A stability indicating UPLC method for the rapid...

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CHAPTER-5

A stability indicating UPLC method forthe rapid separation of relatedcomponents of Gemcitabinehydrochloride

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5.1 Introduction

Gemcitabine hydrochloride described chemically as 2′-Deoxy-2′,2′-

difluorocytidine monohydrochloride(β-isomer). Gemcitabine is pyrimidine

analog and it is a chemotherapeutic agent that works by slowing or

stopping the growth of cancer cells and marketed as Gemzar by Eli Lilly

and company. Gemcitabine is probably one of the most valuable

cytotoxic drugs for several solid tumors, e.g. pancreatic, lung and breast

cancer [1]. Gemcitabine is officially mentioned in the USP [2].

Gemcitabine hydrochloride chemical structure and the drug information

provided in Fig. 5.1.F1 and Table 5.1.T1 respectively.

Gemcitabine hydrochloride is a white to off-white solid. Gemcitabine

hydrochloride is soluble in water, slightly soluble in methyl alcohol, and

practically insoluble in alcohol and in polar organic solvents. The pH of a

1% solution in water is between 2.0 and 3.0.

Gemcitabine Hydrochloride’s innovator is Eli Lilly and is known

world-wide by the brand name Gemzar. Gemzar is a nucleoside analogue

that exhibits antitumor activity. The clinical formulation is supplied in a

sterile form for intravenous use only. Vials of Gemzar contain either 200

mg or 1 g of gemcitabine hydrochloride (expressed as free base)

formulated with mannitol (200 mg or 1 g, respectively) and sodium

acetate (12.5 mg or 62.5 mg, respectively) as a sterile lyophilized powder.

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Fig. 5.1.F1: Chemical structure of gemcitabine

Table 5.1.T1: Drug information

Molecular weight : 299.69

Molecular formula : C9H11F2N3O4. HCl

CAS Registry Number : 122111-03-9

Therapeutic category : Chemotherapy

Ultra-performance liquid chromatography (UPLC) is a new category of

separation technique based upon well established principles of liquid

chromatography, which utilizes sub-2 µm particles for stationary phase.

These particles operate at elevated mobile phase linear velocities to affect

dramatic increase in resolution, sensitivity and speed of analysis. UPLC

enables significant reduction in separation and solvent consumption. The

literature indicates that the UPLC system allows about nine fold

decrease in analysis time as compared to the conventional HPLC system

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using 5 μm particle size analytical columns, and about three fold

decrease in analysis time in comparison with 3 μm particle size

analytical columns without compromise on overall separation [3-4].

Because of its speed and sensitivity, this technique is gaining

considerable attention in recent years for pharmaceutical and biomedical

analysis. In the present work, this technology has been applied to the

method development and validation study of related substance and assay

determination of Gemcitabine hydrochloride API.

The present drug stability test guideline Q1A (R2) issued by

international conference on harmonization (ICH) suggests that stress

studies should be carried out on a drug to establish its inherent stability

characteristics, leading to identification degradation products and hence

supporting the suitability of the proposed analytical procedures. It also

requires that analytical test procedures for stability samples should be

stability indicating and they should be fully validated.

In the literature, limited LC methods were reported for the

determination of gemcitabine in pharmaceutical preparations, which

include “The determination of gemcitabine in plasma samples and in

biological fluids by HPLC [5-10] and LCMS/MS [11,12], a stability

indicating method for the determination of gemcitabine HCl in bulk

samples and in pharmaceutical formulations [13], “The degradation of

the antitumor agent gemcitabine hydrochloride in an acidic aqueous

solution at pH 3.2 and identification of degradation products” [14]. The

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methodologies described in the literature and in USP pharmacopoeia

cannot separate all the degradation impurities especially the degradant

coming after main peak which is visible only at 210 nm. The available

literature methods cannot quantify the related impurities of gemcitabine

hydrochloride where as those methods are specifically for the estimation

of gemcitabine in human plasma. Hence, it is felt necessary to develop a

precise, accurate, specific and stability-indicating chromatographic

method for the quantitative determination of gemcitabine hydrochloride,

the three impurities content and degradation products formed during

stress conditions. The aim of the present work is to develop a stability

indicating UPLC method for gemcitabine hydrochloride bulk drug. We

intend to opt for a faster chromatographic technique, UPLC for the said

study. An attempt was made on determining whether UPLC can reduce

analysis times without compromising the resolution and sensitivity. More

intensive stress studies in our laboratory were carried out on

gemcitabine hydrochloride. Described here is a fully validated sensitive

UPLC method for the quantitative determination of gemcitabine

hydrochloride, the three impurities namely Imp-A, Imp-B, Imp-C (Fig.

5.3.F2(a),(b),(c),(d)) content and its possible degradation products

simultaneously. The developed method is stability indicating and the

method is validated as per the ICH guidelines [15].

5.2 Experimental

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5.2.1 Materials:Samples of gemcitabine hydrochloride and its three impurities

standards were received from Process Research Department of Integrated

product development operations of Dr. Reddy’s Laboratories Limited,

Hyderabad, India. LC grade methanol, potassium dihydrogen ortho

phosphate and phosphoric acid were purchased from Merck, Schuchardt

OHG, Germany. High pure water was prepared by using Millipore Milli Q

plus purification system (Bedford, MA, USA).

5.2.2 Equipment:

The UPLC system, used for method development, forced degradation

studies and method validation was Waters Acquity UPLCTM system

equipped with a PDA detector (Waters Corporation, Milford, USA). The

out put signal was monitored and processed using Empower software

(Waters Corporation, Milford, USA) on a Pentium computer (Digital

Equipment Co).

5.2.3 Chromatographic Conditions:

The chromatographic column used was Waters Acquity UPLC HSS T3

(2.1 x 100 mm, 1.8 µm) column. A mobile phase contains a mixture of

buffer and methanol in the ratio of 90: 10. Buffer consists of 20 mM

potassium dihydrogen orthophosphate, pH adjusted to 2.5 using

phosphoric acid. The mobile phase was filtered through a nylon

membrane filter (pore size 0.2m). The flow rate of the mobile phase was

kept at 0.25 mL min-1. The LC column was maintained at ambient and

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the wavelength was monitored at 210 nm. The injection volume was 1L.

Water was used as diluent for the standard and test samples

preparation.

5.2.4 LC-MS/MS conditions:

LC-MS/MS system (Agilent 1200 series) liquid chromatograph

coupled with Applied Biosystems 4000 Q Trap triple quadrupole mass

spectrometer with Analyst 1.4 software, MDS SCIEX, USA) was used for

the unknown compounds formed during forced degradation studies. The

chromatographic column used was YMC PRO C18, 150 mm x 4.6 mm,

3 m particles (YMC, Schermbeck, Germany). A mobile phase contains a

gradient mixture of solvent A and solvent B. The solvent A consists of 10

mM ammonium acetate (Merck, Darmstadt, Germany), pH adjusted to

2.5 using diluted trifluoroacetic acid. Mobile phase B consists methanol.

The mobile phase was filtered through a nylon membrane filter (pore size

0.45m). The gradient program was set as: time (min)/% solvent B : 0/4,

8/4, 15/40, 22/40, 25/4, 30/4. The flow rate of the mobile phase was

kept at 1.0 mL min-1. The LC column was maintained at ambient and the

wavelength was monitored at 210 nm. The injection volume was 10L.

Mobile phase-A was used as diluent during the standard and test

samples preparation. The analysis was performed in positive electro

spray positive ionization mode. Ion Source voltages was 5000 V. Source

temperature was 450°C. GS1 and GS2 are optimized to 30 and 35 psi

respectively. Curtain gas flow was 20 psi.

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5.2.5 Sample preparation:

Based on the solubility of gemciabine hydrochloride API and all

process related impurities, diluent was selected as water. A stock

solution of gemcitabine hydrochloride (2000 g mL-1) was prepared by

dissolving appropriate amount in the diluent. Working solutions of 1000

and 200 g mL-1 were prepared from above stock solution for related

substances determination and assay determination respectively. A stock

solution of impurity (mixture of Imp-A, Imp-B and Imp-C) at 1000 gmL-1

was also prepared in water.

5.2.6 Generation of stress samples:

One batch of gemcitabine hydrochloride was selected for stress

testing. From the ICH Stability guideline: “stress testing is likely to be

carried out on a single batch [16]”. Different kinds of stress degradation

conditions (like heat, humidity, acid, base, oxidative and light) were

performed on one batch of gemcitabine hydrochloride API based on the

guidance available from ICH Stability Guideline (Q1AR2). The details of

the stress conditions applied are as follows:

a) Acid hydrolysis: Sample solution in 0.1N HCl at 70° C for 24 h.

b) Base hydrolysis: Sample solution in 0.1N NaOH at 70°C for 24 h.

c) Water hydrolysis: Sample solution in water at 70°C for 24 h

d) Oxidative stress: Sample solution in 3% hydrogen peroxide at 70°C

for 1 h.

e) Thermal stress: Sample was subjected to dry heat at 60°C for 10 days.

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f) Photolytic degradation: Sample was exposed to UV and visible light for

10 days.

5.3 Method development and optimization of chromatographic

conditions:

In this section elaborately described the method development

strategies which finally resulted in achieving a robust analytical method

for the determination of impurities in gemcitabine hydrochloride. Forced

degradation studies were performed to develop a stability indicating

UPLC for quantification of related impurities, gemcitabine and

degradation products.

Before starting the experiments reviewed the synthetic pathway of

gemcitabine hydrochloride [Fig. 5.3.F1] to understand the molecule

nature and to predict the possible related impurities [Fig. 5.3.F2].

In first stage of gemcitabine hydrochloride synthesis, the hydroxyl

group is mesylated with methane sulfonyl chloride to get 2-deoxy-2,2-

difluro-D-ribofuranosyl-1-methanesulfonate. This on coupling with Bis

trimethylsilyl N-acetyl cytosine, in the presence of the Trimethyl silyl

trifluoromethane sulfonate gives 2,2-Difluoro-2'-deoxycytidine-3',5'-

dibenzoate as the intermediate product. In the next step the benzoyl

groups, Acetyl group in the intermediate will be de-protected with the

ammonia, the formed free base is treated with the hydrochloric acid to

give gemcitabine hydrochloride.

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Fig. 5.3.F1: Brief synthetic pathway of gemcitabine hydrochloride

The possible process impurities were listed below.

Fig. 5.3.F2: Possible related impurities chemical structures

a) Impurity-A:

Chemical name: 4-aminopyrimidin-2(1H)-one

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b) Impurity-B:

Chemical name: 4-Amino-1-(2-deoxy-2,2-difluro-α-D-erythro pentofur-

anosyl)pyrimidin-2(1H)-one

c) Impurity-C:

Chemical name: 2′-deoxy-2′,2′-difluorouridine.

By reviewing the brief synthetic scheme it is understood that the

impurity B is alpha anomer of gemcitabine. Impurity-A and Impurity-C

are degraded impurities.

5.3.1 Selection of wavelength:

All the three related impurities and gemcitabine spectrums were

collected using Water PDA system. The degradation products at RRT 1.1,

1.2 & 2.0 are having UV absorption maxima at 210 nm as gemcitabine

and related impurities are having UV absorption maxima at 210 and 275

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nm. Since all the impurities (process related and degradents) are having

UV max at 210nm, the detection at 210 nm was selected for method

development purpose. The spectrums of Imp-A, Imp-B, gemcitabine and

Imp-C were shown in Fig. 5.3.F3.

Fig. 5.3.F3: Typical UV spectra of Imp-A, gemcitabine, Imp-B

and Imp-C

1.025 Imp-A209.3 276.0

355.8 399.4

AU

0.000

0.005

0.010

1.400 Imp-B

212.4276.0

329.1 363.3 393.2

AU

0.000

0.002

0.004

0.006

1.900 Gemcitabine209.3 276.0

AU

0.00

1.00

2.00

4.133 Imp-C203.9 259.4

326.0 351.5 372.0

AU

0.000

0.001

0.002

nm250.00 300.00 350.00 400.00

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The available hplc method is not capable for separating the

degradation products from gemcitabine. The conventional gradient HPLC

methods run over approximately 30 minutes when tried to separate all

related degradation products using YMC Pack Pro C-18 150 x 4.6, 3 µm

column [Fig. 5.3.F4]. Trails were carried out for reducing the run time

and increasing the method efficiency by using ULPC column.

Fig. 5.3.F4: Acid degraded chromatogram on YMC Pro C-18 column

Imp-A-3.286

4.799

Gemcitabine-5.781

Degpeak1-6.536

Degpeak2-7.319

Degpeak3-11.640

Imp-C-12.168

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30 .0 0

X-axis: Retention time in min and Y-axis: Peak response in AU

5.3.2 Column Selection:

The main difficulty of the chromatographic method was to get the

separation of closely eluting degradation products, mainly at 0.9 RRT

and 1.1 RRT from the gemcitabine peak. The degradation samples were

run using different stationary phases (Acquity UPLC BEH C8 2.1 x 100

mm, 1.7 µm, Acquity UPLC BEH C18 2.1 x 100 mm, 1.7 µm, Acquity

UPLC HSS T3 2.1 x 100 mm, 1.8 µm and Acquity UPLC Phenyl 2.1 x 100

mm, 1.7 µm) [17] and different mobile phases containing buffers like

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phosphate, sulphate and acetate with different pH (2–7) and using

organic modifiers like acetonitrile and methanol in the mobile phase.

During initial experiments, gradient elution was used to ensure

that all degradation products are eluted, and to determine the

total number of major degradation products. After confirming the

number of major degradation products, development of isocratic method

was undertaken.

When UPLC BEH C8 100 x 2.1mm, 1.7µ column was used with the

mobile phase consists buffer (0.02 M of potassium dihydrogen

phosphate, pH adjusted 2.5 with phosphoric acid) and methanol in the

ratio of 90:10 at 0.25 mL min-1 flow rate, it was observed that known

impurities, degradation products at 0.9 RRT and 1.1 RRT were separated

from gemcitabine [Fig. 5.3.F5]. But resolution has to be improved

between the 1.1 RRT degradation peak and gemcitabine. And also partial

resolution was observed between the degradation peak at 1.9 RRT and

impurity-C.

Fig. 5.3.F5: Trial chromatogram on UPLC BEH C8 column

Imp-A-0.762

Imp-B-1.234

1.748

Gemcitabine-2.074

Deg-1-2.477

Deg-2-4.218

Imp-C-4.429

AU

0.00

0.20

0.40

0.60

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00


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When UPLC BEH C18 100 x 2.1mm, 1.7µ column used with same

chromatographic conditions, the resolution was increased between the

1.1 RRT degradation peak and gemcitabine. But resolution was not

improved between degradation peak at 1.9 RRT and impurity-C [Fig.

5.3.F6].

Fig. 5.3.F6: Trial chromatogram on UPLC BEH C18 column

Imp-A-0.964

Imp-B-1.839

Peak3-2.665

Gemcitabine-3.100

Peak5-3.563

Peak6-4.938

Imp-C-5.104

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00


When UPLC Phenyl 100 x 2.1mm, 1.7µ column was used with same

chromatographic conditions, increased the resolution between the

degradation peak at 1.9 RRT and impurity-C [Fig. 5.3.F7]. But the

resolution was decreased between gemcitabine and degradation peak at

1.1 RRT.

Fig. 5.3.F7: Trial chromatogram on UPLC Phenyl column

Imp-A-0.762

Imp-B-1.234

Gemcitabine-2.074

Peak4-2.477

Peak5-4.218

Imp-C-4.429

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

Minutes0.00 1.00 2.00 3.00 4.00 5.00


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In the further trail the Waters Acquity UPLC HSS T3 100 x 2.1 mm,

1.8 microns column was used. The resolution was increased between

the 1.1 RRT degradation peak and gemcitabine [Fig. 5.3.F8] and also very

good resolution was achieved between degradation peak at 1.9 RRT and

impurity-C. In this trail all related impurities and degradent products

were well separated. Hence Aquity UPLC HSS T3 100 x 2.1 mm, 1.8

microns column was finalized.

Fig. 5.3.F8: Trial chromatogram on Waters Acquity UPLC HSS T3column

Imp-A-0.940

Imp-B-1.582

Peak3-2.203

Gemcitabine-2.581

Peak5-2.951

Peak6-4.379

Imp-C-4.831

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00


5.3.3 Effect of Organic solvent:

The mobile phase in composition of buffer and acetonitrile was not

found suitable, as some of the degradation products were not resolved

from each other and also from gemcitabine peak. The mobile phase in

composition of buffer and methanol was suitable for separation of related

impurities, degradation products and gemcitabine.

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5.3.4 Effect of pH:

The method was checked at different pHs (from 2.0 to 7.0) of buffer

for optimizing the buffer pH. The pH 2.5 was found to be more

appropriate, for good peak shape and allowing well separation of

gemcitabine, all process related impurities (Imp-A, Imp-B and Imp-C)

and degradation products.

Finally, the mobile phase consisting of buffer (0.02 M of potassium

dihydrogen phosphate, pH adjusted 2.5 with phosphoric acid) and

acetonitrile in the ratio of 90: 10 (v/v) as mobile phase at a flow rate of

0.25 mL min-1 using Waters Acquity UPLC HSS T3 (2.1 x 100 mm, 1.8

µm) column was found to be appropriate, allowing good separation of

gemcitabine hydrochloride and process-related impurities [Fig. 5.3.F8].

In the optimized conditions degradation products, Imp-A, Imp-B and

Imp-C were well separated each other and the typical retention times of

Imp-A, Imp-B, gemcitabine and Imp-C were about 1.0, 1.4, 1.9 and 4.0

min, respectively. The resolution between Imp-B and gemcitabine

hydrochloride is more than 4.0. The tailing factor and the number of

theoretical plates for gemcitabine hydrochloride peak are 1.1 and 8650

respectively.

5.3.5 Optimized liquid chromatographic conditions:

Based on development trials, solution stability and degradation

studies the below chromatographic conditions were finalized for the

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determination of gemcitabine hydrochloride API related impurities [Table

5.3.T1].

Table 5.3.T1: Chromatographic conditions

Column Waters Aquity HSS 100 mm, 2.1 mm, 1.7 µm

Mobile phase Mixture of buffer and methanol in the ratio of

90: 10

Buffer 20 mM potassium dihydrogen

orthophosphate, pH adjusted to 2.5 using

phosphoric acid

Flow rate 0.25 ml/min

Column temperature Ambient

Wavelength of detection 210 nm

Injection volume 1l

Run time 5 min

Concentration 1 mg mL -1 in diluent

Diluent Water

5.3.6 Degradation studies:

Degradation studies are more important in defining the stability

indicating HPLC method. Under degradation studies the gemcitabine

hydrochloride sample will be forcibly subjected to acidic, basic, peroxide,

thermal and photolytic conditions, and to ensure that the degradants

which are formed due to these stress conditions are well separated from

the known related substances and gemcitabine peak. All the degradation

samples were injected in the optimized method to further prove that the

method is stability indicating.

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The current regulatory guidelines do not indicate detailed degradation

conditions in stress testing. However, the used forced degradation

conditions were found to effect a degradation preferably not less than 5%

and not complete degradation of API [18]. Intentional degradation of the

drug substance was attempted to stress conditions of UV light (254 nm),

thermal degradation (drug substance exposed to 60 C), acid hydrolysis

(using 0.1 N HCl), base hydrolysis (using 0.1 N NaOH), water hydrolysis

and oxidative degradation (using 3% H2O2) to evaluate the ability of the

proposed method to separate gemcitabine hydrochloride from its

degradation products.

Degradation was not observed in gemcitabine hydrochloride bulk

sample during stress conditions like thermal and UV light degradations.

Considerable degradation was observed during the acid, base and water

hydrolysis stress conditions. The gemcitabine was drastically degraded in

oxidation condition. Gemcitabine was degraded into Imp-B and Imp-C

under base, oxidative conditions and Imp-C under acid hydrolysis, water

hydrolysis it was confirmed by co-injection with qualified standards of

Imp-B and Imp-C and by LC-MS/MS analysis. LC-MS/MS analysis was

performed as per experimental conditions and mass of the impurity was

111, 263 and 264 which was corresponding to the mass of Imp-A, Imp-B

and Imp-C. Peak purity test results confirm that the gemcitabine peak is

homogeneous and pure in all the analyzed stress samples. Assay studies

were carried out for stressed samples against qualified reference

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standard having potency 99.8% and the mass balance (% assay+ %

degradation) was calculated. The mass balance of stressed samples was

more than 99%. The results were recorded in Table 5.3.T2 and Table

5.3.T3. Typical chromatograms of degradation study were presented in

Fig. 5.3.F9 to Fig. 5.3.F14. The assay of gemcitabine is unaffected in the

presence of Imp-A, Imp-B and Imp-C which confirms the stability-

indicating power of the method.

5.3.4.2 Acid degradation:

Gemcitabine hydrochloride sample prepared in 0.1N HCl and refluxed

at 70 °C for 24 hours under constant stirring. The sample was injected

in the optimized conditions to further confirm the method suitability. All

the degradation products were well separated from the principal peak

and the gemcitabine hydrochloride impurities, indicating the method

specificity [Fig.5.3.F9 (a) and (b)].

Fig. 5.3.F9 (a): Typical blank chromatogram of acid hydrolysis

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


Fig. 5.3.F9 (b): Typical HPLC chromatogram of acid hydrolysis

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Imp-A-0.976

1.235

Un-knownpeak-1.747

Gemcitabine-1.874

2.247

2.704

3.590

Imp-C-4.054

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


Peak Purity Results

Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity

Gemcitabine 1.87 1.58 2.18 No pass

The purity angle value is less than purity threshold value indicates

that the gemcitabine hydrochloride peak is pure and homogenous. The

method is specific with respect to acid degradation.

5.3.4.3 Base degradation [0.1 N NaOH]:

Gemcitabine hydrochloride sample prepared by using 0.1 N NaOH and

refluxed at 70 °C for 24 hour. The sample was injected in the optimized

conditions to further confirm the method suitability. All the degradation

products were well separated from the principal peak and the

gemcitabine hydrochloride impurities, indicating the method specificity

[Fig. 5.3.F10 (a) and (b)].

Fig. 5.3.F10(a): Typical blank chromatogram of alkali hydrolysis

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AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


Fig. 5.3.F10(b): Typical HPLC chromatogram of alkali hydrolysis

Imp-A-1.042

1.118

1.190

Imp-B-1.394

Un-knownpeak-1.743

Gemcitabine-1.869

2.694

2.786

Imp-C-4.037

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


Peak Purity Results

Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity


The purity angle value is less than purity threshold indicates that the

gemcitabine hydrochloride peak is pure and homogenous. The method is

specific with respect to base degradation.

5.3.4.4 Peroxide degradation [3 % H2O2]:

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The peroxide degradation samples were injected in the optimized

conditions. All the degradants were well separated from the principal

peak and the gemcitabine hydrochloride impurities, indicating the

method specificity. The gemcitabine hydrochloride peak was pure and

homogenous [Fig. 5.3.F11(a) and (b)].

Fig. 5.3.F11(a): Typical blank chromatogram of oxidativedegradation

1.005

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


Fig. 5.3.F11 (b): Typical HPLC chromatogram of oxidativedegradation

Imp-A-1.1331.167

1.249

Imp-B-1.350

1.540

Un-knownpeak-1.701

Gemcitabine-1.877

2.048

2.187

2.448

2.573

3.255

3.608

Imp-C-4.085

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


Peak Purity Results

214

Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity




specific with respect to peroxide degradation.

5.3.4.4 Water hydrolysis:

The water hydrolysis samples were injected in the optimized

conditions. All the degradation products were well separated from the

principal peak and the gemcitabine hydrochloride impurities, indicating

the method specificity. The gemcitabine hydrochloride peak was pure

and homogenous [Fig. 5.3.F12 (a) and (b)].

Fig. 5.3.F12 (a): Typical blank chromatogram of water hydrolysis

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes0.00 0.50 1. 00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


215

Fig. 5.3.F12 (b): Typical chromatogram of water hydrolysis

Imp-A-1.002

1.229

Un-knownpeak-1.755

Gemcitabine-1.883

2.255

3.602

Imp-C-4.074

AU

0.00

0.02

0.04

Minutes0.00 0.50 1. 00 1.50 2.00 2.50 3.00 3.50 4.00 4. 50 5.00

X-axis: Retention time in min and Y-axis: Peak response in AUPeak Purity Results

Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity




specific with respect to water hydrolysis.

5.3.4.5 Thermal degradation [at 60 °C]:

The thermal degradation samples were injected in the optimized

conditions. No significant degradation is observed. The gemcitabine

hydrochloride peak was pure and homogenous [Fig. 5.3.F13].

Fig. 5.3.F13: Typical chromatogram of thermal degradation

Imp-B-1.397

Un-knownpeak-1.751

Gemcitabine-1.879

Imp-C-4.083

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

216


Peak Purity Results

Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity


5.3.4.6 Photolytic degradation [at 254 nm]:

The photolytic degradation samples were injected in the optimized

conditions. No degradation was observed in photolytic conditions. The

gemcitabine hydrochloride peak was pure and homogenous [Fig.

5.3.F14].

Fig.5.3.F14. Typical HPLC chromatogram of photolytic degradation

Imp-B-1.395

Un-knownpeak-1.750

Gemcitabine-1.877

Imp-C-4.084

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


Peak Purity Results

Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity


217

5.3.4.7 Degradation results:

Consolidated results of degradation studies were provided in below

table-5.3.T2 & T3.

Table 5.3.T2: Stress study final conditions and mass balance

Stress conditions Time % Degradation% Assay of

Gemcitabine

Mass

balance

Thermal (60° C) 10 days Nil 99.8 99.7

Photolytic Deg. 10 days Nil 99.6 99.7

Acid hydrolysis 24 h 2.54 97.1 99.6

Base hydrolysis 24 h 5.14 94.4 99.5

Water hydrolysis 24 h 2.14 97.2 99.3

Oxidation(3%H202) 1 h 11.65 87.7 99.4

Table 5.3.T3: Specificity results

Stress condition Purity angle Purity threshold

Thermal (60°C) 1.91 2.14

Photolytic degradation 1.90 2.22

Acid hydrolysis 1.58 2.18

Base hydrolysis 1.53 1.97

Water hydrolysis 1.65 2.21

Oxidation (3% H202) 1.04 1.63

218

5.4 Method validation

The developed and optimized HPLC method was taken up for

validation. The analytical method validation was carried out in

accordance with ICH guidelines.

5.4.1 System Suitability Test:

A mixture of gemcitabine hydrochloride standard, Imp-A, Imp-B, and

Imp-C were injected into HPLC system and good resolution was obtained

between impurities and gemcitabine hydrochloride [Fig. 5.4.F1].

Fig. 5.4.F1: Typical system suitability chromatogram

Imp-A-1.020

1.208

Imp-B-1.392

Un-knownpeak-1.748

Gemcitabine-1.891

Imp-C-4.103

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


5.4.2 Precision:

The precision of an analytical procedure expresses the closeness of

agreement between a series of measurements obtained from multiple

sampling of the same homogenous sample under the prescribed

conditions. The precision of the method was checked by injecting six

individual preparations of gemcitabine hydrochloride (1.0 mg mL-1)

spiked with 0.10% of Imp-A, Imp-B and Imp-C with respect to

219

gemcitabine hydrochloride analyte concentration. The % RSD of area

Imp-A, Imp-B and Imp-C for six consecutive determinations was

tabulated in Table 5.4.T1 and the typical representative chromatogram

shown in Fig. 5.4.F2.

Assay method precision was evaluated by carrying out six

independent assays of test sample of gemcitabine hydrochloride against

qualified reference standard and calculated the % RSD. The RSD of assay

of gemcitabine hydrochloride during assay method precision study was

well within 0.5%. The results were presented in Table 5.4.T2.

Table 5.4.T1: Precision results

Preparation Imp-A Imp-B Imp-C

1 25179 8544 7881

2 24829 8796 7993

3 25066 8732 7804

4 24945 8860 7837

5 24987 8749 7648

6 24470 8715 7683

Average 24913 8733 7808

%RSD 0.9 1.2 1.6

220

Table 5.4.T2: Assay method precision results

Preparation Gemcitabine hydrochlorideAssay

1 99.78

2 99.65

3 99.47

4 99.83

5 99.59

6 99.32

Average 99.61

%RSD 0.19

Fig. 5.4.F2: Typical chromatogram of precision

Imp-A-1.020

1.208

Imp-B-1.392

Un-knownpeak-1.748

Gemcitabine-1.891

Imp-C-4.103

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


5.4.3 Limit of detection (LOD) and Limit of quantification (LOQ):LOQ and LOD established for Imp-A, Imp-B and Imp-C based on

s/n ratio method.

5.4.3.1 Limit of detection (LOD):The detection limit of an individual analytical procedure is the

lowest amount of analyte in a sample, which can be detected but not

221

necessarily quantitated as an exact value. The LOD values were

represented in Table 5.4.T3.

Table 5.4.T3: LOD values of the impurities

S.No Impurity NameLOD Conc. in %w.r.t gemcitabine

hydrochloride

1 Impurity-A 0.003

2 Impurity-B 0.010

3 Impurity-C 0.016

5.4.3.2 Limit of quantification (LOQ):The Limit of Quantification (LOQ) of an analytical procedure is the

lowest amount of analyte in a sample, which can be quantitatively

determined with suitable precision and accuracy. The limit of

quantification results were represented in Table 5.4.T4.

Table 5.4.T4: LOQ values of the impurities

S.No Impurity NameLOQ Conc. in %w.r.t gemcitabine

hydrochloride1 Impurity-A 0.012 Impurity-B 0.033 Impurity-C 0.05

5.4.4 Precision at limit of quantification level:Prepared six individual solutions containing Imp-A, Imp-B and Imp-

C at the limit of quantification level. Injected each solution once and

calculated the % RSD for the areas of each impurity. The precision at

limit of quantification for Imp-A, Imp-B and Imp-C was less than 10.0%,

222

confirming good precision of the method at LOQ [Table 5.4.T5] and the

typical representative chromatogram shown in Fig. 5.4.F3.

Table 5.4.T5: Precision at LOQ level

S.No ImpurityName Prep-1 Prep-2 Prep-3 Prep-4 Prep-5 Prep-6 %RSD

1 Imp-A 3749 3694 3675 3694 3649 3671 0.92

2 Imp-B 2469 2450 2466 2454 2418 2457 0.75

3 Imp-C 4084 4003 4012 3915 4076 3926 1.79

Acceptance criteria: The % RSD should not be more than 15

Fig. 5.4.F3: Typical chromatogram of LOQ precision

Imp-A-1.020

1.208

Imp-B-1.392

Un-knownpeak-1.748

Gemcitabine-1.891

Imp-C-4.103

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


5.4.5 Accuracy at limit of quantification level:Prepared three different solutions containing Imp-A, Imp-B and

Imp-C at the limit of quantification level and injected each solution once.

Sample preparation: Weighed accurately 50.0 mg of gemcitabine

hydrochloride sample into a 50mL volumetric flask, dissolved and diluted

223

to the volume with diluent. Prepared the test solution for three times

from the same homogeneous sample.

Prepared three different sample solutions containing Imp-A, Imp-B

and Imp-C at the limit of quantification level and injected each solution

once, calculated % Recovery for the impurities [Table 5.4.T6].

Table 5.4.T6: Accuracy at LOQ levelS.No Impurity Name % Recovery

1 Impurity -A 101.1

2 Impurity -B 112.3

3 Impurity -C 99.3

Acceptance criteria: The percentage recovery should not be less than

70.0 and should not be more than 130.0

5.4.6 Linearity:The linearity of an analytical procedure is its ability to obtain test

results, which are directly proportional to the concentration of analyte in

the test sample. Linearity experiments were carried out by preparing the

gemcitabine hydrochloride sample solutions containing Imp-A, Imp-B

and Imp-C from LOQ to 200% (i.e. LOQ, 25%, 50%, 75%, 100%, 125%,

150%, 175% and 200%) with respect to their specification limit (0.10%).

Calibration curve was drawn by plotting average area of the impurity

(Imp-A, Imp-B and Imp-C) on the Y-axis and concentration on the X-axis

[Table 5.4.T7 to 5.4.T9].

224

Table 5.4.T7: Linearity results of Imp-A

Concentration (µg/ml) Imp-A Peak area

0.01 3572

0.25 7364

0.50 12526

0.75 18814

1.00 25113

1.25 28818

1.50 34738

1.75 40443

2.00 46683

Correlation Coefficient(r) 0.999

Slope 21801Intercept 2404

Fig 5.4.F4: Linearity plot for Imp-A

X-axis: Concentration in µg mL-1 and Y-axis: Peak area mAU

225

Table 5.4.T8: Linearity results of for Imp-B

Concentration (µg/ml) Imp-B Peak area

0.01 1407

0.25 2607

0.50 4370

0.75 6749

1.00 8612

1.25 10595

1.50 12388

1.75 14738

2.00 16615


Slope 7829

Intercept 837

Fig 5.4.F5: Linearity plot for Imp-B


Table 5.4.T9: Linearity results of for Imp-C

226

Concentration (µg/ml) Imp-C Peak area

0.01 1597

0.25 2815

0.50 4722

0.75 6207

1.00 7728

1.25 9892

1.50 11436

1.75 13070

2.00 14820


Slope 6736

Intercept 1287

Fig 5.4.F6: Linearity plot for Imp-C


Linearity test solutions for assay method were prepared from stock

solution at five concentration levels from 50% to 150% of assay analyte

227

concentration (100, 150, 200, 250 and 300 g mL-1). The peak area

versus concentration data was subjected to least-squares linear

regression analysis. The calibration curve was drawn by plotting

gemcitabine hydrochloride average area for triplicate injections and the

concentration expressed in percentage. Linear calibration plot for assay

method was obtained over the calibration ranges tested, i.e. 100 g mL-1

to 300 g mL-1 and the correlation coefficient obtained was greater than

0.999 [Table 5.4.T10].

Table 5.4.T10: Linearity results of gemcitabine

Concentration (µg/ml) gemcitabine area

100 744580

150 1123300

200 1514105

250 1893426

300 2252571


Slope 7572

Intercept -8846

228

Fig 5.4.F7: Linearity plot for gemcitabine


5.4.6.1 LOQ level linearity:Impurities Imp-A, Imp-B and Imp-C were spiked at LOQ level to

gemcitabine hydrochloride. Representative chromatogram was shown in

Fig. 5.4.F8.

Fig. 5.4.F8: Representative chromatogram of LOQ level linearity.

Imp-A-1.015

Imp-B-1.391

Un-knownpeak-1.746

Gemcitabine-1.889

Imp-C-4.094

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


229

5.4.6.2 Linearity at 25 % level:Impurities Imp-A, Imp-B and Imp-C were spiked at 25% level to


Fig. 5.4.F9.

Fig. 5.4.F9: Representative chromatogram of 25% level linearity.

Imp-A-1.015

Imp-B-1.391

Un-knownpeak-1.746

Gemcitabine-1.889

Imp-C-4.094

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

X-axis: Retention time in min and Y-axis: Peak response in AU5.4.6.3 Linearity at 50 % level:

Impurities Imp-A, Imp-B and Imp-C were spiked at 50 % level to


Fig. 5.4.F10.

Fig. 5.4.F10: Representative chromatogram of 50 % level linearity

Imp-A-1.012

Imp-B-1.384

Un-knownpeak-1.738

Gemcitabine-1.883

Imp-C-4.093

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


5.4.6.4 Linearity at 75 % level:

230



Fig. 5.4.F11.


Imp-A-1.018

Imp-B-1.390

un-knownpeak-1.745

Gemcitabine-1.889

Imp-C-4.101

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


5.4.6.5 Linearity at 100 % level:Impurities Imp-A, Imp-B and Imp-C were spiked at 100 % level to


Fig. 5.4.F12.


Imp-A-1.018

Imp-B-1.390

un-knownpeak-1.745

Gemcitabine-1.890

Imp-C-4.103

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


5.4.6.6 Linearity at 125 % level:

231



Fig. 5.5.F13.

Fig.5.4.F13: Representative chromatogram of 125 level % linearity

Imp-A-1.015

Imp-B-1.387

un-knownpeak-1.742

Gemcitabine-1.886

Imp-C-4.099

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

X-axis: Retention time in min and Y-axis: Peak response in AU5.4.6.7 Linearity at 150 % level:



Fig. 5.4.F14.


Imp-A-1.020

Imp-B-1.392

un-knownpeak-1.747

Gemcitabine-1.891

Imp-C-4.103

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00



232


Fig. 5.4.F15.


Imp-A-1.018

Imp-B-1.390

un-knownpeak-1.744

Gemcitabine-1.889

Imp-C-4.101

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00




Fig. 5.4.F16.


Imp-A-1.019

Imp-B-1.391

un-knownpeak-1.745

Gemcitabine-1.889

Imp-C-4.096

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

X-axis: Retention time in min and Y-axis: Peak response in AU5.4.7 Accuracy:

The accuracy of an analytical procedure expresses the closeness of

agreement between the value, which is accepted either as a conventional

true value or an accepted reference value and the value found.

233

5.4.7.1 Accuracy of the method:

The accuracy study of impurities was carried out in triplicate at

0.05, 0.10 and 0.15% of the gemcitabine hydrochloride analyte

concentration (1000 g/mL). The percentage recoveries for impurities

were calculated.

The accuracy of the assay method was evaluated in triplicate at

three concentration levels i.e. 100, 200 and 300 g mL-1 in bulk drug

sample. The percentage recoveries for gemcitabine hydrochloride were

calculated.

