A stability indicating UPLC method for the rapid

In first stage of gemcitabine hydrochloride synthesis, ... group is mesylated with methane sulfonyl chloride to get 2-deoxy-2,2- ... In the next step ...

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191

CHAPTER-5 A stability indicating UPLC method for the

rapid

components hydrochloride

separation of

of

related

Gemcitabine

192

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.

193

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

194 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

195 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

196 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 UPLC TM 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 0.2m). The flow rate of the mobile phase was kept at 0.25 mL min-1. The LC column was maintained at ambient and

197 the wavelength was monitored at 210 nm. The injection volume was 1L. 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 0.45m). 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 10L. 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.

198 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.

199 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-2,2difluro-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.

200 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

201 b) Impurity-B:

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

202 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-A

209.3

276.0

0.010

U A

0.005

355.8

0.000 0.006

1.400 Imp-B

399.4

276.0

212.4 0.004 U A

0.002

329.1

0.000 1.900 Gemcitabine

363.3

393.2

276.0

209.3 2.00 U A

1.00

0.00 4.133 Imp-C

203.9

259.4

0.002 U A

0.001

326.0

0.000 250.00

300.00 nm

351.5 372.0 350.00

400.00

203 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 0.10

1 8 7 .5 e n i b a it c m e G

0.08 0.06 U A

0.04 0.02 0.00 -0.02 0.00

6 8 2 .3 -A p Im

9 9 7 . 4

6 3 .5 6 1 ka e p g e D

08 6 4 6 .1 .1 2 11 - 3C k -p a e pm I g e D

9 1 .3 7 2 k a e p g e D

5.00

10.00

15.00

20.00

25.00

30 .0 0

Minutes

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

204 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 0.60

0.40

U A

0.20

2 6 .7 0 A -p m I

8 4 .7 1

0.00

0.00

4 7 .0 2 e n ib a ti c m e G

4 3 .2 1 -B p m I

1.00

2.00

8 1 2 .4 -2 g e D

7 7 4 .2 -1 g e D

3.00

X-axis: Retention time in min

4.00 Minutes

9 2 4 . 4 -C p m I

5.00

6.00

7.00

and Y-axis: Peak response in AU

8.00

205 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 0.10 0.08 0.06 U A

0.04 0.02

4 6 9 . 0 A -p m I

9 3 .8 1 B p m I

0.00 -0.02 0.00

1.00

2.00

5 6 6 .2 3 k a e P

0 0 1 .3 e n i b a ti c m e G

8 3 .9 4 6 k a e P

3 6 .5 3 5 k a e P

3.00

4.00

4 0 1 .5 C p m I

5.00

6.00

7.00

8.00

Minutes

X-axis: Retention time in min

and Y-axis: Peak response in AU

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 0.10 0.08 0.06 U A

2 6 .7 0 A p m I

4 3 .2 1 B p m I

0.04

4 7 .0 2 e n i b a ti c m e G

0.02

8 1 2 . 4 5 k a e P

7 7 4 . 2 4 k a e P

9 2 .4 4 C p m I

0.00 -0.02 0.00

1.00

2.00

3.00

4.00

5.00

Minutes

X-axis: Retention time in min

and Y-axis: Peak response in AU

206 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 T3 column 0.10 0.08 0.06 U A

0.04 0.02

0 4 .9 0 A -p m I

2 8 5 .1 B p m I

3 0 2 . 2 3 ka e P

0.00 -0.02 0.00

1.00

2.00

X-axis: Retention time in min

1 8 5 .2 e n ib a it c m e G

9 7 .3 4 6 k a e P

1 5 9 . 2 5 k a e P

3.00 Minutes

4.00

1 3 8 .4 -C p m I

5.00

6.00

and Y-axis: Peak response in AU

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.

207 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

208 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

1l

Run time

5 min

Concentration

1 mg mL

Diluent

Water

-1

in diluent

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.

209 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% H 2O2) 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

210 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 stabilityindicating 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

0.04

U A

0.02

0.00 0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

Minutes

X-axis: Retention time in min

and Y-axis: Peak response in AU

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

5.00

211

7 4 7 . 1 k a e p n w o n kn U

0.04

U A

6 7 9 .0 -A p m I

0.02

0.00

0.00

0.50

5 3 2 . 1

1.00

1.50

4 7 .8 1 e n i b a ti c m e G

7 4 .2 2

2.00

0 9 .5 3

4 0 7 . 2

2.50 Minutes

3.00

3.50

4 5 0 .4 -C p m I

4.00

4.50

5.00

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

Retention Time

Purity Angle

Purity Threshold

Purity Flag

Peak Purity

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

212 0.050 0.040 0.030 U A

0.020 0.010 0.000 0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4. 00

4.50

5. 00

Minutes

X-axis: Retention time in min and Y-axis: Peak response in AU Fig. 5.3.F10(b): Typical HPLC chromatogram of alkali hydrolysis 3 4 .7 1 k a e p n w o n kn U

0.04

U A

2 4 0 . 1 A 80 - 1 p 19 . .1 m I 11

0.02

0.00

0.00

0.50

4 9 3 . 1 B p m I

1.00

1.50

9 6 8 .1 e n i b a ti c m e G

7 3 0 .4 -C p m I 4 6 9 8 6 . 7 2 .2

2.00

2.50

3.00

3.50

4.00

4.50

5. 00

Minutes

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

Retention Time

Purity Angle

Purity Threshold

Purity Flag

Peak Purity

Gemcitabine

1.87

1.53

1.97

No

pass

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]:

213 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 oxidative degradation 0.050

5 0 0 . 1

0.040 0.030 U A

0.020 0.010 0.000 0.00

0.50

1.00

1.50

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

X-axis: Retention time in min and Y-axis: Peak response in AU Fig. 5.3.F11 (b): Typical HPLC chromatogram of oxidative degradation 0.04

U A

79 64 1 . .2 11 3 3 .1 1 A p m I

0.02

0.00 0.00

0.50

1.00

0 4 .5 1 0 5 3 . 1 -B p m I 1. 50

1 0 7 . 1 k a e p n w o n kn U

7 7 8 . 1 e n 8 i b 4 .0 ta i c 2 m e G

2.00

7 8 1 . 2 3 7 5 . 2 8 4 4 . 2

2.50 Minutes

5 5 .2 3 3.00

8 0 .6 3 3.50

5 8 0 . 4 -C p m I 4.00

4.50

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

5.00

214

Name

Retention Time

Purity Angle

Purity Threshold

Purity Flag

Peak Purity

Gemcitabine

1.87

1.04

1.63

No

pass

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 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 0.050 0.040 0.030 U A

0.020 0.010 0.000 0.00

0.50

1. 00

1.50

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

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

5.00

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

5 5 7 .1 ka e p n w o n kn U

0.04

U A

2 0 0 . 1 A p m I

0.02

0.00 0.00

0.50

9 2 .2 1

1. 00

3 8 .8 1 e n i b a itc m e G

1.50

2 0 6 . 3

5 5 .2 2

2.00

2.50 Minutes

3.00

3.50

4 7 0 .4 C p m I

4.00

4. 50

5.00

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

Retention Time

Purity Angle

Purity Threshold

Purity Flag

Peak Purity

Gemcitabine

1.88

1.65

2.21

No

pass

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 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

1 5 .7 1 k a e p n w o n kn U

0.04

U A

7 9 .3 1 -B p m I

0.02

0.00 0.00

0.50

1. 00

1.50

9 7 8 .1 e n i b a it c m e G

3 8 .0 4 -C p Im 2.00

2.50 Minutes

3.00

3.50

4.00

4. 50

5.00

216 X-axis: Retention time in min and Y-axis: Peak response in AU Peak Purity Results Name

Retention Time

Purity Angle

Purity Threshold

Purity Flag

Peak Purity

Gemcitabine

1.88

1.91

2.14

No

pass

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

0 5 7 . 1 k a e p n w o n kn U

0.04

U A

5 9 3 .1 -B p m I

0.02

0.00 0.00

0.50

1.00

1.50

7 7 8 . 1 e n i b a ti c m e G

4 8 0 .4 -C p m I 2.00

2.50

3. 00

3.50

4. 00

4.50

Minutes

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

Retention Time

Purity Angle

Purity Threshold

Purity Flag

Peak Purity

Gemcitabine

1.88

1.90

2.22

No

pass

5.00

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 % Assay of

Mass

Gemcitabine

balance

Nil

99.8

99.7

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

1h

11.65

87.7

99.4

Stress conditions

Time

% Degradation

Thermal (60° C)

10 days

Photolytic Deg.

Oxidation(3%H202)

Table 5.3.T3: Stress condition Thermal (60°C)

Specificity results Purity angle Purity threshold 1.91

2.14

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

Photolytic degradation

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

0.04

U A

0 2 .0 1 -A p m I

0.02

0.00

0.00

0.50

8 0 2 . 1

1.00

8 4 .7 1 k a e p n w o n kn U

2 9 3 . 1 -B p m I

1.50

1 9 .8 1 e n ib a itc m e G

3 0 1 . 4 -C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

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

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

0.04

U A

0 2 .0 1 A p m I

0.02

0.00

0.00

0.50

1.00

8 0 2 . 1

8 4 .7 1 k a e p n w o n -k n U

2 9 .3 1 B p m I

1.50

1 9 .8 1 e n i b a ti c m e G

2.00

3 0 .1 4 C p m I

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

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

LOD 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 LOQ Conc. in % S.No Impurity Name w.r.t gemcitabine hydrochloride Impurity-A 1 0.01 Impurity-B 2 0.03 3 Impurity-C 0.05 5.4.4 Precision at limit of quantification level: Prepared six individual solutions containing Imp-A, Imp-B and ImpC 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 Impurity Prep-1 Prep-2 Prep-3 Prep-4 Prep-5 Prep-6 %RSD Name Imp-A 3749 3694 3675 3694 3649 3671 0.92

S.No 1

Imp-B

2 3

Imp-C

2469

2450

2466

2454

2418

2457

0.75

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

0.04

0 2 .0 1 A -p m I

U 0.02 A

0.00

0.00

0.50

1.00

8 0 .2 1

2 9 3 .1 B p m I

1.50

8 4 7 . 1 k a e p n w o n kn U

1 9 .8 1 e n i b a ti c m e G

2.00

3 0 1 .4 C p m I

2.50

3.00

3.50

4.00

4.50

5.00

Minutes

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

S.No

Table 5.4.T6: Accuracy at LOQ level Impurity Name % Recovery

1 2 3

Impurity -A Impurity -B Impurity -C

101.1 112.3 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

21801

Intercept

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

Correlation Coefficient(r)

0.999

Slope

7829

Intercept

837

Fig 5.4.F5: Linearity plot for Imp-B

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

Correlation Coefficient(r)

0.999

Slope

6736

Intercept

1287

Fig 5.4.F6: Linearity plot for Imp-C

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

Correlation Coefficient(r)

0.999

Slope

7572

Intercept

-8846

228 Fig 5.4.F7: Linearity plot for gemcitabine

X-axis: Concentration in µg mL-1 and Y-axis: Peak area mAU 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. 6 4 .7 1 k a e p n w o n kn U

0.04

U A

5 1 .0 1 A -p m I

0.02

0.00

0.00

0.50

1.00

1 9 3 . 1 B p m I

1.50

9 8 8 . 1 e n i b a ti c m e G

4 9 .0 4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

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

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 gemcitabine hydrochloride. Representative chromatogram was shown in Fig. 5.4.F9. Fig. 5.4.F9: Representative chromatogram of 25% level linearity. 6 4 .7 1 k a e p n w o n kn U

0.04

U A

5 1 .0 1 A p m I

0.02

0.00

0.00

0.50

1 9 3 . 1 B p m I

1.00

9 8 8 . 1 e n i b a it c m e G

1.50

4 9 .0 4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

X-axis: Retention time in min and Y-axis: Peak response in AU 5.4.6.3 Linearity at 50 % level: Impurities Imp-A, Imp-B and Imp-C were spiked at 50 % level to gemcitabine hydrochloride. Representative chromatogram was shown in Fig. 5.4.F10. Fig. 5.4.F10:

Representative chromatogram of 50 % level linearity 8 3 .7 1 k a e p n w o n kn U

0.04

U A

2 1 0 .1 -A p m I

0.02

0.00

0.00

0.50

1.00

4 8 3 . 1 -B p m I

1.50

3 8 8 . 1 e n ib a ti c m e G

3 9 .0 4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

X-axis: Retention time in min and Y-axis: Peak response in AU 5.4.6.4 Linearity at 75 % level:

5.00

230 Impurities Imp-A, Imp-B and Imp-C were spiked at 75 % level to gemcitabine hydrochloride. Representative chromatogram was shown in Fig. 5.4.F11. Fig. 5.4.F11: Representative chromatogram of 75 % level linearity 5 4 7 . 1 k a e p n w o n -k n u

0.04

U A

8 1 .0 1 A -p m I

0.02

0.00 0.00

0.50

0 9 3 . 1 -B p m I

1.00

9 8 .8 1 e n i b a ti c m e G

1.50

1 0 1 .4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

X-axis: Retention time in min and Y-axis: Peak response in AU 5.4.6.5 Linearity at 100 % level: Impurities Imp-A, Imp-B and Imp-C were spiked at 100 % level to gemcitabine hydrochloride. Representative chromatogram was shown in Fig. 5.4.F12. Fig. 5.4.F12:

Representative chromatogram of 100 % level linearity 5 4 7 . 1 k a e p n w o n kn u

0.04

U A

8 1 .0 1 A p m I

0.02

0.00 0.00

0.50

1.00

0 9 .3 1 B p m I

1.50

0 9 8 . 1 e n i b a ti c m e G

3 0 1 .4 -C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

X-axis: Retention time in min and Y-axis: Peak response in AU 5.4.6.6 Linearity at 125 % level:

5.00

231 Impurities Imp-A, Imp-B and Imp-C were spiked at 125 % level to gemcitabine hydrochloride. Representative chromatogram was shown in Fig. 5.5.F13. Fig.5.4.F13: Representative chromatogram of 125 level % linearity 0.04

5 1 .0 1 A p m I

U 0.02 A

0.00

0.00

0.50

1.00

2 4 .7 1 k a e p n w o n -k n u

7 8 .3 1 B p m I

1.50

6 8 .8 1 e n i b a ti c m e G

9 9 .0 4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

X-axis: Retention time in min and Y-axis: Peak response in AU 5.4.6.7 Linearity at 150 % level: Impurities Imp-A, Imp-B and Imp-C were spiked at 125 % level to gemcitabine hydrochloride. Representative chromatogram was shown in Fig. 5.4.F14. Fig. 5.4.F14:

0.04

U A

Representative chromatogram of 150 % level linearity

0 2 0 . 1 -A p m I

0.02

7 4 7 . 1 k a e p n w o n kn u

2 9 .3 1 -B p m I

0.00 0.00

0.50

1.00

1.50

1 9 8 . 1 e n i b a ti c m e G

3 0 .1 4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

X-axis: Retention time in min and Y-axis: Peak response in AU 5.4.6.8 Linearity at 175 % level: Impurities Imp-A, Imp-B and Imp-C were spiked at 175 % level to

