degradation products. Each 1 mL of the drug solution contained 20 mg of Pralatrexate (PTXT), sufficient sodium chloride to achieve an isotonic (280-300 mOsm) solution, and sufficient sodium hydroxide, and hydrochloric acid if needed, to adjust and maintain the pH at 7.5-8.5 [6].

2.2. Equipments

High precision water bath (Vision lab equipments, Hyderabad, India) equipped with digital temperature capable of controlling the temperature with in ± 2 oC was used for generating hydrolytic and oxidation degradation products. Hot air oven (Cintex industrial corporation, Mumbai, India) equipped with digital temperature capable of controlling the temperature with in ± 2 oC was used for generating thermal degradation products. Photo degradation was carried out in a photo stability chamber (Thermo lab Scientific equipments, Vasai, India) equipped with a light bank consisting of two black light UV lamps and six white fluorescent lamps and capable of controlling temperature and humidity in the range of ± 2 oC and ± 3% RH, respectively. The chamber was set at a temperature of 25 oC and at relative humidity of 60% RH. The light system compiled with option 2 of the ICH guideline Q1B [7]. The forced degradation samples were analyzed on a Shimadzu 2010 integrated HPLC system (Shimadzu corporation, Kyoto, Japan) equipped with binary pump, auto liquid sampler, column compartment and prominence diode array detector (SPD-M20A), controlled by Lab solutions software (Version 6.4). Mobile phase was degassed using Ultrasonic bath (PCI analytics, Mumbai, India). The LC-MS analyses was performed on Agilent 1200 series (Agilent technologies Inc., CA, USA) equipped with variable wavelength detector G1314B,quaternary pump G1311A, auto liquid sampler G1329A, column compartment G1316A and degasser G1322A.The MS studies were carried out using positive as well as negative electro spray ionization (+ESI and –ESI) modes on Agilent 6310 ion trap mass spectrometer instrument (Agilent technologies Inc., CA, USA) interfaced with multimode source (ESI and APCI), controlled by Chemstation software (version 6.2).

2.3. Forced degradation study 5

Stress studies of PTXT DP were conducted to identify its degradation products. Acidic and alkaline hydrolysis of PTXT DP was conducted in 0.1 M HCl and 0.1 M NaOH respectively. The drug product was diluted with acidic and alkaline solutions to obtain a concentration of 4 mg/ml and hydrolytic studies were carried out at 80 °C for 3 h. For oxidative stress study, the drug product sample was diluted with 3% peroxide solution to obtain a concentration of 4 mg/ml and then exposed at 80 °C for 3 h. Photo degradation was performed by exposing DP to light at an illumination of 1.2 million lux h and integrated near UV energy of 200 Wh/m2 [7]. A parallel set of the drug product solutions was stored in the dark chamber under similar conditions for the same period of time to serve as a control. Thermal studies were conducted on drug product, which was heated at 80 °C for 7 d in hot air oven.

2.4. LC-UV method and sample preparation

The chromatographic separation was achieved on Capillarypak C18 (250 x 4.6 mm; 5 µ) column (Shiseido Co., Ltd., Tokyo, Japan), at 30 ºC column temperature using a gradient elution. Mobile phase A consisted of methanol and ammonium formate (0.01M, pH 3.0) (10:90 v/v) and phase B consisted of methanol and ammonium formate (0.01M, pH 3.0) (80:20 (v/v)). The gradient program was set as follows (Time (min)/ A: B (v/v)): T0 90:10, T5 90:10, T30 50:50, T40 20:80, T55 20:80, T58 90:10 and T70 90:10. The mobile phase was delivered at a flow rate of 1.0 ml/min. The injection volume and detection wavelength were fixed at 20 µL and 242 nm respectively. Each degraded drug solution was diluted with water prior to the injection to give a final concentration of 1 mg/ml. The HPLC analysis of each samples was preceded by corresponding blank.

2.5. LC-PDA, LC-MS and MS studies:

LC-PDA analysis of degraded drug solution containing PTXT and all degradation products was carried out to establish the purity and to extract the UV absorption spectrum of each peak in the chromatogram. The chromatographic conditions for LC-MS study were same as those for the LC-UV method. The operating conditions for MS scan of PTXT in electro spray ionization were optimized as follows. High-purity Nitrogen was used as nebulizing gas, with a nebulizer pressure set to 65 psi and as drying gas with a flow rate of 12 L min

– 1

and the temperature set to 3500C. The other working

parameters include vaporizer temperature of 2000C, end plate off set voltage of -500 V , Octopole RF amplitude 183.5 vpp, capillary voltage for +ESI mode is -2500 V and -ESI mode is +2500 V. The spectrometer was operated in scan mode (100 m/z-1000 m/z) using both positive and negative 6

electro spray ionization an (ESI) for PTXT and its degradation products and the mass scans of each analyte peak detected in the total ion chromatography (TIC) were recorded. 3. Results and Discussion

3.1. Stress decomposition behavior and LC-PDA study About nine degradation products (I-IX) of PTXT were observed in the stress conditions (Fig. 2). Eight out of nine degradation products (II-IX) were observed under photolytic conditions (Fig. 3f). The degradation products, V and VII, were also observed under peroxide stress degradation conditions (Fig. 3e). PTXT was degraded to product I in acid, alkaline and thermal stress conditions (Fig. 3b, 3c, 3d). The degradation product II was formed under acid stress condition. Nevertheless, the product II was also observed in trace amounts under photolytic conditions (Fig. 3f). LC-PDA studies revealed that the UV absorption spectra of products II, III, V, and VI- IX were similar to λmax of PTXT which suggest that there is no change in the basic chromophore of the degradation products, II, III, V, and VI – IX (Supplementary Fig. 1). However, the products I and IV differed in the absorptions distinctively from PTXT. The product I showed an additional UV maximum at 272 nm and the product IV exhibited the UV maximum at 338 nm with very low intensity when compared with PTXT UV spectrum. This observation suggested that the chromophore position was altered in product IV and there might be minor changes in the basic structure of product I from PTXT DP. The chromatographic profile of LCUV analysis (Fig. 3f) revealed that the product III was relatively a more polar compound. Even though the spectral profile of III and PTXT are similar, the product III was eluted faster than PTXT which also gave a further insight on the polarity difference between these two molecules.

3.2. Mass fragmentation of PTXT A three stage mass spectra (MS3) of PTXT were recorded to study its mass fragmentation pattern for facilitating the characterization of the degradation products. PTXT was detected at m/z 478.2 (M) in positive mode and 476.0 (MI) in negative mode as a parent ion in MS1 spectrum. The +ESI MS2 and MS3 fragmentation of PTXT showed the fragment ions at m/z 349.1 (F1) and 322.1 (F2) respectively (Table 1). An intense peak observed in MS2 spectrum at m/z 349.1 in +ESI and 347.0 (F1I) in -ESI is due to cleavage of N-C bond connecting to aromatic ring followed by rearrangement (Fig. 4) and a mass difference of 129 amu was observed between parent ions M/MI and fragment ions F1/F1I of PTXT. The fragment observed at m/z 322.1 was explained based on the 7

resonance stabilized propargyl carbocation formed by the loss of .CH=CH2 radical (27 amu) from the precursor ion at m/z 349.1.The MS3 fragment at m/z 303.0 (F3) in -ESI mode was possible to form by the elimination of CO2 (44 amu) from the precursor ion at m/z 347. The fragmentation pattern of PTXT in both positive and negative modes of ESI was proposed in Fig. 4. 3.3 . Characterization of degradation products

Based on the fragmentation pattern of PTXT, the structural characterization of its degradation products was carried out. The degradation products I, II, III and IV were detected as [M-H]- and [M+H]+ precursor ions. The other degradation products (V-IX) were detected as [M+H]+ precursor ions. However, the degradation products V-IX were not ionized in negative mode (Table 1). The precursor ions of PTXT corresponded to molecular mass of 477 amu while the molecular masses of the products I-IX were established to be 478, 348, 206, 295, 347, 431, 491, 361, and 448 respectively. The precursor ions of PTXT and its degradation products at m/z values corresponding to more than 99.45% of their actual mass values (Supplementary Table 1). The degradation products IIX (Fig. 2) were characterized through comparison of mass fragmentation pattern of PTXT and degradation products in +ESI and –ESI modes (Fig. 4, Fig. 5 and Fig. 6)

