HPLC Methods For The Determination of Simvastatin and Atorvastatin

Trends
Trends in Analytical Chemistry, Vol. 27, No. 4, 2008
HPLC methods for the

determination of simvastatin and
atorvastatin
Lucie Novakova, Dalibor Satnsky, Petr Solich
We review high-performance liquid chromatography (HPLC) methods for the determination of two major statins used in clinical
treatment simvastatin and atorvastatin in various fields of application, including bio-analytical assays, pharmaceutical assays
and environmental applications.
Statin molecules are known to be susceptible to interconversion of the lactone and acidic forms, so it is necessary to consider
this phenomenon during method development. We highlight liquid chromatography coupled to tandem mass spectrometry (LCMS/MS) methods, as they have become a method of choice in bio-analytical and environmental applications. We compare the
methods from the point of view of sensitivity. We discuss selection of the precursor ion for performing selected reaction
monitoring (SRM) in MS detection and sample preparation.
2008 Elsevier Ltd. All rights reserved.
Keywords: Atorvastatin; Bio-analytical method; Environmental analysis; High-performance liquid chromatography; HPLC; Interconversion;
Pharmaceutical analysis; Simvastatin; Tandem mass spectrometry
Abbreviations: [13CD3], Stable-isotope labeling using 13C and three atoms of deuterium; [d5], Stable isotope labeling using five atoms of deuterium;
2-OH-AT, 2-hydroxyatorvastatin; AcAc, Acetic acid; ACN, Acetonitrile, AmAc, Ammonium acetate; AT, Atorvastatin; AT-L, Atorvastatin lactone;
DFAT, Desfluoro-atorvastatin; DSAT, Diastereomer-atorvastatin; ESI, Electrospray ionization; Et-Ac, Ethylacetate; FAc, Formic acid; FD,
Fluorescence detection; GC, Gas chromatography; HLB, Hydrophilic lipophilic balance; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A;
HPLC, High-performance liquid chromatography; IS, Internal standard; LDL, