Identification, separation, isolation and characterization...

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Chapter-IV

Identification, separation, isolation and

characterization of impurities present in capecitabine

active pharmaceutical ingredient#

4.1.0 Introduction

Capecitabine, chemically known as pentyl[1-(3,4-dihydroxy-5-methyl-

tetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl] aminomethanoate is an

orally administered fluoropyrimidine carbamate drug which has been approved by the

FDA (Food and Drug Administration) for use as first-line therapy in patients with

metastatic colorectal cancer [1-3]. The drug is also commonly used as a single agent

in the treatment of metastatic breast cancer which is resistant to both anthracycline

and paclitaxel based treatments where further anthracycline treatment is

contraindicated. It is also used in combination with docetaxel after failure of the prior

anthracycline-based chemotherapy. The drug (when administered individually or in

combination with other anti-tumor drugs) has also shown benefits in the patients with

prostate, pancreatic, renal, and ovarian cancers [4]. The chemical structure of

Capecitabine is:

# This work has been published in ACTA CHROMATOGRAPHICA 20, 4 (2008) 609-624.

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NH

O OH

OHCH3

NH

O

F

OCH3

O

IUPAC name: pentyl[1-(3,4-dihydroxy-5-methyl-tetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-

pyrimidin-4-yl] aminomethanoate

Capecitabine is a prodrug, which is converted to 5-fluorouracil by thymidine

phosphorylase in the tumors, where it inhibits the DNA (Deoxyribonucleic acid is a

nucleic acid that contains the genetic instructions used in the development and

functioning of all known living organisms and some viruses) synthesis and slows

down growth of the tumor tissue. Recent investigations on oral fluoropyrimidines are

aimed at achieving pharmaco-economic advantages and, possibly, to improve the

quality of life of the cancer patients [4-7]. Consequently, there are several literature

reports on the determination of capecitabine and its metabolites in biological fluids

using HPLC [8-10], LC-MS/MS [11], and NMR [12-14]. These methods involve

quantification of capecitabine, 5’-deoxy-5-fluorocytidine (5’DFCR) (peak 1), 5’-

deoxy-5-fluorouridine (5’DFUR) (peak 2) from the biological fluids and isolation of

unknown metabolite during the metabolic study of capecitabine [13-14]. Literature

search revealed that there is no report on the impurity profiling of the drug. Hence, it

was the interest of author to carry out such experiments. During one such experiment

on the drug, the HPLC chromatogram showed five small peaks in addition to the main

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peak due to capecitabine. The area percent of all the five peaks ranged from 0.05 to

0.12%. The peak 1 and peak 2 were identified by mass and NMR spectral data as 5’-

deoxy-5-fluorocytidine (5’DFCR) and 5’-deoxy-5-fluorouridine (5’DFUR),

respectively, which were already reported [13-14].The present investigation was

carried out to confirm whether these additional peaks are due to the related

substances/impurities of capecitabine. The compounds due to these peaks were also

characterized since the regulatory requirement from ICH on impurities was mandatory

that all peaks having more than 0.05% area in the chromatogram should be

characterized [15].

This chapter describes separation and identification of the three impurities present

in capecitabine drug substance by analytical HPLC; and also their isolation by

preparative HPLC followed by characterization by NMR and mass spectral

techniques.

4.2.0 Experimental details

4.2.1 Reagents and samples

Capecitabine drug substance and standards for two impurities (intermediates)

were supplied by M/S Cipla Ltd., Bangalore, India. Sodium hydroxide pellets and

hydrochloric acid were procured from Rankem, New Delhi, India. Ammonium

acetate, trifluoroacetic acid (TFA), HPLC grade methanol and dichloromethane were

purchased from Spectrochem, Mumbai, India. All the chemicals were of analytical

reagent (AR) grade. HPLC grade water was obtained from a Milli-Q water

purification system (Millipore, MA, USA).