3.3.1. Product I The heaviest ion in LC-MS ESI spectrum I was noted at m/z 479.2. amu (M1) in positive mode and 477.0(M1I) amu in negative mode (Fig.5) corresponding to its molecular mass of 478 amu. An even actual molecular mass of product I indicated that one nitrogen atom was lost during the formation of I from PTXT. The fragmentation pattern of I and PTXT appear to be similar. A mass difference of 129 amu between MS2 fragment ion (m/z 350.2 in +ESI and at m/z 348.0 in -ESI) and precursor ion suggested that the former was formed due to the cleavage of N-C bond connected to aromatic ring followed by rearrangement at position 6I , similarly as F1/F1I was formed from PTXT. The daughter ion produced at m/z 305.0 in -ESI MS3 spectrum due to the elimination of carbon dioxide (44 amu) from the precursor ion at m/z 348.0. The formation of fragment ion at m/z 323.1 in +ESI MS3 spectrum was rationalized by the loss of ethylene radical (27 amu) from the precursor ion at m/z 350.2. This is an evidence that product I was having similar kind of structural motif except hydroxyl group present on pteridine moiety. The UV absorption spectrum of I showed an additional strong absorption band at λmax 272 nm indicating that it had an additional UV absorption than the other degradation products (Supplementary Fig.1) which was due to the extended conjugation of pteridine by sharing with non bonding electrons on hydroxyl (-OH) group. There was a mass 8

difference of 1 amu between PTXT and I observed. This observation provided further support for the nucleophilic hydroxyl substitution (17 amu) with amine (16 amu) at 4th position in PTXT (Fig. 1) under basic conditions. Similar kind of degradation was reported earlier on folate analogues [8]. Based on the literature, UV absorption spectrum and mass spectral fragmentation pattern (Fig. 5), the product I (Fig. 2) was established as (S)-2-(4-(1-(2-amino-4-hydroxypteridin-6-yl) pent-4-yn-2-yl) benzamido) pentanedioic acid.

3.3.2. Product II Product II showed its parent ion at m/z 349.2 amu (M2) in positive mode and 347.0 amu (M2 I) in negative mode corresponding to its actual mass of 348 amu. The even value of actual mass indicated that one nitrogen atom was lost during the formation of product II from PTXT. Incidentally, M2/M2I was also noted as a MS2 fragment ion of PTXT. A mass difference of 129 amu between PTXT and M2/M2I suggested that the product would be F1/F1I.Further, a fragment peak at m/z 322.1 (F2) in + ESI MS2 spectrum of product II is same as the fragment F2 of the drug. The +ESI MS3 spectrum showed the base peak at m/z 305.1 and the same fragment was observed in –ESI MS2 spectrum also with a relative intensity of 40.7%. From +ESI MS3 and –ESI MS2 spectra of product II, a mass difference of 44 amu in between molecular ion and fragment ion suggested that the latter was formed due to loss of carbon dioxide molecule from II similarly as F3 was formed from F1I of PTXT (Fig. 4 and Fig. 6). Based on the mass fragmentation studies (Fig. 6), the structure of product II (Fig. 2) was established as 4-(1-(2,4-diaminopteridin-6-yl)pent-4-yn-2-yl) benzoic acid.

3.3.3. Product III Product III was a polar degradation product (Fig. 3f) and its major ion was detected at m/z 207.1 amu (M3) in +ESI spectrum and 204.8 amu (M3I) in -ESI spectrum corresponding to its actual mass of 206 amu. The +ESI MS2 spectrum showed a base peak at m/z 163.0 and the same fragment ion was observed in -ESI MS 2 spectrum of III at m/z 160.9 with a relative intensity of 51.3% .The even valued molecular mass indicated an even number of nitrogen atoms in it. A mass difference of 44 amu in +ESI and -ESI modes between molecular ion and fragment ion suggested that the latter was formed due to loss of carbon dioxide which indicated that the molecular ion had the -COOH group. A mass difference of 271 amu between PTXT and III indicating that III was formed due to cleavage of C14I-C15I bond (tertiary carbon) [9]. It was also reported that the photo degradation was responsible for the formation of product III from other drugs such as folic acid, methotrexate and talotrexan [8-10]. Based on the literature, chromatographic profile (Fig. 3f) and mass spectral data (Fig. 5), product III (Fig. 2) was characterized as 2, 4-diaminopteridine-6-carboxylic acid. 9

