American Journal of Analyt ical Chemistry, 2010, 2, 83-90
doi:10.4236/ajac.2010.12011 Published Online August 2010 (
Copyright © 2010 SciRes. AJAC
Degradation Pathway for Pitavastatin Calcium by
Validated Stability Indicating UPLC Method
Antony Raj Gomas1,2, Pannala Raghu Ram1,2, Nimmakayala Srinivas1, Jadi Sriramulu2
1Shasun Pharmaceuticals Limited, Chennai, India
2Department of Chemistry, Sri Krishna Devaraya University, Anan tapur, India
Received July 7, 2010; revised July 30, 2010; accepted August 3, 2010
Degradation pathway for pitavastatin calcium is established as per ICH recommendations by validated and
stability indicating reverse phase liquid chromatographic method. Pitavastatin is subjected to stress condi-
tions of acid, base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base
stress conditions. Four impurities are studied among which impurity-4 is found prominent degradant. The
stress samples are assayed against a qualified reference standard and the mass balance is found close to
99.5%. Efficient chromatographic separation is achieved on a BEH C18 stationary phase with simple mobile
phase combination delivered in gradient mode and quantification is carried at 245 nm at a flow rate of 0.3
mL min-1. In the developed UPLC method the resolution between pitavastatin calcium and four potential
impurities is found to be greater than 4.0. Regression analysis shows an r value (correlation coefficient) of
greater than 0.998 for pitavastatin calcium and four potential impurities. This method is capable to detect the
impurities of pitavastatin calcium at a level of 0.006% with respect to test concentration of 0.10 mg/mL for a
2-µL injection volume. The developed UPLC method is validated with respect to specificity, linearity &
range, accuracy, precision and robustness for impurities determination and assay determination.
Keywords: Column Liquid Chromatography; Pitavastatin Calcium; Forced Degradation; Validation; Stability
1. Introduction
Pitavastatin: (E)-7-[2-cyclopropyl-4-(4-fluorophenyl)qui-
nolin-3-yl]-3,5-dihydroxy-hept-6-enoic acid. Pitavastatin
(usually as a calcium salt) is a novel member of the
medication class of statins. Like the other statins, it is an
inhibitor of HMG-CoA reductase, the enzyme that ca-
talyses the first step of cholesterol synthesis. It has been
available in Japan since 2003, and is being marketed
under license in South Korea and in India. It is likely that
pitavastatin will be approved for use in hypercholester-
olaemia [1-2]. There are some mass detection methods
reported for determination of pitavastatin in plasma and
biological fluids and two methods for pitavastatin quan-
tification in tablets by HPLTC were reported [3-6]. As
far as we are aware there is no stability-indicating LC
method for determination of related substances and assay
determination of pitavastatin calcium. In this paper we
describe validation of related substances and assay meth-
ods for accurate quantification of four potential process
impurities in pitavastatin calcium samples as per ICH
recommendations. Intensive stress studies are carried out
on pitavastatin calcium; accordingly a stability-indicating
method is developed, which could separate various deg-
radation produ c t s.
The present active pharmaceutical Ingredient (API)
stability test guideline Q1A (R2) issued by international
conference on harmonization (ICH) [7] suggests that
stress studies should be carried out on active pharmaceu-
tical ingredient (API) to establish its inherent stability
characteristics, leading to separation of degradation prod-
ucts and hence supporting the suitability of the proposed
analytical procedures. It also requires that analytical test
procedures for stability samples should be stability indi-
cating and they should be fully validated. Accordingly,
the aim of present study is to establish degradation
pathway of pitavastatin calcium through stress studies
under a variety of ICH recommended test conditions
Copyright © 2010 SciRes. AJAC
2. Experimental Design
2.1. Chemicals
Samples of pitavastatin calcium with purity more than
99.8% and its related impurities having purity more than
99.0% are received from Shasun research centre, Chennai,
India (Figure 1). HPLC grade acetonitrile is purchased
from Merck, Darmstadt, Germany. Analytical reagent
grade orthophosphoric acid and is purchased from Merck,
Darmstadt, Germany. High purity water is prepared by
using Millipore Milli-Q plus water purification system.
2.2. Procedure
2.2.1. Equipments
The LC system, used for method development and method
validation is Waters-Acquity UPLC. The output signal is
monitored and processed using Empower 2 software on
Pentium computer (Digital equipment Co). UPLC is
equipped with Binary gradient pump, Auto Sampler,
thermostatted column compartmen t, Tunable U V D etec to r,
Auto sampler thermostatted, Computer with windows
based Empower 2 Metho d vali dat i on manager software.
2.2.2. Chromatogr aphic Con ditions
The chromatographic column used is Waters BEH C18
(100 × 2.1 mm) with 1.7µm particles. The mobile phase- A
contains a 0.03% of orthophosphoric acid buffer (0.3mL/L ).
Acetonitrile is used as mobile phase-B. The flow rate of
the mobile phase is 0.3 mL/min with a gradient program
of 0/45, 2/45, 2.5/100, 4/100, 4.5/45 and 5/45 (time
The column temperature is maintained at 40°C and the
detection is monitored at wavelength of 245 nm. The
injection volume is 2 µL. Diluent consists water and
acetonitrile in the ratio 90:10.
2.2.3. Preparation of Solu tio ns
All the impurities are dissolved initially by adding 5 mL
of acetonitrile then make up to the volume with diluent. A
Stock solution of pitavastatin calcium (0.10 mg/mL) is
prepared by dissolving appropriate amount in the diluent.
Working so lution 10 µg/mL is prepared from above stock
solution for assay determination.
2.3. Method development and optimization
Impurities and pitavastatin calcium solutions are prepared
in diluent at a concentration of 100 ppm and scanned in
UV-visible spectrometer; all the 4 impurities and pitavas-
tatin calcium are having UV maxima at around 245 nm
which is selected for method development purpose. Mo-
bile phase of ammonium acetate (0.05 M) and acetonitrile
(60:40, v/v) is selected for initial tria l on a C18 stationary
phase column [150 × 4.6 mm, 5 µm] and flow rate at
Figure 1. Chemical Structures and labels of pitavastatin
calcium and its impurities. Pitavastatin calcium: (E)-7-[2-
hept-6-enoic acid; Impurity-1: Methyl 4-(4’ -fluorophenyl)-
2-cyclopropyl-quinolin-3-yl-carboxylate; Impurity-2: 3-
hydroxymethyl-2-cycloprop yl-4-(4-fluoroph enyl)-quinoline;
Impurity-3: 3-Bromomethyl-2-cyclopropyl-4-(4-fluoroph-
enyl)quinoline; Impurity-4: (4R,6S)-6-{(E)-2-[2-cyclopropyl-
Copyright © 2010 SciRes. AJAC
1 mL/min. Spike sample analysis revealed that principal
peak RT is late and impurities 1 and 3 are not resolved
properly. Similar results are obtained with other C18
columns length with the 250 mm. To further resolve the
impurity-1 and impurity-3 triethyl- amine is added to
buffer then impurity-1 and impurity-3 are separated but
the retention time of pitavastatin calcium is late.
Gradient program is introduced for better resolution
between Impurity-1 and impurity-3, 0.03% H3PO4 buffer
used as mobile phase-A and 100% acetonitrile is used as
mobile phase-B. Initial ratio of buffer: acetonitrile tried
as 80:20 in this case impurity-1 and impurity-3 are well
resolved but peak shapes are not good for all components
and retention time of pitavastatin calcium is not de-
creased. Shorter length columns are selected like 50mm
and 100 mm with the 4.6 mm diameter of symmetry C18
and C8 for decreasing of retention time for pitavastatin
calcium peak in this case pitavastatin calcium retention is
decreased but impurities all are not resolved well. Keep-
ing these disadvantages the shorter length and less inter-
nal diameter column like waters BEH C18 column is
selected with the dimensions 50 × 2.1 mm 1.7µm in this
column all components are separated with the minimum
resolution 1.5.
