Spectroscopic characterization and quantitative determination of atorvastatin calcium impurities by novel HPLC method

Article abstract of DOI:10.1016/j.saa.2012.06.037

Seven process related impurities were identified by LC-MS in the atorvastatin calcium drug substance. These impurities were identified by LC-MS. The structure of impurities was confirmed by modern spectroscopic techniques like ¹H NMR and IR and physicochemical studies conducted by using synthesized authentic reference compounds. The synthesized reference samples of the impurity compounds were used for the quantitative HPLC determination. These impurities were detected by newly developed gradient, reverse phase high performance liquid chromatographic (HPLC) method. The system suitability of HPLC analysis established the validity of the separation. The analytical method was validated according to International Conference of Harmonization (ICH) with respect to specificity, precision, accuracy, linearity, robustness and stability of analytical solutions to demonstrate the power of newly developed HPLC method.

Full text of DOI:10.1016/j.saa.2012.06.037

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 495–501
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectroscopic characterization and quantitative determination of atorvastatin
calcium impurities by novel HPLC method
⇑
Lokesh Kumar Gupta
Department of Chemistry, Zakir Husain College, University of Delhi, JLN-Marg, New Delhi 110 002, India
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
"
"
"
"
Identification of possible impurities
in atorvastatin calcium drug
substance.
Synthesis and spectroscopic
characterization of atorvastatin
related compounds.
Development of single analytical
method for quantitative
determination with HPLC.
Method validation, as per
international pharmaceutical
guidelines.
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven process related impurities were identified by LC–MS in the atorvastatin calcium drug substance.
These impurities were identified by LC–MS. The structure of impurities was confirmed by modern spectro-
scopic techniques like 1H NMR and IR and physicochemical studies conducted by using synthesized authen-
tic reference compounds. The synthesized reference samples of the impurity compounds were used for the
quantitative HPLC determination. These impurities were detected by newly developed gradient, reverse
phase high performance liquid chromatographic (HPLC) method. The system suitability of HPLC analysis
established the validity of the separation. The analytical method was validated according to International
Conference of Harmonization (ICH) with respect to specificity, precision, accuracy, linearity, robustness and
stability of analytical solutions to demonstrate the power of newly developed HPLC method.
Ó 2012 Elsevier B.V. All rights reserved.
Received 14 February 2012
Received in revised form 17 June 2012
Accepted 25 June 2012
Available online 10 July 2012
Keywords:
Atorvastatin calcium
HPLC
Spectroscopic determination
Validation of analytical method
Introduction
synthesized by an American scientist: Dr. Bruce D. Roth in 1985
when he was working as Senior Scientist with Parke-Davis of
Atorvastatin calcium: (3R,5R)-7-[2-(4-Fluoro-phenyl)-5-isopro-
pyl-3-phenyl-4-phenyl carbamoyl-pyrrol-1-yl]-3,5-dihydroxy-
Warner-Lambert Company (later on in 2000 it was merged with
Pfizer). The method of synthesis prescribed in this article is not
concordant of original one; it is alternate, cost effective and com-
paratively better synthone pathway. The atorvastatin drug sub-
stance popularized with the name of ‘‘Lipitor’’ as a drug product
is very commonly used cholesterol reducing drug, being used
around the globe. The manufacturers of atorvastatin purify the
drug substance and evaluate the quality either on TLC or HPLC.
Although, however, the United States Pharmacopoeia (USP) had
incorporated the monograph of atorvastatin in September 2009,
but still impurity related information are deficient and till now
there is no literature that describes any analytical technology
heptanoic acid calcium salt, is an established drug [1,2] under
the category of cardio-vascular therapeutic use. Previously the
majority of formulations were being made by using ‘‘Simvastatin’’,
‘‘Lovastatin’’ or ‘‘Fluvastatin’’ for the same purpose. But due to a
number of side-effects and less effectiveness of these molecules,
atorvastatin (calcium salt) was developed and found effective
against several cardiac diseases [3]. The drug atorvastatin was first
⇑
Tel.: +91 9673994052; fax: +91 2525 273092.
E-mail address: lokesh_kg@rediffmail.com
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.06.037

496
L.K. Gupta / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 495–501
Step-1:
H C
3
CH
3
H C
3
CH
3
C
C
O
O
O
O
O
O
CH
3
CH
3
Ni/H
2
NC
H N
2
O
CH
3
Methanolic Ammonia
O
CH
3
CH
3
CH
3
(AT-Oil)
(Amino Derivative)
Step-2:
H C
3
CH
3
C
O
O
O
O
CH
3
+
H N
2
O
O
CH
3
NHC H
6
5
CH
3
F
(Amino Derivative)
(Diketo Compound)
H C
3
CH
3
O
C
O
O
O
CH
3
NH
N
O
CH
3
CH
3
(Atorva A)
F
Step-3:
H C
3
CH
O
3
O
O
C
O
O
OH
OH
O
CH
CH
3
3
CH
CH
NH
3
NH
N
O
CH
3
N
O
CH
3
(Atorva A)
3
CH OH/HCl
3
NaOH/H O
2
F
F
2
O
O
OH
OH
O
OH
OH
O
NH
NH
N
O-
N
ONa+
(Sodium Salt)
++
Ca
(Atorvastatin Calcium)
F
F
Fig. 1. The synthesis scheme of atorvastatin calcium.
which is able to quantify ‘‘Seven’’ process related impurities. The
method presented in this article gives sufficient information about
possible impurity and their evaluation to help the chemists in puri-
fying the drug in a better way. Atorvastatin has been formulated as
an individual and in several combinations [4].
British Pharmacopoeia [7,8] describes a thin layer chromatography
(TLC) method for the quantitative assessment of atorvastatin re-
lated substances. Atorvastatin is cholesterol reducing drug and acts
as HMG-CoA reductase inhibitory agent, like other drugs i.e. Lova-
statin, Simvastatin, etc. and has some common side effects. This
article describes the possibility of process related impurities, their
structure and HPLC technology to quantify them, by which these
A few bio-analytical methods are reported in the literature for
the quantitative determination of atorvastatin calcium [5,6]. The

L.K. Gupta / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 495–501
497
Sr.
Name of related
Structure
No.
compound
+2
Ca
OH OH
O
O
C
O
N
N
H
1.
2.
ATVRS-1
ATVRS-2
2
+2
Ca
OH
O
O
O
O
C
N
N
H
F
2
+2
Ca
OH OH
O
O
C
O
N
N
H
3.
4.
ATVRS-3
ATVRS-4
F
2
+2
Ca
OH
OH
O
O
C
N
N
H
O
F
F
2
OH
O
O
C
O
N
N
H
5.
6.
7.
ATVRS-5
ATVRS-6
ATVRS-7
F
OH OH
O
O
C
N
N
O
CH3
H
F
+2
Ca
O
O
O
O
O
C
N
N
H
F
2
Fig. 2. The structure of atorvastatin calcium related substances.

498
L.K. Gupta / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 495–501
Table 1
Mass, melting point and elemental analysis data of the compounds.