3.3.4. Product IV The LC-MS ESI spectrum of IV showed the precursor ion peak at m/z 296.1 amu (M4) in positive mode and 293.9 amu (M4I) in negative mode with a molecular mass of 295 amu. The odd valued molecular mass indicated an odd numbers of nitrogen atoms in it. The +ESI MS2 spectrum showed the peaks at m/z 278 amu with a relative intensity of 26.6% along with the fragment ion at m/z 149.0 with an intensity of 100% (base peak). The -ESI MS2 spectrum showed the base peak at m/z 275.9 amu along with the fragment ion at m/z 231.9 with a relative intensity of 98.3%. ESI spectrum of both positive and negative modes showed a mass difference of 18 amu between M4/M4 I and fragment ion at m/z 278 amu /275.9 amu suggested that the latter was formed by the loss of water from product IV. A mass difference of 147 amu between M4 and MS2 fragment ion at m/z 149 suggested that the latter was formed due to the loss of glutamic acid moiety from product IV. Apart from these, a mass difference of 44 amu in –ESI MS

2

between product ions at m/z 275.9 and 231.9 indicated that the

latter was formed due to the loss of carbon dioxide moiety from the product ion at m/z 275.9. This was further confirmed by the PDA/UV absorption spectrum IV (Supplementary Fig. 1). The disappearance of UV absorption band at 338 nm for the product IV indicating pteridinyl part of PTXT molecule was lost during formation of IV from PTXT. Hence, based on the UV absorption spectrum and mass spectral data (Fig. 5), the product IV (Fig. 2) was identified as (S)-2-(4-(2-hydroxyethyl) benzamido) pentanedioic acid. 3.3.5. Product V Product V was detected as parent ion at m/z 348.2 (M5) amu with an actual mass of 347 amu. The odd value of actual mass indicated that all the nitrogen atoms of PTXT might be intact in product V. A mass difference of 130 amu between M5 and PTXT and nitrogen rule suggested that product was an amide compound formed due to the loss of glutaric acid moiety. Further, a base peak at m/z 321.1 in + ESI MS2 spectrum of product V is similar to the fragment F2 of the drug produced from precursor ion F1. The fragmentation mechanism of product V and product II was found similar (Fig. 6). A mass difference of 44 amu between the fragment ion at m/z 304.1 amu from + ESI MS 3 spectrum and the [M+H]+ precursor ion (m/z 348.2) indicated that the former was formed by the loss of amide moiety from the latter. Hence, the structure of product V (Fig. 2) was established as 4-(1(2,4-diaminopteridine-6-yl) pent-4-yn-2-yl)benzamide.

3.3.6. Product VI

10

The molecular mass of LC-MS + ESI spectrum of VI, showed the parent ion peak at m/z 432.2 amu (M6) along with fragment ion at m/z 348.2 amu with an intensity of 83.6% .The mass value of daughter ion VI (m/z 348.2) is same as the mass of parent ion V indicating that VI is an amide fragment. The +ESI MS2 spectrum and MS3 spectrum of product VI showed the base peaks at m/z 349.2 amu (F1) and 322.1amu (F2) respectively. This suggested that the fragmentation mechanism of VI (Fig. 6) was same as fragmentation of drug (Fig 4).The odd value of an actual mass 431 amu indicated the presence of an odd number of nitrogen atoms. This suggested that all the nitrogen atoms of PTXT might be intact in product VI. A mass difference of 46 amu between M6 and PTXT suggested that the former was formed due to the loss of hydrogen at position 3 1 and carbon dioxide moiety at position 4I simultaneously. Based on the fragmentation pattern , the structure of the product VI (Fig. 2) was established as (E)-4-(4-(1-(2,4-diaminopreridin-6-yl)pent-4-yn-2-yl)benzamido)but-3enoic acid.