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4.2.2 High performance liquid chromatography (Analytical)

Two analytical HPLC systems were used in the studies. One was Agilent 1200

Series system consisting of an on-line degasser, G1311A quaternary pump, G1329A

auto liquid sampler, G 1316A thermostat column controller and G1314B variable

wavelength UV detector (VWD). The data acquired on this system were processed

through the use of Chemstation software version B-02-01-SR1 (260). Another HPLC

system consisted of Waters Alliance 2695 separations module and 2996 Photo-diode

array (PDA) detector. Empower 2 software (version 6.10.00.00) was used to process

the data obtained from Waters system. The separations were carried out on an Inertsil

ODS-3V (250 mm x 4.6 mm, 5 µ) column (G L Sciences Inc., Torrance, USA). For

the analytical separations, a solvent gradient program was used, wherein solvent A

consisted of 20 mM ammonium acetate buffer (pH 4.6 adjusted with TFA) and

methanol (67:33 v/v); and solvent B contained methanol. The initial mobile phase

ratio adjusted as 100 % v/v of solvent A. Subsequently the percentage of solvent B

(methanol) in the mobile phase was increased from 0 to 26 up to 7 mins. The same

ratio was held for 18 mins, and the percentage of methanol in the mobile phase was

increased from 26 to 70 up to 7 mins. The same ratio was held for 13 mins, and

brought back to the initial condition within 5 mins. The column was allowed to get

equilibrated for 10 min before performing the next injection. The injection volume

was 20µL. The mobile phase was pumped at a flow rate of 1.2 ml/min. The column

oven was set at ambient conditions and the detection was made at 290 nm.

4.2.3 High performance liquid chromatography (preparative)

For the collection of isolated fractions, a preparative HPLC system (Agilent

1200 Series) equipped with G1361A binary pump, G2260A auto liquid sampler,

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G1364B fraction collector and G1315B Diode Array Detector (DAD) (Agilent

Technologies, USA) was used. The Data were processed through Chemstation

software version B-02-01 (244). For the same, a semi-preparative column (250 mm x

20 mm, 5µm) from Inertsil ODS-3 column ((G L Sciences Inc., Torrance, USA).)

was employed.

For collection of the two early eluting fractions, a different gradient program

was used with mobile phase A containing 20 mM ammonium acetate buffer and

methanol (67:33 v/v) at pH 4.6 and mobile phase B containing methanol. The pH of

the mobile phase A was adjusted with TFA. The initial mobile phase ratio was

adjusted to 100 % v/v of mobile phase A for 10 mins. Subsequently the percentage of

mobile phase B in the mobile phase was increased from 0 to 80 up to 5 mins. The

same ratio was held for 5 mins, and brought back to the initial condition with in 4

mins. The column was allowed to get equilibrated for 4 min before performing the

next injection. The injection volume was 900µL. Flow rate was kept at 20.0 ml/ min

and column eluent was monitored at 290 nm. For collection of a late eluting fraction,

a different gradient program was used with mobile phase A containing 20 mM

ammonium acetate buffer and methanol (80:20 v/v) at pH 4.6 and mobile phase B

containing methanol. The pH of the mobile phase A was adjusted with TFA. Where

the initial mobile phase ratio was a 50:50, v/v of mobile phase A and mobile phase B

for 5 mins. Subsequently the percentage of methanol in the mobile phase was

increased from 50 to 80 up to 5 mins. The same ratio was held for 5 mins, and brought

back to the initial condition within 3 mins. The column was allowed to get

equilibrated for 5 min before performing the next injection.

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4.2.4 FT-IR

IR spectra of the olanzapine and its impurities were recorded in the solid state

as KBr dispersion using Perkin Elmer spectrum 100 series FT-IR spectrophotometer

with DRS (Diffuse Reflectance Sampler) technique.

