a stability indicating uplc method for the rapid...
TRANSCRIPT
191
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
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
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
X-axis: Retention time in min and Y-axis: Peak response in AU
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
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
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
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
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 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
X-axis: Retention time in min 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.
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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
X-axis: Retention time in min and Y-axis: Peak response in AU
Fig. 5.3.F9 (b): Typical HPLC chromatogram of acid hydrolysis
211
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
X-axis: Retention time in min and Y-axis: Peak response in AU
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
212
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
X-axis: Retention time in min and Y-axis: Peak response in AU
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
X-axis: Retention time in min and Y-axis: Peak response in AU
Peak Purity Results
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
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 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
X-axis: Retention time in min and Y-axis: Peak response in AU
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
X-axis: Retention time in min and Y-axis: Peak response in AU
Peak Purity Results
214
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
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
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
X-axis: Retention time in min and Y-axis: Peak response in AU
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
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
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
X-axis: Retention time in min and Y-axis: Peak response in AU
Peak Purity Results
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
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
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
X-axis: Retention time in min and Y-axis: Peak response in AU
Peak Purity Results
Name RetentionTime
PurityAngle
PurityThreshold
PurityFlag
PeakPurity
Gemcitabine 1.88 1.90 2.22 No pass
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
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 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
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 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
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].
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
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.
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 AU
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.
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
gemcitabine hydrochloride. Representative chromatogram was shown in
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
X-axis: Retention time in min and Y-axis: Peak response in AU
5.4.6.4 Linearity at 75 % level:
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
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
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
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
X-axis: Retention time in min and Y-axis: Peak response in AU
5.4.6.6 Linearity at 125 % level:
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
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:
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: Representative chromatogram of 150 % level linearity
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
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
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
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: Representative chromatogram of 200 % level linearity
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
X-axis: Retention time in min and Y-axis: Peak response in AU
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
X-axis: Retention time in min and Y-axis: Peak response in AU
Fig. 5.4.F19: 100 % accuracy typical chromatogram
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
X-axis: Retention time in min and Y-axis: Peak response in AU
Fig. 5.4.F20: 150 % accuracy typical chromatogram
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
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 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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