Compounds
MW (calculated)
MW (observed)
Color
M.P. (°C)
Elemental analysis % found (calculated)
C
H
N
ATVRS-1 (C66H70N4O10Ca)
1118
1150
1155
1190
540
1078
1110
1114
1151
541
White powder
White powder
White powder
White powder
Off-white powder
Off-white powder
White powder
White powder
163
191
193
188
159
145
187
160
70.92
(70.84)
68.72
(68.87)
68.51
(68.63)
66.70
(66.55)
73.19
(73.33)
71.38
(71.33)
70.17
(70.02)
68.52
6.18
(6.26)
5.43
(5.57)
5.99
(5.89)
5.68
(5.55)
6.00
(6.11)
6.43
(6.47)
6.22
(6.16)
6.89
5.13
(5.01)
5.02
(4.87)
4.99
(4.85)
4.85
(4.71)
5.36
(5.19)
5.01
(4.90)
4.63
(4.54)
4.92
ATVRS-2 (C66H64N4O10F2 Ca)
ATVRS-3 (C66H68N4O10F2 Ca)
ATVRS-4 (C66H66N4O10F4 Ca)
ATVRS-5 (C33H33N2O4F)
ATVRS-6 (C34H37N2O5F)
572
572
ATVRS-7 (C72H76N4O10F2 Ca)
Atorvastatin calcium (C66H68N4O10F2Ca)
1234
1155
1194
1114
(68.63)
(6.76)
(4.85)
performed over the ‘‘Bruker’’ instrument. The microanalysis (CHN)
was measured on CHN analyzer which was of ‘‘Perkin Elmer’’ make.
Table 2
Gradient program of the HPLC analysis.
Synthesis of atorvastatin calcium
Sr. No.
Time (minutes)
Mobile phase A%
Mobile phase B%
Curve
1
2
3
4
5
6
0
10
20
35
47
50
100
100
85
15
100
100
0
0
15
85
0
6
6
6
6
6
6
Atorvastatin was made in three steps [9] (Fig. 1) by using two
intermediates, i.e. AT-oil and Diketo compound. These intermediates
were readily available in market and were outsourced from China.
0
Step-1: AT-oil to amino derivative
AT-oil was hydrogenated with hydrogen gas in presence of nickel
catalyst in ammonical methanol. After reaction completion, catalyst
was filtered off and methanol distilled out, oily product (amino
derivative) was obtained which was taken as such, for next stage.
can be properly evaluated and an impurity free drug can be man-
ufactured. The evaluation of toxicological impacts of these impuri-
ties and their biological effects can be an encouraging part for
further research, which is a separate chemistry. Since the impurity
profile study of any pharmaceutical substance is a crucial part of
process development, it was quite desirable and necessary to de-
velop a reliable method for quantitation of all identified impurities
in atorvastatin calcium, in a single shot.
In process development several impurities were detected in
crude and pure sample of atorvastatin calcium by using a newly
developed reverse phase gradient HPLC method. To get a control
on this synthesis process related impurities it was quite compul-
sory to identify these impurities first. In this regard the LC–MS
spectra of atorvastatin samples were recorded and the synthesis
process was reviewed. It was observed that there is a possibility
of seven impurities to be present in the atorvastatin samples. For
the complete identification and confirmation, these all seven
impurities have been synthesized, separately and characterized.
By using these impurities as reference material the HPLC method
has been validated for their quantification.
Step-2: atorva A series
Product amino derivative (output of step-1) and diketo com-
pound (outsourced) were allowed for condensation in the presence
of soft acid in the organic solvent after reaction completion, reac-
tion mixture was washed with alkaline water and then sodium
chloride solution. Solvent distilled out, product was crystallized
in alcoholic solvent and DM water than filtered, dried & further
re-crystallized in the same solvent.
Step-3: atorva A to atorvastatin calcium
Atorva A was stirred in methanol and hydrochloric acid solution
then this methanolic solution was concentrated and then again
stirred. After reaction completion, adjust pH alkaline with sodium
hydroxide solution and stirred for several (10–12) h, after reaction
completion, methanol was distilled off under reduced pressure.
Product is taken in methanol water mixture and washed with or-
ganic solvent. Aqueous calcium acetate solution was added, prod-
uct (atorvastatin calcium) was precipitated out which was filtered,
washed with de-mineralized water and dried under vacuum.
Experimental
Atorvastatin calcium samples (crude and pure, both) were re-
ceived from An API manufacturing company. Other chemicals were
purchased from Sigma Aldrich and Lancaster/Merck. Solvents used
were of Spectroscopic/Chromatographic grade and were used as re-
ceived. The study has been performed with HPLC (Water Alliance
2695system equipped with UVdetector, quaternary gradient flowing
pump, column and sample cooler/heater), Perkin Elmer FT–IR BX-II
spectrometer. The MS studies were carried out on Waters Quadrapole
ion trapmassspectrophotometer attached withthe HPLC system. The
melting point of the compounds was measured on ‘‘Labindia’’ made
automatic melting point recorder. The used UV spectrophotometer
was of ‘‘Schimadzu’’ make while the NMR studies have been
Characterization atorvastatin related compounds
All impurities (atorvastatin related substances), synthesized in
laboratory (Fig. 2) have been characterized by using physicochem-
ical (color, yield, melting point and CHN analysis) and modern
spectroscopic techniques (Mass, IR, 1H NMR, UV and HPLC). The
observed value of the CHN%, which found very close to the calcu-
lated values, Table 1, mass and IR spectra confirms the proposed
structure of these compounds. UV (maxima at 210–240 nm) and
NMR analysis further supports the elucidated structure. The HPLC
analysis gives the idea of the purity of the compounds. Elucidated

L.K. Gupta / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 495–501
499
Fig. 3. HPLC chromatogram; system suitability of atorvastatin calcium.
phases, A and B, were used in timed gradient flow of 1.5 mL per/
minute. The mobile phase A consists the phosphate buffer with
pH 5.4 and the mobile phase B have the HPLC grade Acetonitrile:
Tetrahydrofuran:: 90:10 (v/v). The injection volume was 20 L.
The column and sample temperature was maintained at 40 and
6 °C, respectively. The detector was set at 220 nm, throughout
the analysis. Gradient program was set shown in Table 2.
Table 3
R2 values of all impurities evaluated over the concentration linear curve.
Sr.
No.
Name of the
component
R2 value (limit not less than
0.99)
Linear
equation
1
2
3
4
5
6
7
ATVRS-1
ATVRS-2
ATVRS-3
ATVRS-4
ATVRS-5
ATVRS-6
ATVRS-7
0.9958
0.9955
0.9977
0.9988
0.9921
0.9975
0.9980
y = 144909x
y = 43605x
y = 17980x
y = 35988x
y = 75112x
y = 59178x
y = 34013x
Liquid chromatography–mass spectrophotometry
During mass studies the in instrument (as described above), the
source voltage was maintained at 3.0 kV and capillary temperature
at 240 °C. Helium gas was used as both sheath and auxiliary gas. The
mass to charge ratio was scanned across the range of m/z 70 to 1500 u.
structure of the possible impurities in atorvastatin calcium has
been shown in Fig. 2.
High performance liquid chromatography
Infra-red spectrophotometry
All samples were analyzed on a HPLC by using C-18 250 mm
(long) Â 4.6 mm (ID), 3.5-micron particle size analytical column
was used for chromatographic separation. There were two mobile
The IR spectra of the newly synthesized API and its related sub-
stances were recorded in the solid phase as KBr pallets on FTIR
spectrometer.

500
L.K. Gupta / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 495–501
Table 4
Concentration linearity data of all impurities.
Linearity of atorvastatin calcium related substances (impurities)
Concentration
Level
ATVRS-1
ATVRS-2
ATVRS-3
ATVRS-4
ATVRS-5
ATVRS-6
ATVRS-7
Conc. Average
area
Conc. Average
area
Conc. Average
area
Conc. Average
area
Conc. Average
area
Conc. Average
area
Conc. Average
area
70.0%
90.0%
100.0%
110.0%
130.0%
1.40
1.80
2.00
2.20
2.60
206,697.72 1.40
61,618.65 1.40
25,390.77 1.40
50,447.48 1.40
110,270.36 1.40
83,863.84 1.40
47,583.72
60,912.68
69,062.00
75,070.39
87,667.30
254,860.30 1.80
286,682.00 2.00
320,797.16 2.20
379,566.97 2.60
76,862.46 1.80
85,784.00 2.00
96,935.92 2.20
114,435.86 2.60
32,418.25 1.80
35,314.00 2.00
39,904.82 2.20
46,791.05 2.60
65,460.76 1.80
71,153.00 2.00
79,406.75 2.20
93,495.04 2.60
133,814.57 1.80
149,014.00 2.00
163,617.37 2.20
195,804.40 2.60
107,592.30 1.80
116,316.00 2.00
129,808.66 2.20
154,467.65 2.60
Precision
Results and discussion
The system precision was examined by using six replicate injec-
tions of a standard solution (composite of all impurities along with
atorvastatin calcium). The relative standard deviation (RSD) was
calculated for the response area of each component, individually.