4.2.5 Mass spectrometry (LC-MS/MS)

LC part of the LC/MS/MS system consisted of an Agilent-1100 series

quaternary gradient pump with a degasser, an auto sampler and a column oven

(Agilent Technologies, USA). MS/MS part of the system contained API-2000 system

(Applied Bio-Systems, Concord, Canada). The chromatographic conditions used for

the LC separations were same as described in section 3.2.2. The HPLC effluent was

introduced (30 % of the total effluent) into electron spray ionization (ESI) positive

mode and negative mode at a flow rate of 1.2 ml/min with split ratio of 3:7. The ion

source voltage was maintained at 5500 volts and the capillary temperature was 350

°C. Nitrogen was used both as nebulizer and turbo spray gas. Mass range was tuned to

50-1000 amu. MS/MS studies were carried out by maintaining normalized collision

energy of 35 eV with the m/z range of 50-600 amu. The MS and MS/MS data

obtained for capecitabine and its impurities are given in the Table 4.1.

Table 4.1: MS and MS/MS data for capecitabine and the other peaks on the

chromatogram

Drug/Impurity (M+1)/(M-1) m/z value for parent molecule and fragments

Capecitabine (M+1) 360.4, 244.2, 173.9, 130.0, 102.8, 70.9 Peak 1 (M+1) 245.9, 130.0, 128.9, 117.3, 72.9

Peak 2 (M-1) 245.1, 227.0, 170.9, 107.9, 83.8, 79.9, 65.8, 62.0

Peak 3 (M+1) 330.1, 201.3, 141.1, 129.9, 113.1, 99.0 Peak 4 (M+1) 444.1, 244.2, 201.2, 173.9, 141.1, 99.0 Peak 5 (M+1) 558.2, 444.1, 358.3, 314.0, 244.2, 201.2, 173.7, 140.9, 98.9

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4.2.6 NMR spectroscopy

The 1H, 13C and DEPT experiments were performed on OXFORD AS 400

NMR (Varian, Palo Alto, USA) with dual broad band. 1H chemical shift values were

reported on the δ scale in ppm relative to TMS (δ = 0.000 ppm) and 13C chemical

shifts values were reported relative to DMSO-d6 (δ = 49.5 ppm) and CDCl3 (δ = 72.5

ppm). The NMR data for 1H, 13C and DEPT experiments for all the peaks along with

capecitabine are presented in Table 4.2. The NMR spectrum for Capecitabine and

impurity peaks1 to 5 are shown in the figure 4.1 to 4.12.

Table4. 2: NMR data for 1H, 13C and DEPT experiments.

Drug/Impurity 1H No. of protons/ppm (Position No.)

13C ppm (Position No.)

DEPT ppm (Position No.)

Capecitabine 3H/0.88 (20), 4H/1.31 (18, 19), 3H/1.31 (11), 2H/1.60 (17), 1H/3.66 (9),1H/3.89 (8), 1H/3.9 (10), 2H/4.01 (16), 1H/5.07 (12), 1H/5.44 (13), 1H/5.67 (7),1H/8.07 (6), 1H/10.54 (14)

14.37 (20), 18.71 (11), 22.32 (19) 27.96 (18), 28.39 (17), 66.23 (16) 74.19 (9), 74.49 (8), 79.83 (10) 91.55 (7), 129.8 (6), 137.0 (5), 152.2(15), 153.1 (2), 153.8 (4)

14.35 (20), 18.75 (11), 22.3 (19) 27.97 (18), 28.4 (17), 66.25 (16) 74.25 (9), 74.63 (8), 79.78 (10) 91.58 (7), 130.0 (6)

Peak 1 3H/1.27 (11), 1H/3.66 (9), 1H/3.81 (8), 1H/3.99 (10), 1H/5.0 (12), 1H/5.28 (13),1H/5.68 (7), 2H/7.72 (14), 1H/7.75 (6)

19.13 (11), 73.96 (9), 74.91 (8) 79.33 (10), 90.65 (7), 126.6 (6) 137.5 (5), 154.2 (2), 158.0 (4)

19.1 (11), 74.0 (9), 74.9 (8) 79.4 (10), 90.7 (7), 126.0 (6)

Peak 2 3H/1.28 (11), 1H/3.70 (9), 1H/3.84 (8), 1H/4.10 (10), 1H/5.09 (12), 1H/5.35(13), 1H/5.68 (7), 1H/7.92 (6), 1H/11.90(3)

19.23 (11), 73.14 (9), 74.69 (8) 80.04 (10), 89.63 (7), 126 (6) 140.5 (5), 149.9 (2), 157.6 (4)

199.22 (11), 73.14 (9), 74.7 (8) 80.05 (10), 89.64 (7), 125.9 (6)

Peak 3 3H/1.44 (11), 3H/2.08 (13), 3H/2.11(15), 1H/4.22 (10), 1H/4.99 (9), 1H/5.32(8), 2H/5.96, 8.65 (16), 1H/5.97 (7), 1H/7.39 (6)

18.58 (11), 20.7 (13), 20.72 (15) 73.84 (9), 74.13 (8), 77.51 (10) 88.87 (7), 124.5 (6), 137 (5) 154 (2), 158.5 (4), 169.8 (12)

18.58 (11), 20.65 (13), 20.67 (15) 73.85 (9), 74.13 (8), 77.5 (10) 88.86 (7), 124.5 (6)

Peak 4 3H/0.91 (22), 2H/1.31 (21), 2H/1.31 (20)3H/1.46 (11), 2H/1.74 (19), 3H/2.09 (13)3H/2.12 (15), 2H/4.17 (18), 1H/4.17 (10)1H/5.01 (9), 1H/5.29 (8), 1H/5.95 (7) 1H/7.40 (6), 1H/12.02 (16)

14.12 (22), 18.74 (11), 20.58 (13) 20.63 (15), 22.4 (21), 28 (20), 28.4 (19),66.8 (18), 73.1 (10), 74 (9), 78.3 (8),88.2 (7), 124 (6), 139.5 (5), 146.4 (17),15.3 (2), 153.5 (4), 163.6 (12), 169.9 (14)

14.12 (22), 18.75 (11), 20.58 (13) 20.63 (15), 22.4 (21), 28.02 (20) 28.41 (19), 66.8 (18), 73.1 (10) 74 (9), 78.31 (8), 88.2 (7), 124.2 (6)

Peak 5 6H/0.86 (22, 28), 4H/1.26 (21, 27) 4H/1.26 (20, 26), 4H/1.71 (19, 25) 4H/4.13 (18, 24), 3H/1.39 (11) 3H/2.11 (13), 3H/2.15 (15), 1H/4.15 (10)1H/4.99 (9),1H/5.22 (8)1H/5.89 (7) 1H/7.45 (6)

14.11 (22, 28), 22.36 (21, 27), 28.01 (20, 26), 28.38 (19, 25), 66.78 (18, 24), 146.41 (17, 23) 18.74 (11), 20.6 (13), 20.62 (15), 73.12(10), 74.03 (9), 78.32 (8), 88.23 (7),124.1 (6), 139.48 (5), 153.35 (2),153.61 (4), 163.62 (12)

14.1 (22, 28), 22.38 (21, 27) 28.02 (20, 26), 28.4 (19, 25) 66.79 (18, 24), 18.74 (11), 20.59 (13), 20.63 (15), 73.11 (10) 74.02 (9), 78.31 (8), 88.22 (7), 124.2(6)

For numbering refer Figure 4.14

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Figure 4.1: 1H NMR spectrum of Capecitabine recorded in d6-DMSO

Figure 4.2: 13C NMR spectrum of Capecitabine recorded in d6-DMSO

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Figure 4.3: 1H NMR spectrum of impurity Peak1 recorded in d6-DMSO

Figure 4.4: 13C NMR spectrum of impurity Peak1 recorded in d6-DMSO

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Figure 4.5: 1H NMR spectrum of impurity Peak2 recorded in d6-DMSO

Figure 4.6: 13C NMR spectrum of impurity Peak2 recorded in d6-DMSO

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Figure 4.7: 1H NMR spectrum of impurity Peak3 recorded in CDCl3

Figure 4.8: 13C NMR spectrum of impurity Peak3 recorded in CDCl3

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110



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4.2.7 Trials to enrich the impurities

For isolation of the impurities in a quantity, which is sufficient enough to

confirm their structure through various spectroscopic techniques; attempts were made

to increase the content of the impurities through acid and base treatment of the drug.

For acid treatment, 1 g of the drug (capecitabine) was taken in 25 ml of 0.1M HCl and

stored at ambient conditions for 1 h. Subsequently, the solution was diluted further 2

folds with a mixture of acetonitrile:water (1:1 v/v). Similarly, for the base treatment, 1

g of the drug was taken in 25 ml of 0.1M NaOH and kept at ambient conditions. Also,

the trials were made by refluxing the drug with 0.1M NaOH for 1 h. In both the cases,

the respective solutions were further diluted 2 folds with a mixture of

acetonitrile:water (1:1 v/v). The resulting solutions were checked for the content of

different impurities on HPLC.

4.3.0 Results and discussion

4.3.1 Detection of impurities in capecitabine by reverse phase HPLC

Capecitabine (as obtained) sample solutions were prepared in the mobile

phase A to get the required concentration of about 1 mg/mL. The solutions were

analyzed using the solvent system as described in the section 4.2.2. In the

chromatograms, along with the peak due to capecitabine, five other peaks were

observed. These were appearing at RRT (relative retention time) of 0.18 (peak 1),

0.20 (peak 2), 0.41 (peak 3), 1.74 (peak 4) and 2.13 (peak 5), respectively. The

typical chromatogram (Figure 4.13) showing all the peaks appeared distinctly.

Percent area on the chromatogram for the five peaks (other than drug) was ranging

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from 0.05%-0.12%. Resolution for each peak was more than 2.0 from any other

nearest peak. Further, the peaks were checked for purity to ascertain the absence

of any co-eluting peaks and it was found that all the peaks were pure. Table 4.3

gives further details on the data for purity angle, purity threshold and resolution.

This indicated that there are five potential impurities that may be present in

capecitabine.

Table 4.3: Retention time (RT), purity angle, purity threshold and resolution for the different peaks. Peak Retention time

(Minutes)

Purity

Angle

Purity

Threshold

Resolution

Peak 1 3.3 1.500 1.981 -

Peak 2 3.7 1.018 1.382 2.6

Peak 3 7.6 2.202 2.835 18.5

Capecitabine 18.5 0.067 0.282 24.6

Peak 4 32.2 1.403 1.746 27.0

Peak 5 39.5 3.873 4.166 22.5

Pea

k1 -

3.31

2P

eak2

- 3.

747

Pea

k3 -

7.67

6 Cap

ecita

bine

- 18

.542

Pea

k4 -

32.2

23

Pea

k5 -

39.4

58

AU

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Wavelength: 290 nm

Figure 4.13: HPLC chromatogram of Capecitabine showing five other peaks in addition to the main peak due to Capecitabine.

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4.3.2 Analysis of acid and base treated samples by reverse phase HPLC

The samples mentioned under the section 4.2.5 were injected into analytical

LC using the solvent gradient system as described in section 4.2.2. The acid treated

sample resulted in two major peaks, out of which one was of the drug and the other

was at RRT 0.20 (peak 2), area percent of which was 39.7%. In the case of base

treated sample too, a peak other than drug was observed at RRT 0.18 (peak 1). Area

percent of this peak was ~17.6%. Incidentally, the two new peaks observed in the acid

and the base treated samples were at the same RRT as that of peaks 2 and 1,

respectively, in the chromatogram for capecitabine (Figure 4.13).

4.3.3 Characterization of impurity peaks 3 and 4

Attempts were made to characterize all the new peaks (other than that

of the drug) appearing in the chromatogram for Capecitabine. Standard of the two

process related impurities, which are the intermediates for synthesis of the drug [17],

i.e., (2-(4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)-5-methyltetrahydro furan-3, 4-

diyl diacetate (intermediate I) and 2-(5-fluoro-2-oxo-4-(pentyloxycarbonyl amino)

pyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3, 4-diyl diacetate (intermediate II),

were available in the laboratory. The same were spiked with the drug to get an idea

whether any of the new peaks corresponded to the process related impurities.

Intermediate I eluted at the same RRT as that of peak 3, while intermediate II had the

same RRT as that of peak 4.

Furthermore, this observation was confirmed through LC-MS

experiments and details of the results are shown in Table 2. As depicted, M+1 value

for the peak 3 was 330.1 amu, which corresponds to that of Intermediate I. Also, M+1

value for the peak 4 was 444.1 amu, and the same is as for Intermediate II.

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For the remaining three peaks, no impurity standards were available.

Therefore, it was decided to isolate the other three peaks by preparative HPLC for

further characterization by different spectroscopic techniques.

4.3.4 Isolation of impurity peaks 1, 2 and 5 by preparative HPLC

Approximately 3.0 g of the acid treated sample was loaded onto the

preparative HPLC coloumn to collect the fraction corresponding to peak 2 in the

chromatogram. The fractions were collected at retention time from 4.5 to 5.5 minutes,

and were pooled together. The purity of the pooled fractions was checked on

analytical HPLC by using the method described in the section 4.2.2. The same was

~98.8% on area percent basis. The collected and pooled fractions were subjected to

rotary evaporator under high vacuum. The remaining aqueous solution containing

ammonium acetate was subjected to liquid-liquid extraction using dichloromethane

(in 1:1 v/v ratio with the aqueous solution) by shaking in a separator funnel for about

2 minutes. The organic layer was collected and concentrated on the rotary evaporator

under high vacuum to obtain the isolated product in solid form. The solid thus

obtained was again scrutinized on analytical HPLC and the same was found to be of

98.4% pure (on area percent basis), which was relatively good enough for carrying

out subsequent spectroscopic experiments.

Similarly, approximately 5.0 g of the base treated sample was also loaded on

to the preparative HPLC coloumn to collect the fractions corresponding to the peak 1

in the chromatogram. The fractions were collected at retention time from 3.8 to 4.9

minutes, and were pooled together. The purity of the pooled fractions on analytical

HPLC was found to be ~ 99.1% on area percent basis. The fractions were treated in

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the similar way as that of the acid treated samples. The obtained solid product was

found to be ~ 98.2% pure (on area percent basis), when checked on HPLC.

As peak 5 was present only upto a level of 0.12% in the original drug and also

the level of this was not increased even after acid/base treatment, the same was

isolated from the obtained drug sample. As peak 5 was very late eluting on analytical

column (Figure 4.13), for its isolation, sufficient modifications were done to the

gradient elution programme used for isolation of peaks 1 and 2. Approximately 50 g

of the capecitabine sample was loaded on to preparative HPLC and fractions were

collected from 16.7 to 17.2 minutes. The pooled fractions showed a purity value of

97.8% on area percent basis. The collected fractions were processed further as

discussed above. The obtained solid in the final step was again subjected to HPLC

analysis and purity of the same was found to be about 97.2 (on area percent basis).

4.3.5 Structural elucidation of the isolated compounds (impurity Peak 1)

All the isolated solid compounds collected from fractions of peaks 1, 2 and

5, respectively, were subjected to various spectroscopic analyses (Mass, IR, and

NMR) for the structure elucidation. Tables 4.2 and 4.3 show m/z values for all the

isolated and characterized compounds. Although the peaks 3 and 4 were not isolated,

data for them are also shown in the table for comparison purpose. It is shown that

compound collected from fraction of peak 1 exhibited (M+1) peak at 245.9, indicating

a mass of 245. The other information provided by the mass spectra was the possibility

of presence of odd number of nitrogen atoms (due to odd mass value).