3.3.7. Product VII Product VII was detected as a parent ion at m/z at 492.2 amu (M7) with an actual mass of 491 amu. The +ESI MS2 and MS3 spectra of product VII showed the base peaks at m/z 363.1 amu and 336.1 amu respectively. The m/z value and odd number of nitrogen atoms suggested that all the nitrogen atoms in PTXT were expected to be intact in product VII. M7 was found to be heavier than the parent ion of PTXT. The mass difference of 14 amu between M7 and PTXT may be due to oxidation at 15I carbon position which would introduce a C=O functionality. The + ESI MS2 fragment ion was formed at m/z of 363.1 amu due to the cleavage of N-C bond attached to aromatic ring similar to that observed in the formation of F1 from PTXT. Further, a fragment peak at m/z 336.1 in +ESI MS3 spectrum of product VII indicated that the fragmentation pattern of product VII and that of PTXT appear to be similar (Fig. 4 and Fig. 6). The +ESI spectrum of VI exhibited the mass peak at m/z 432.1 amu (M6) with an intensity of 1.5% suggested that M7 might also be a precursor ion of M6. Hence, based on the fragmentation mechanism, the structure of the product VII (Fig. 2) was characterized as (S)-2-(4-(1-(2,4-diaminopteridin-6-yl)-1-oxopent-4-yn-2-yl) benzamido)pentanedioic acid.

3.3.8. Product VIII Product VIII was detected at m/z at 362.1 amu as a parent ion (M8) with an actual mass of 361 amu. The +ESI MS2 and MS3 spectrum showed the base peaks at m/z 215.0 amu and 173.1 amu respectively. The even valued M8 corresponding to odd number of nitrogen atom suggested that all the nitrogen atoms of PTXT were proposed to be intact in product VIII. The mass difference of 116 11

amu between M8 and PTXT suggested that the former was formed by loss of glutaric acid moiety and simultaneously undergo oxidation at 15I carbon position from PTXT. This can be further confirmed from product VII. The mass difference of 130 amu between VII and M8 suggested that the later might have similar kind of structure as VII with out glutaric acid moiety. The fragment ion was formed at m/z of 215.0 amu which might be due to loss of pteridine moiety from M8 and formation of 4-(2-oxohex-5yn-3-yl) benzamide. The fragment ion formed at m/z of 173.1 amu might be due to loss of -CONH2 (amide) moiety from the +ESI MS2 fragment ion at m/z 215.0. Based on the mass analysis (Fig. 5), the structure of product VIII (Fig. 2) was characterized as 4-(1-(2,4-diaminopteridin-6-yl)-1-oxopent-4yn-2-yl) benzamide.

3.3.9. Product IX Product IX was detected at m/z at 448.2 amu as a parent ion (M9) with an actual mass of 447 amu. The fragment ions of M9 produced at m/z 348.2 amu and 321.1 amu in + ESI MS2 and MS3 spectrum respectively. A mass difference of 30 amu between PTXT and M9 suggested that the parent ion of IX might have been formed by decarboxylation on glutamic acid moiety and oxidation at 15th carbon position simultaneously. A mass difference of 100 amu between M9 and a fragment peak at m/z 348.2 indicated the loss of pentanoic acid moiety from M9. This suggested that the precursor ion had the pentanoic acid moiety. The fragment ion observed at m/z of 348.2 amu was characterized as 4-(1-(2, 4-diaminopteridine-6-yl) pent-4-yn-2-yl) benzamide (product V). A mass difference of 27 amu in between MS2 and MS3 fragment ions suggested that the latter was formed due to loss of .

CH=CH2 radical ion from MS2 fragment similarly as MS2 fragment ion formed from product V (Fig. 6).

The even valued M9 indicated an odd number of nitrogen atoms suggested that all the nitrogen atoms of PTXT might be intact in product IX. Hence, based on the mass analysis the structure of product

IX

(Fig.

2)

was

established

as

4-(4-(1-(2,4-diaminopteridin-6-yl)-1-oxopent-4-yn-2-

yl)benzamido) butanoic acid.

3.4. Postulated Degradation pathway mechanism The most probable mechanistic explanation for the formation of degradation products I-IX from PTXT is depicted in scheme (Fig. 7).The product I was proposed to form by alkaline/ acid hydrolysis of the 4-amino group of PTXT as part of an amidine system. However, it was a major degradation product in alkaline medium. Product ions II,V and VIII was proposed to form after breakdown of the tertiary amine bond at position 6I, which means the substitution at 6I caused the loss of C5H6O4 from position 1I-5I. The product II was formed as a result of hydrolysis of PTXT in acidic medium. It was 12

formed from PTXT due to cleavage of N-C bond at position 6I and rearrangement via ring structure [11]. The protonation of amide carbonyl in presence of acidic medium leads the formation of product II. PTXT on photolytic oxidation resulted in the cleavage of C14I-C15I (tertiary carbon) to produce product III. Product IV was formed due to photolysis and hydrolysis resulted in the formation of alcohol due to dealkylation of C14I-C18I and cleavage at C-6 position. The conversion of PTXT to product V is occurred due to the photolysis followed by the alkaline hydrolysis which resulted in the formation of amide compound (V) due to cleavage of N-C bond to form amide [11]. The product VI was proposed as an olefin derivative of PTXT formed due to elimination of hydrogen at position 31 and carbon dioxide moiety at position 4I simultaneously. Product VII proposed to form by photo catalysed oxidation at C15I of PTXT. Reaction involves the proton transfer from alkane (C15’) to oxygen and formation of alkyl hydro-peroxides emerging from the recombination of alkyl and OOH. The elimination of H2O yields product VII [12]. The product VIII was formed as a result of by photolytic oxidation followed by hydrolysis of PTXT. The product IX was proposed to form by oxidation and subsequently loosing carbon dioxide from PTXT.

4. Conclusions:

Forced degradation studies on PTXT were conducted in accordance with ICH guidelines. The drug product was found to be sensitive to acid, alkali, peroxide, heat and light. The degradation product I was detected upon hydrolysis and heating. The product II was acid and photolytic degradation product. The products III-IX were formed under photolytic conditions. All the degradation impurities were optimally resolved by LC-MS compatible LC-UV/PDA method. The molecular masses of degradation products were established by recording LC-MS scans in +ESI mode and characterised through LC-MS and LC-PDA data.

Acknowledgements:

The authors are thankful to the management of Gland Pharma Limited for providing necessary facilities and extending their cooperation and support to carry out the present investigation. They are also grateful to JNTUH for giving an opportunity to pursue this research work. 13

References [1] Australian Public Assessment Report for Pralatrexate, August 2013

https://www.tga.gov.au/auspar/auspar-pralatrexate [2] Enrica Marchi and Owen A. O’Connor, Safety and efficacy of pralatrexate in the treatment of patients with relapsed or refractory peripheral T-cell lymphoma, Ther Adv Hematol. (2012) 227-235 [3] ICH Q1A(R2), Stability testing of new drug substances and products (step4), in: international conference on harmonization, Geneva, 2003 [4] ICH Q3A(R2), impurities in new drug substances (step4), in: international conference on harmonization, Geneva, 2006

[5] ICH Q3B (R2), impurities in new drug products (step4), in: international conference on harmonization, Geneva, 2006 [6] DailyMed-FOLOTYN- pralatrexate injection https://dailymed.nlm.nih.gov/dailymed/ [7] ICH Q1B, Stability testing: Photostability testing of new drug substances and products (step4), in: international conference on harmonization, Geneva, 1996 [8] Dulal C.Chatterji and Joseph F. Gallelli, Thermal and Photolytic Decomposition of Methotrexate in Aqueous Solutions, J.Pharm.Sci.(1977) 526-530 [9]

M.M.Araujo, E.Marchioni, M.Zhao, F.Kuntz, T.DiPascoli, A.L.C.H.Villavicencio, M.Bergaentzle, LC/MS/MS identification of some folic acid degradation products after E-beam irradiation, J.Radiat.Phys.Chem. (2011) 1-4

[10] Chapter 3 Photochemical Transformations of Talotrexin and Xipamide

http://shodhganga.inflibnet.ac.in/bitstream/10603/28509/8/08_chapter%203.pdf [11] Cai-Sheng Wu, Yuan-Feng Tong, Peng-Yuan Wang, Dong-Mei Wang, Song Wu and Jin-Lan Zhang, Identification of impurities in methotrexate drug substances using high-performance liquid chromatography coupled with a photodiode array detector and Fourier transform ion cyclotron resonance mass spectrometry, J.Rapid commun. mass spectrum. (2013) 971-978 [12] Heinz Frei, Berkeley, CA (US); Fritz Blatter, Basel (CH); Hai Sun, Saint, Charles, MO (US).Selective Thermal and Photooxidation of Hydrocarbons in Zeolites by Oxygen, in: United States Patent Frei et al Patent N0.:(45) Date of Patent: US 6,329,553 B1(2001)

14

12' NH2 5 15' 4 10 N 6 11' 3N 14'

8'

1

OH 5'

6'

2'

N 4' 3' H

7'

9'

10'

H2N 2 N 9 N 7 16' 17' 8

O

O

13'

1' OH

O

18'

Fig. 1. Pralatrexate (PTXT) structure

O O OH

H2N

N

OH

NH2 OH

N H

N

N

O

O

H2N

OH N

N N

N

N Product-II Product-I

O O

NH2 N

N H2N

N

CO2H

O

OH

N H HO

N

NH2 OH O

H2N

N

Product-IV

Product-III

NH2 N

N

N Product-V

O NH2 N

N H2N

O

OH

N H

NH2

O

N

H2N

N

Product-VI

OH OH

N H

N

N N

O

O

O

N Product-VII

O O NH2

NH2

N

H2N

N

O N

N

N

N H2N

O

NH2 N

N H

OH O

N Product-IX

Product-VIII

Fig. 2. Pralatrexate (PTXT) DP degradation products

15

16

Fig. 3. Chromatogram of control sample of PTXT (a) and PTXT subjected to acid hydrolysis (b) alkaline hydrolysis (c) thermal (d) peroxide (e) and photolysis (f)

17

O NH2 N H2N

N

CO2H N H

N N

CO2H

Molecular mass 477 amu

PTXT

+ MS1

-MS1

O NH2 N H2N

N

N H

N

O

CO2H

O

NH2 N

O H2N

N

N

CO2H CO2H

N H

N

H

N m/z 478.2 (M)

m/z 476.0 (MI)

O NH2 N H2N

N

CO2H

N H HO

N

CO2H O

N

N-C bond cleavage followed by rearrangement

+MS2

-MS2

O

O NH2 N H2N

N

NH2

O N

N H2N

N

N

OH N N

m/z 347.0 (F1I) -MS3

m/z 349.1 (F1) +MS3

CO2 decarboxylation

.

CH=CH2

O NH H2N

NH2

N

N N

N m/z 303.0 (F3)

H

N H2N

N

+ .

OH N N

m/z 322.1 (F2)

Fig. 4. Proposed mass fragmentation pattern of Pralatrexate in +ESI mode and –ESI mode

18

O O OH OH N H O

OH N

N

H2N N N

H O

OH

N-C bond cleavage followed by rearrangement

-MS2 +MS2

N HOH

OH N

N H2 N

N

C O

N

H OH

H OH

O

-MS3

N N

m/z 149 base peak

O m/z 278.0 (+ESI) m/z 275.9 (-ESI) O

OH

OH O

H2 O

N H

N

H2 N

O O OH

-MS2 +MS2

H2N

O OH

m/z 296.1 (M4) (+ESI) m/z 293.9 (M4I) (-ESI)

O O OH

N CO2

m/z: 350.2 (+ESI) m/z: 348.0 (-ESI)

H2N

CO2

N

N N

O

N

N H O m/z 231.9

m/z 305.0

.

CH=CH2

O

MS3 +.

O OH H2N

OH N

N N

N

C CH2 m/z:323.1

O NH2

H NH2

O N

N H2N NH2 N

N H2N III

N

COOH

H

N N m/z 362.1 (M8) VIII

N

H2N

m/z 207.1 (M3) (+ESI) m/z 204.8 (M3I) (-ESI)

O

CO2

N

N H2N

N

N

H

m/z 215.0 MS3

NH2

N

N

NH2

O -MS2 +MS2

NH2 N

MS2

elimination of amide group

H O

H

N

m/z 163.0 (+ESI) m/z 160.9 (-ESI)

+

HO

O

OH IV

O

MS2

H OH

N H

m/z479.2 (M1) (+ESI) m/z:477.0 (M1I) (-ESI)

I

O

m/z 173.1

Fig. 5. Proposed mass fragmentation pattern of degradation products I,III,IV and VIII

19

MS2

O NH2

N-C bond (connecting to aromatic ring)cleavage / rearrangement

OH N

N H2N

H

N

NH2 N N

m/z: 349.2 (M2)(+ESI) m/z: 347.0 (M2I)(-ESI)

II 2

-MS / +MS

MS2

CO2

m/z: 432.2 (M6)

MS3

CH=CH2

+.