232 gemcitabine hydrochloride. Representative chromatogram was shown in Fig. 5.4.F15. Fig. 5.4.F15:

Representative chromatogram of 175 % level linearity 8 1 0 .1 A p m I

0.04

0.02

U A

4 4 7 .1 k a e p n w o n kn u

0 9 3 . 1 B -p m I

0.00

0.00

0.50

1.00

9 8 .8 1 e n i b a ti c m e G

1.50

1 0 1 .4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

X-axis: Retention time in min and Y-axis: Peak response in AU 3.4.6.9 Linearity at 200 % level: Impurities Imp-A, Imp-B and Imp-C were spiked at 125 % level to gemcitabine hydrochloride. Representative chromatogram was shown in Fig. 5.4.F16. Fig. 5.4.F16:

9 1 0 . 1 A p m I

0.04

U A

Representative chromatogram of 200 % level linearity

0.02

5 4 7 . 1 k a e p n w o n kn u

1 9 .3 1 B p m I

0.00

0.00

0.50

1.00

1.50

9 8 8 . 1 e n i b a ti c m e G

6 9 .0 4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

X-axis: Retention time in min and Y-axis: Peak response in AU 5.4.7 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, ImpB 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 Gemcitabine

Recovery levels

% Imp-A

% Imp-B

% Imp-C

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

hydrochloride

234 Fig. 5.4.F17:

100% accuracy authentic typical chromatogram

0.04

7 1 0 . 1 A -p m I

U 0.02 A

0.00

0.00

0.50

1.00

4 8 .8 1 e in b a it c m e G

7 8 .3 1 -B p m I

1.50

5 6 .0 4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

X-axis: Retention time in min and Y-axis: Peak response in AU Fig. 5.4.F18:

50 % accuracy typical chromatogram 0 4 .7 1 k a e p n w o n -k n U

0.04

U A

7 1 .0 1 -A p Im

0.02

0.00

0.00

0.50

7 8 3 .1 -B p m I

1.00

2 8 8 .1 e n ib a ti c m e G

1.50

4 7 .0 4 -C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

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

Fig. 5.4.F19:

100 % accuracy typical chromatogram 2 4 .7 1 k a e p n w o n kn U

0.04

U A

7 1 .0 1 -A p m I

0.02

0.00 0.00

0.50

1.00

8 8 .3 1 -B p m I

1.50

4 8 .8 1 e n ib a ti c m e G

5 8 0 . 4 C p m I

2.00

2.50 Minutes

3.00

3.50

4.00

4.50

5.00

235 X-axis: Retention time in min and Y-axis: Peak response in AU Fig. 5.4.F20: 0.04

U A

0 2 .0 1 -A p m I

0.02

150 % accuracy typical chromatogram

3 9 3 .1 -B p m I

0.00 0.00

0.50

1.00

9 4 7 .1 k a e p n w o n kn U

1.50

4 9 8 .1 e n ib a ti c m e G

8 1 1 . 4 C p m I

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes

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

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 ImpC 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 Temperature

Flow rate (+ 0.02 mL

pH of buffer

Parameter

(+ 5°C of 25°C)

Variation

20°C

30°C

0.23

0.27

2.3

2.7

4.1

4.3

4.2

4.4

4.1

4.3

1.1

1.0

1.2

1.1

1.2

1.2

min-1of 0.25)

(+0.2 of 2.5)

The resolution between Impurity-B and gemcitabine USP Tailing factor for gemcitabine 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 Related substances by HPLC

B.No Imp-A

Imp-B

Imp-C

Single maximum impurity

Total impurity

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 Conclusion The

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 References: [1]

S. Noble, K. Goa, Gemcitabine-A review of its pharmacology and clinical in non-small cell lung cancer and pancreatic cancer. Drugs 54 (1997) 447-472.

[2]

The United States Pharmacopeia, USP29 NF24, Asian Edition; 990.

[3]

S.

Wren,

P.

Tchelitcheff,

Use

of

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