The observed values were well within the generally acceptable
limit of not more than 10%. The method precision was determined
by analyzing samples of atorvastatin calcium using six different
preparations. The calculated RSD of these results were found to
be 1.17%. The intermediate precision was established by perform-
ing the method precision with two columns of different lot and by
using the solvents of two different lots. The cumulative RSD of both
the results was 1.43%.
HPLC analysis using the above method revealed the presence of
seven impurities (ATVRS-1 to ATVRS-7) at RRTs: 1.35, 1.42, 1.05,
1.10, 0.22, 0.82 and 1.15 with respect to principle peak of atorva-
statin calcium. The target impurities, under study, are marked as
ATVRS-1, ATVRS-2, ATVRS-3, ATVRS-4, ATVRS-5, ATVRS-6 and
ATVRS-7, respectively. The typical chromatogram of (composite
of all seven impurities and atorvastatin calcium) system suitability,
highlighting the retention times of all impurities and their resolu-
tion is shown in Fig. 3. The evaluation of spectroscopic results con-
firms the structure of the compounds as prescribed in respective
figures (Figs. 1 and 2).
Validation of HPLC analytical method
Accuracy and recovery
The newly developed method for atorvastatin calcium and its
related substances was validated as per the ICH guidelines [10]
and the guidelines described in United State Pharmacopoeia
(USP) [11].
The validation studies [12] were carried out for the analysis of
all possible seven impurities in atorvastatin calcium. The system
suitability (Fig. 3) was established to demonstrate and verify the
chromatographic separation between all the components.
The entire activity of the analytical method validation was car-
ried out under the following heads:
The accuracy of the method was determined for the related sub-
stances by spiking known amounts of the impurity in atorvastatin
calcium standard solution. At three levels that is 80%, 100% and
120%. The observed recovery of impurities (96.3–103.6%) was well
within the specified limit of 90–110%.
Linearity
To examine the linearity of the method and to set the range
for analysis the composite solution of all components in a range
of concentration (70–130%, 5 concentration levels) was injected
onto the HPLC in triplicate for each level. To calculate different
statistical parameters the regression curve was plotted between
the area response and concentration for each component, sepa-
rately. Linear calibration curve for the related substances method
were obtained over the range of 70–130% of standard concentra-
tion. Method showed the linear response for all the impurities.
The values of calculated co-relation co-efficient (R2) along-with
the linear equation has been shown in Table 3 and corresponding
data in Table 4.
(a) Specificity.
(b) Precision (Intermediate Precision, Method Precision and Sys-
tem Precision).
(c) Accuracy and recovery.
(d) Linearity and range.
(e) Robustness.
(f) Stability of analytical solutions.
Specificity
The ability of analytical method to unequivocally assess the
analyte in the presence of other components can be demonstrated
by evaluating specificity (system suitability). The specificity of this
HPLC method was determined by injecting individual impurity
samples and the composite solution of all impurities along-with
the atorvastatin calcium drug substance, wherein no interference
was observed for any of the components. The chromatograms were
checked for the presence of any other/extra peak. The peak purity
of these samples was checked on Waters 2998 photodiode array
(PDA) detector. The purity of the principle peak and other peaks
due to impurities found satisfactory (Observe peak purity was
>98%). It suggests that there is no merging of any unknown peak
with any known peak. This study confirmed the stability indicating
power of the method.