O N

N

N H2N

N

m/z 305.1 (+ESI) m/z 303.0 (-ESI) (II)

OH

OH

NH2

N N

O

VI

NH2 H2 N

OH

N H

H2N N N

3

H

N

H

O

N

O

C CH2

N

m/z 322.1

O NH2 N

N H2N

MS3

H NH2

N

N

elimination of amide moiety

V m/z: 348.2 (M5)

O

H2N

N N

H

O

NH2 N

MS2

OH

H2N

N H

N

N

MS2

N

O

NH2

CH=CH2

N

OH O

m/z 448.2 (M9)

N

IX

m/z 304.1 +.

O NH2 H2N

NH2 N

N N

N

MS3

C CH2

m/z 321.1 MS2 O O OH NH2 O N N

N H

N-C bond (connecting to aromatic ring)cleavage / rearrangement

H2N N N VII

NH2 O N N

OH O

OH

H2N N N

m/z 363.1 m/z 492.2 (M7) MS3

.

CH=CH2

deoxidation/-CO2

N H

N

OH O

+.

O

H

O NH2 N N

H

O

H

H2 N

NH2 O N C CH2 N N

OH

H2N N N m/z 432.1(VI)

m/z 336.1

fragment ion

Fig. 6. Proposed mass fragmentation pattern of degradation products II,V,VI,VII and IX.

20

NH2 O NH2

OH

H 2N

N

O OH

CO2H N H

N N

O

N

elimination of O OH OH N hv H O moiety

acid hydrolysis/ hv

H2N

N

H OH

CO2H

Product I m/z: 479.2

HO

hv

O

N

NH2 N

N

N H2 N

N

oxidation and elimination of glutaric acid moiety

N Product IX m/z 448.2

O NH2

hv

hv

N

N

Product VIII m/z 362.1

Product V (m/z 348.2)

hv O NH2

H 2N

N H

N

N

oxidation

N

N Product VI m/z 432.2

O

H NH2

NH2

N

N H 2N

O

N

elimination of glutaric acid moiety

dehydro genation and decarboxy lation

H O

N

PTXT m/z 478.2

OH

N H

H NH2

hv CO2H

hv / oxidation O

H2N

CO2H N H

N

N H 2N

O

N

N

-CO2

O

Product-IV m/z 296.1 cleavage of pteridine ring and elimination of alkyl chain

H

acid /base hydrolysis

H CO2H

NH2

NH2

N

CO2H N H

Product III m/z 207.1

N Product II (m/z 349.2)

N

O N

N

N

N H2N

H

N

N H2 N

O

N

CO2H N H

CO2H

H

N

Product VII m/z 492.2

Fig. 7. PTXT and degradation products (I-IX) degradation pathway mechanism.

21

CO2H H

Table1 Measured masses of precursor and fragment ions of PTXT and its degradation products (I-IX) in different MS conditions PTXT degradation products MS condition

PTXT I

II

III

IV

V

VI

VII

VIII

IX

+ESI/MSI a (RI %) c

478.2(M) 479.2 (100) (100)

349.2 (100)

207.1 (100)

296.1 (100)

348.2 (100)

432.2 (100) 348.2 (83.6)

492.2 (100) 432.1 (1.5)

362.1 (100)

448.2 (100)

+ ESI/MS2 (RI %)

349.1(F1) 350.2 (100) (100)

322.1 (100)

163.0 (100)

278.0 (26.6) 149.0 (100)

321.1 (100)

349.2 (100)

363.1 (100)

215.0 (100)

348.2 (100)

+ ESI/MS3 (RI %)

322.1(F2) 323.1 (100) (100)

305.1 (100)

NF d

304.1 (100)

322.1 (100)

336.1 (100)

173.1 (8.4)

321.1 (100)

-ESI/MSI b (RI %)

476.0(MI) 477.0 (100) (100)

347.0 (100)

204.8 (100) 160.9 (51.3)

293.9 (100)

NIe

NI

NI

NI

NI

-ESI/MS2 (RI %)

347.0(F1I) 348.0 (100) (100)

303.0 (40.7)

NF

275.9 (100) 231.9 (98.3)

NI

NI

NI

NI

NI

- ESI/MS3 (RI %)

303.0(F3) 305.0 (94.2) (100)

NF

NF

NF

NI

NI

NI

NI

NI

a

[M+H] +

b

[M-H] -

c

Relative intensity

d

No further ionization

e

No ionization

22