To increase the resolution between each component
100 × 2.1 mm column is selected. After several other
trails satisfactory results (Retention time of pitavastatin
calcium is ~1.16 min and the resolution between all the
impurities is > 4.0) are obtained with optimized condi-
tions. In the optimized conditions pitavastatin calcium,
Impurity-1, Impurity-2, Impurity-3 and Impurity-4 are
well separated with a resolution greater than 4.0 and the
typical retention times of pitavastatin calcium, Impu-
rity-1, Impurity-2, Impurity-3 and Impurity-4 are about
1.169, 3.763, 1.763, 3.984 and 2.361 min respectively
meeting the chromatographic system suitability require-
ments (Table 1).
2.4. Analytical Method Validation
The developed chromatographic method is validated for
specificity and stress studies, sensitiv ity, linearity & range,
precision, accuracy, and robustness and system suitability
2.4.1. Specificity and Stress Studies
Specificity is the ability of the method to measure the
analyte response in the presence of its potential impuri-
ties. The specificity [10-11] of the develop ed LC method
for pitavastatin calcium is determined in the presence of
its impurities namely Impurity-1, Impurity-2, Impurity-3
and Impurity-4 at a concentration of 0.15 µg/mL and
Table 1. System suitability report.
Component USP
(RS )
cal plates
%RSD at
calcium -- 1.2 3752 1.04
Impurity -27.1 1.2 6554 1.50
Impurity -46.1 1.1 8024 1.43
Impurity -117.6 1.1 86618 1.91
Impurity -34.2 1.1 90439 2.19
degradation products. The stress conditions employed for
degradation study includes photolytic (carried out as per
ICH Q1B), thermal (100°C), acid hydrolysis (1N HCl),
base hydrolysis (1N NaOH), hydrolysis and oxidation
(10% H2O2). All stressed samples of pitavastatin calcium
are analysed for an extended run time of 10 min to check
the late eluting degradants. Assays are carried out for
stress samples against qualified reference standard and
the mass balance (%assay + %of impurities + %of deg-
radation products) is calculated for all the samples.
2.4.2. Precision
The precision of the related substance method is checked
by injecting six individual preparations of (100 µg mL-1)
pitavastatin calcium spiked with 0.02% each impurity.
The %RSD for percentage of each impurity is calculated.
The intermediate precision (ruggedness) of the method
is evaluated by different analyst using different column,
different day and di fferent a n al y st i n t he sam e l a boratory .
The precision of the assay method is evaluated by car-
rying out six independent assay of test sample of pitavas-
tatin calcium against a qualified reference standard. The
%RSD of six obtained values is calculated.
2.4.3. Sensitivity
Sensitivity was determined by establishing the Limit of
detection (LOD) and Limit of quantification (LOQ) for
each component estimated by based on the Signal to noise
ratio method. The precision study was also carried out at
the LOQ level by injecting six replicates and calculated
the % RSD for the area of each component.
2.4.4. Linearity and Range
Linearity test solutions from LOQ to 150% with respect
to test concentration are prepared by diluting the impu-
rity stock solution to the required concentrations. For
assay method test solutions from 50% to 150% with re-
spect to test concentration are prepared by diluting the
stock solution to the required concentrations. The corre-
lation coefficient, slope and Y-intercep t of the calibration
curve are calculated for the both related substances and
assay methods.
2.4.5. Accuracy
A known amount of the impurity stock solutions are
Copyright © 2010 SciRes. AJAC
spiked to the previously analysed samples at LOQ, 100
and 150% of the analyte concentration (100 µg/mL). The
percentage of recoveries for Impurity-1, Impurity-2, Im-
purity-3 and Impurity-4 are calculated. A known amount
of pitavastatin calcium stock solution spiked to the su-
crose at 50%, 100% and 150% of the analyte concentra-
tion (10 µg/mL). Each concentration level is prepared for
three times. The percentage of recoveries is calculated.
2.4.6. Robustness
To determine the robustness of the developed method,
experimental conditions are deliberately changed and the
resolution between each component is evaluated. The
flow rate of the mobile phase is 0.3 mL/min. To study the
effect of flow rate on the resolution, 0.03 units changed
i.e. 0.27 and 3.3 mL/min. The effect of column tempera-
ture on resolution is studied at 35°C and 45°C instead of
40°C. In the all above varied conditions, the components
of the mobile phase are hel d constant.
2.4.7. Solution Stability and Mobile Phase Stability
The solution stability of pitavastatin calcium and its re-
lated impurities are carried out by leaving spiked sample
solution in tightly capped volumetric flask at room tem-
perature for 48 h. Impurity content is determined for
every 6 h interval up to the study period. Mobile phase
stability is also carried out for 48 h by injecting the
freshly prepared sample solutions for every 6 h interval.
Impurity content is checked in the test solutions. Mobile
phase prepared is kept const an t duri n g t he stu dy peri od .
3. Results and Discussion
3.1. Specificity and Stress studies
Stress studies on pitavastatin calcium under different
stress conditions suggested the following degradation
behavior (Table 2).
3.1.1. Degradation i n Aci d St ress Condi ti on
Pitavastatin calcium gradually undergone degradation
with time in 1 N HCl upon heating for 2 h and prominent
degradation is obser ved as i m puri t y -4.
3.1.2. Degradation i n B ase Stress Condi ti on
Pitavastatin calcium is gradually undergone degradation
with time in 1 N NaOH upon heating for 2 h and promi-
nent degradation is observed as impurity-2 and impu-
3.1.3. Degradation in Peroxide Stress Condition
Pitavastatin calcium is gradually undergone degradation
with time in 10% H2O2 upon heating for 2 h and mild
degradation is obser ved as i m puri t y -4.
3.1.4. Degradation in Neutral (Water) Stress Condition
Pitavastatin calcium is exposed water heating for 2 h, no
degradation is obser ved.
3.1.5. Photolyt i c Stress Condition
Pitavastatin calcium is exposed to light for an overall il-
lumination of 1.2 million Klux hours and an integrated
near ultraviolet energy of 200-watt hours/square meter
(w/mhr) (in photo stability chamber), mild degradation is
3.1.6. Thermal Stress Condition
Pitavastatin calcium exposed to dry heat at 100°C for 48
hours, no degradation is observed.
The mass balance of stressed samples is close to 99.5%.
The assay of pitavastatin calcium is unaffected in the
presence of four impurities and its degradation products
confirm the stability indicating power of the developed
3.2. Method Validation
3.2.1. Precision
The %RSD of area of pitavastatin calcium, Impurity-1,
Impurity-2, Impurity-3 and Impurity-4 and %RSD of
area% of each impurity in precision study are within 5.0%
confirming the good precision of the developed analytical
method. The %RSD obtained in intermediate precision
study for pitavastatin calcium, Impurity-1, Impurity-2,
Impurity-3 and Impurity-4 are well within 5.0%, con-
firming the intermediate precision of the method. The
%RSD obtained in precision and intermediate precision
studies for pitavastatin calcium are well within 1.0% of
assay determination m et hod.
3.2.2. Sensitivity
The limit of detection of pitavastatin calcium, impurity-1,
impurity-2, impurity-3 and impurity-4 is 0.006% (of ana-
lyte concentration, i.e. 100 µg/mL) for 2 L injection
volume. The limit of quantification of pitavastatin cal-
cium, Impurity-1, Impurity-2, Impurity-3 and Impurity-4
is 0.02% (of analyte concentration, i.e. 100 µg/mL) for 2
L injection volume. The % RSD for area of pitavastatin
calcium, Impurity-1, Impurity-2, Impurity-3 and Impu-
rity-4 are below 5 for precision at LOQ level.
3.2.3. Linearity and Range
Calibration curve obtained by the least square regression
analysis between average peak area and concentration
showed linear relationship with a correlation coefficient
of 0.998 over the calibration ranges tested. Linear calibra-
tion plot for related substance method is obtained over the
calibration ranges tested, i.e. LOQ to 0.225% for Impu-
rity-1, Impurity-2, Impurity-3 and Impurity-4 and LOQ to
0.15% for pitavastatin calcium. The correlation coeffi-
cient obtained is g reater than 0.998 for all fou r impurities
and pitavastatin calcium. The result shows an excellent
correlation existed between the peak area and concentra-
tion of pitavastatin calcium and all impurities. Linear
calibration plot for assay determination method is ob-
Copyright © 2010 SciRes. AJAC
tained over the calibration ranges tested, i.e. 50 to 150%
for pitavastatin calcium and found the correlation coeffi-
cient more than 0.999. The results shows an excel- lent
correlation existed between the peak area and con- cen-
tration of pitavastatin calcium in assay determination
method (Figur e 3, Table 3).