Robustness
The robustness of the method has also been established by per-
forming the exercise of method precision with making minor but
significant changes in the specified chromatographic conditions.
The changes tried were: change in column temperature (2.0 °C),
change in flow rate (0.1 mL per minute) and the change in the
pH of mobile phase A (0.2). The results obtained with the standard
method and altered methods were not significantly differing and
were within the acceptance criteria.

L.K. Gupta / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 495–501
501
Stability of analytical solutions
ogy, New Delhi (SR/FTP/CS-67/2005) and DRDO are thankfully
acknowledged for financial assistance.
The freshly prepared analytical standard solution (composite of
all impurities and the atorvastatin calcium), as prepared for system
precision was injected onto the HPLC and after the intervals of 3, 6,
12 and 24 h. The cumulative RSD of the area of each component,
separately, was calculated. The observed value was 1.8% (limit
not more than 10%). So it was concluded that the analytical solu-
tions are stable for 24 h at 6 °C.
References
[1] W. Xing, L. Daowei, Li-Guafi, Z. Zhiyi, Eur. J. Pharmacol. 636 (2010) 114–120;
S. Tom, Eur. J. Health Eco. 11 (2010) 267–277.
[2] J.L. Venneerstrom, W.Y. Eliss, A.J. Ager, S.L. Andersen, L. Gerena, W.K. Milhous, J.
Med. Chem. 35 (1992) 2129–2134;
A. Young, K.J. Yaun, S. Hak, J. Cardiovascular Pharmacol. Therm. 15 (2010) 167–
174.
[3] L. Roberto, C. Domenico, M. Roberta, B. Silvia, C. Alessandra, B. Cesare, Int. J.
Cardiol. 141 (2010) 68–74.
[4] C. Alvaro, G. Fabiana, D.V. Grasi, S. Simone, H. Marioh, D. Egidiol, Clin. Chim.
Acta 411 (2010) 631–637.
Conclusion
A new HPLC method was developed for the separation and
quantification of possible impurities in atorvastatin calcium bulk
drug sample. For the identification and confirmation these impuri-
ties were synthesized and characterized. The newly developed
HPLC method has also been validated as per the regulatory guide-
lines; it can be conventionally used for the quantitative determina-
tion of related substances in atorvastatin calcium API sample. The
analytical method was found to be specific, accurate and precise,
linear and robust. It can be used for the routine analysis as well
as to monitor the stability studies of the drug substance.
[5] S. Erturk, E.S. Aktas, L. Ersoy, S. Ficiciolu, J. Pharm. Biomed. Anal. 33 (2003)
1017–1023.
[6] U. Seshachalam, C.B. Kothapally, J. Liq. Chromatogr. Related Technol. 31 (2008)
714–721.
[7] V.B. Dohalsky, J. Huclova, B. Barrett, B. Nemec, I. Ulc, I. Jelinek, Anal. Bio-anal.
Chem. 386 (2006) 275–285.
[8] Chinese Pharmacopoeia, 2 (2000) 530.
[9] M. Kracun, A. Kocijan, A. Bastarda, R. Grahek, J. Plavec, D. Kocjan, J. Pharm.
Biomed. Anal. 50 (2009) 729–736.
[10] International Conference on Harmonization of Technical Requirements for
Registration of Pharmaceuticals for Human Use, Validation of analytical
procedures text and methodology, section Q2 (R1).
[11] United State Pharmacopoeia 34 (2011).
[12] V.G. Dongre, P.P. Karmuse, P.D. Ghugare, M. Gupta, B. Nerukar, C. Shaha, A.
Kumar, J. Pharm. Biomed. Anal. 43 (2007) 186–195.
Acknowledgements
The authors thank the authorities of the USIC, University of Del-
hi, for instrumental support. The Department of Science & Technol-