Test solution prepared in triplicate (n=3) with impurities (Imp-A, Imp-

B and Imp-C) at 0.05 %, 0.10 % and 0.15 % level w.r.t. analyte

concentration (i.e. 1000 g/mL). Each solution was injected once into

HPLC system. Mean %recovery of impurities calculated in the test

solution using the area of impurities standard at 0.10% level with respect

to analyte. The recovery results were tabulated in Table 5.4.T11 and the

reference chromatograms were shown in Fig. 5.4.F17 to Fig. 5.4.F20.

Table 5.4.T11: Accuracy results

Recoverylevels % Imp-A % Imp-B % Imp-C

Gemcitabine

hydrochloride

50% 100.1 94.2 101.6 100.7

100% 98.4 95.0 97.8 100.4

150% 92.2 93.2 100.4 100.1

234

Fig. 5.4.F17: 100% accuracy authentic typical chromatogram

Imp-A-1.017

Imp-B-1.387

Gemcitabine-1.884

Imp-C-4.065

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


Fig. 5.4.F18: 50 % accuracy typical chromatogram

Imp-A-1.017

Imp-B-1.387

Un-knownpeak-1.740

Gemcitabine-1.882

Imp-C-4.074

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00



Imp-A-1.017

Imp-B-1.388

Un-knownpeak-1.742

Gemcitabine-1.884

Imp-C-4.085

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

235



Imp-A-1.020

Imp-B-1.393

Un-knownpeak-1.749

Gemcitabine-1.894

Imp-C-4.118

AU

0.00

0.02

0.04

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


Acceptance criteria: The % recovery should not be less than 80 and

should not be more than 120.

5.4.8 Solution stability:

Based on the solubility nature of gemcitabine hydrochloride and the

related impurities the diluent was finalized as water. The solution

stability of gemcitabine hydrochloride in the assay method was carried

out by leaving both the test solutions of sample and reference standard

in tightly capped volumetric flasks at room temperature for two days. The

same sample solutions were assayed for 6 h interval up to the study

period. The mobile phase stability was also carried out by assaying the

freshly prepared sample solutions against freshly prepared reference

standard solutions for six hours interval up to two days. Mobile phase

prepared was kept constant during the study period. The % RSD of assay

236

of gemcitabine hydrochloride was calculated for the study period during

mobile phase and solution stability experiments.

The solution stability of gemcitabine hydrochloride and its impurities

in the related substances method was carried out by leaving spiked

sample solution in tightly capped volumetric flask at room temperature

for 48hours. Content of Imp-A, Imp-B and Imp-C were determined for

every 6h interval up to the study performed. Mobile phase was also

carried out for 48 hours by injecting the freshly prepared sample

solutions for every 6h interval. Content of Imp-A, Imp-B and Imp-C were

checked in test solutions. Mobile phase prepared was kept constant

during the study period.

The RSD of assay of gemcitabine hydrochloride during solution

stability and mobile phase stability experiments was within 1.0%. No

significant change was observed in the content of Imp-A, Imp-B and Imp-

C during solution stability and mobile phase stability experiments when

performed using related substances method. The solution stability and

mobile phase stability experiments data confirms that sample solutions

and mobile phase used during assay and related substance

determination were stable up to 48 hours.

5.4.9 Method Robustness:

In order to demonstrate the robustness of the method, system

suitability parameters were verified by making deliberate changes in the

chromatographic conditions, viz, change in flow rate by +0.02 ml/min,

237

change in pH of the buffer +0.2 unit and changing column temperature

to 22°C and 30°C from 25°C(ambient) during the development stage

itself. The method was demonstrated to be robust over an acceptable

working range of its HPLC operational parameters. The tailing factor,

USP resolution between peaks Imp-B and gemcitabine were evaluated.

Results were within the limits illustrating the robustness of the method.

Table 5.4.T12.

Table 5.4.T12: Results of robustness study

Parameter

Temperature

(+ 5°C of 25°C)

Flow rate

(+ 0.02 mL

min-1of 0.25)

pH of buffer

(+0.2 of 2.5)

Variation 20°C 30°C 0.23 0.27 2.3 2.7

The resolution

between Impurity-B

and gemcitabine

4.1 4.3 4.2 4.4 4.1 4.3

USP Tailing factor

for gemcitabine

1.1 1.0 1.2 1.1 1.2 1.2

5.4.10 Batch analysis data:

Using the above validated method, some gemcitabine hydrochloride

samples were analyzed and the data is furnished in Table 5.4.T13.

238

Table 5.4.T13. Gemcitabine hydrochloride samples analysis data

B.NoRelated substances by HPLC

Imp-A Imp-B Imp-C Single maximumimpurity

Totalimpurity

001 ND ND 0.06 0.01 0.07

002 ND ND 0.06 0.01 0.07

003 ND ND 0.07 0.01 0.08

* ND – Not detected

5.5 Summery and ConclusionThe new RP-UPLC method developed for the quantitative

determination of gemcitabine hydrochloride assay, related compounds

and its possible degradation products is precise, accurate and specific to

analyze gemcitabine hydrochloride in bulk active substance. The stability

indicating power of the method was demonstrated by analyzing the stress

studies on samples in the developed method. The method was fully

validated showing satisfactory data for all the method validation

parameters tested. The developed method was found “specific” to the

drug substances, as the peaks of the degradation products did not

interfere with the gemcitabine hydrochloride peak. The developed method

is stability indicating and can be conveniently used for the routine

analysis of production samples and also to check the stability of bulk

samples to establish the retest period for gemcitabine hydrochloride.

Moreover, the lower solvent consumption along with the short analytical

run time of 5.0 min leads to cost effective chromatographic method.

239

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