The 13C NMR spectrum of the isolated fraction of peak 1 displayed signals

due to 9 carbons and DEPT spectrum displayed 6 positive signals (one due to methyl

group and 5 due to –CH- groups, Table 4.3). However, 13C NMR spectrum of

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capecitabine displayed signals due to 15 carbons and DEPT spectrum displayed 4

negative signals due to the presence of 4 methylene groups and 8 positive signals (2

due to methyl groups and 6 due to –CH- groups). The signals in the 1H NMR

spectrum at δ 7.56 and 7.81 ppm are supposed to be present due to two amine protons

(-NH2), but this signal was not observed in the 1HNMR spectrum of capecitabine. In

addition to this observation, characteristic absorption band at around 1718 cm-1 due to

–CO- stretching in the IR spectrum of capecitabine was absent in case of Peak 1. This

observation was supported by the appearance of 4 methylene groups and one methyl

group signal in the 1H NMR spectrum of capecitabine, which was not observed in the

1H NMR spectrum of peak 1. Based on the spectral data, structure of the peak 1 was

assigned as 4-amino-1- (3,4-dihydroxy -5- methyltetrahydrofuran-2-yl) -5-

fluoropyrimidin-2(1H)-one (Figure 4.14).

4.3.6 Structural elucidation of impurity Peak 2

The mass spectrum of peak 2 exhibited a molecular ion at (M-1) 245.1 amu

which was 1 amu more than that of peak 1 and 113 amu less than that of capecitabine.

The even molecular ion of peak 2 indicated the possibility of presence of even number

of nitrogen atoms. 1H NMR spectrum of peak 2 showed a broad singlet signal (brs) at

11.9 ppm due to -NH- proton which was not observed in the 1H NMR spectrum of the

peak 1. The 13C NMR spectrum displayed signals due to 9 carbons, and DEPT

spectrum displayed 6 positive signals (one due to methyl and 5 due to –CH- groups).

This observation was further supported by the appearance of signal at 157.6 ppm (due

to –CO- group) in the peak 2, which was not observed in the corresponding spectrum

of the drug, capecitabine. Other signals in the 1H NMR spectrum of the drug were

displayed at 0.88, 1.31, 1.60 and 3.89 ppm due to methyl and methylene protons

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which were absent in the 1H NMR spectrum of peak 2. In addition to this observation,

two characteristic absorption bands at around 1717 cm-1 and 1688 cm-1 due to two –

CO- stretching were observed in the IR spectrum of peak 2 and this was absent in the

IR spectrum of capecitabine. Based on the above spectral data the structure of the

unknown peak 2 was characterized as 1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-

yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (Figure 4.14).

4.3.7 Structural elucidation of impurity Peak 5

The molecular ion of peak 5 exhibited (M+1) at 558.2 amu (Figure 4.16)

which was 114 amu more than that of peak 4 and 198 amu more than that of

capecitabine (M+1) 360.4 amu (Figure 4.15). This indicated the possibility of

presence of two pentyl groups in the compound due to peak 5. The fragmentation

pattern obtained in MS/MS data was similar to that of the fragmentation pattern

obtained in the MS/MS data for peak 4. The fragmentation pattern obtained in

MS/MS data (Figure 4.17) also supported that one of fragment ion was observed as

m/z 444.4 amu due to cleavage of a pentyloxy group. On the other hand, other

fragment ions at m/z values of 244, 201, 174, 141 and 99 were matching with the

fragment ions of peak 4. The 1H NMR spectrum of peak 4 shows a singlet at 12.02

ppm due to –NH- proton, which was not observed in the 1H NMR spectrum of peak 5.

Based on the above spectral data, the structure of the peak 5 was characterized as 2-

(4-(bis(pentyloxycarbonyl) amino)-5-fluoro-2-oxopyrimidin-(2H)-yl)- 5- methyl

tetrahydro furan -3,4-diyl diacetate (Figure 4.14).

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N

NO

F

O

OH

OH

H3C

NH O

O

20

19

18

17

161514

123

45

7

89

11

3

12

13

6

10

N

NO

F

O

OH

OH

H3C

NH2

123

45

6

78

910

1112

13

14

HN

NO

F

O

OH

OH

H3C

O

12

34

5

6

78

910

11 12

13

N

NO

F

O

O

O

H3C

NH2

O

O

12

34

5

6

78

910

1112

13

14

15

16

N

NO

F

O

O

O

H3C

NH O

O

O

O

123

45

6

7

8

910

1112

13

1415

1617

18

19

20

21

22

N

NO

F

O

O

O

H3C

N O

O

O

O

123

45

6

78

910

11

1213

14

15

16 1718

19

20

21

22

O

O

23

24

25

26

27

28

Capecitabine Peak 1

Peak 2Peak 3

Peak 4 Peak 5

Fig. 4.14: Chemical structures of capecitabine and its five impurities.

119

Figure 4.15.Mass spectrum of Capecitabine.

Figure 4.16.Mass spectrum of impurity peak 5.

120

Figure 4 .17: Fragmentation pathway of the impurity Peak 5

4.4.0 Conclusion

In the present study, a gradient RP-HPLC method was developed for

separation of capecitabine and other five peaks (potential impurities). The mobile

phase consisted of 20 mM ammonium acetate buffer and methanol. Flow rate and

detection wavelength were 1.2 mL/min. and 290 nm, respectively. The developed

method could successfully separate all the peaks with good resolution and all the

peaks were found to be pure. The same method was transferred to LC-MS and mass

values of all the peaks other than capecitabine were determined. Two out of the five

new peaks were found to be due to the process related intermediates (confirmed

through mass spectral data and retention time matching with standards). Stress studies

121

in acidic and basic media showed increase in content of two of the other three peaks

and both of them along with the third one were isolated by preparative HPLC. All the

three isolated fractions were further characterized using MS/MS, FT-IR and NMR

(1H, 13C and DEPT). Based on the spectral data, structures of the three impurities

were proposed as 5’ deoxy-5-fluorocytidine (Peak 1), 5’ deoxy-5-fluorouridine (Peak

2) and 2-(4-(bis(pentyloxycarbonyl)amino)-5-fluoro-2-oxopyrimidin-(2H)-yl)-5-

methyl tetrahydrofuran-3, 4-diyl diacetate (Peak 5).

Out of five impurities observed in the Capecitabine drug substance, only

impurity peak-5 (2-(4-(bis(pentyloxycarbonyl)amino)-5-fluoro-2-oxopyrimidin-(2H)-

yl)-5-methyl tetrahydrofuran-3, 4-diyl diacetate) is novel.

122

4.5.0 References

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Brouwers, Clin. Ther., 26, 579 (2004).

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Weidekamm, B. Roos, L. Banken, M. Utoh, B. Osterwalder, Clin. Cancer

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P. W. Erhardt, J. Chromatogr. A, 1138, 101 (2007).

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11. A. Salvador, L. Millerioux, A. Renou, J. Chromatographia, 63, 609 (2006).

12. F. Desmoulin, V. Gilard, R. Martino, M. Malet-Martino, J. Chromatogr. B,

792, 323 (2003).

13. R. Martino, V. Gilard, F. Desmoulin, M. Malet-Martino, J. Pharm. Biomed.

Anal., 38, 871 (2005).

14. M. Malet-Martino, V. Gilard, F. Desmoulin, R. Martino, Clinica Chimica

Acta, 366, 61 (2006).

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Impurities in New Drug Substances. Q3A(R). Federal Register, 65, 45085

(2000).

123

16. ICH Draft Guidelines on Validation of Analytical Procedures: Definitions and Terminology, Federal Register, vol.60, IFPMA, Switzerland, 11260 (1995). 17. M. Arasaki, H. Ishitsuka, I. Kuruma, M. Miwa, C. Murasaki, N. Shimma, I.

Umeda, United States Patent 5472949. Dec. 5, (1995).

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