3.2.5. Robustness
Close observation of analysis results for deliberately
changed chromatographic conditions (flow rate and col-
umn temperature) revealed that the resolution between
closely eluting impurities, namely impurity-1 and impu-
rity-3 is greater than 4.0, illustratin g the robustness of the
3.2.6. Solution Stability and Mobile phase Stability
The %RSD of assay of pitavastatin calcium during solu-
tion stability and mobile phase stability experiments is
within 1.0. No significant changes are observed in the
content of impurity-1, impurity-2, impurity-3 and impu-
rity-4 during solution stability and mobile phase stability
experiments. The solution stability and mobile phase sta-
Table 2. Summary on forced degradation results.
Stress condition Time % Assay of
active substance
%impurities +
% Degradation
Mass balance
(%Assay + %impurities +
% Degradation products) Remarks
Acid St r es se d s amp le (1 N
HCl) 2 hrs heat-
ing 93.0 6.60 99.6 Formed as Impurity-4
Base St r es se d s amp le (1 N
NaOH) 2 hrs heat-
ing 95.1 4.40 99.5 Form ed as Impurit y- 4
Peroxide stressed sample
(10% H202) 2 hrs heat-
ing 98.8 1.32 100.1
Formed as Im p uri t y - 2 an d
Thermal stressed sample
(Heated at 100˚C) 48 hours 99.7 0.08 99.8 No degradation is observed
Photo light stressed sample 1200 Klux
hours 99.5 0.34 99.8
No prominent degradation is
Standard solution
Copyright © 2010 SciRes. AJAC
Figure 2. Typical chromatogram of blank, standard solution and pitavastatin calcium spiked
with impurities & Stress sample Chromatograms.
le with s
iked im
Acid stress sam
Base stress sam
Oxidation stress sample
Copyright © 2010 SciRes. AJAC
Figure 3. Typical charts and for pitavastatin calcium and
its impurities.
bility experiments data confirms that sample solutions
and mobile phase used related substance determination
are stable up to the study period of 48 h.
Analysis is performed for different samples of pita-
vastatin calcium (n = 3). All the four impurities in these
samples are less than 0.1% and Assay is more than
Table 3. Linearity table.
Component Trendline equation Range
Correlation coeffi-
cient % Intercept Residual sum of
Impurity-1 69037X+239.39 0.02-0.225 0.99843 2.32 75426
Impurity-2 63379X+198.94 0.02-0.225 0.99899 2.12 68828
Impurity-3 77784X+61.46 0.02-0.225 0.99880 0.54 82815
Impurity-4 44536X+48.47 0.02-0.225 0.99910 0.74 47613
Related substances39934X+48.18 0.02-0.15 0.99910 1.22 43281
calcium Assay
determination 22727193X-1044 50%-150% 0.99998 –0.31 24868238
Table 4. Table for accuracy study.
% of Recovery
Assay determination
Amount of impurity added (µg)
to the 100% sample Imp-1 Imp-2 Imp-3 Imp-4 Amount of substance
added %Recovery
(Pitavastatin calcium)
0.02 98.2 101.1 96.5 99.0 50% 99.48
0.10 97.5 102.5 98.0 97.1 100% 100.00
0.15 96.4 98.5 93.6 101.8 150% 100.11
4. Conclusions
The degradation pathway of pitavastatin calcium is es-
tablished as per ICH recommendations. The gradient
UPLC method developed and used for stress studies is
also fit for quantitative, related substance and assay de-
termination of pitavastatin calcium. The method is vali-
dated as per ICH requirements. The developed method is
stability indicating which can be used for the impurity
testing and assay determination in routine analysis of
production samples and also to analyze stability samples.
5. Acknowledgements
The authors wish to thank the management of Shasun
Chemicals & Drugs Limited for supporting this work.
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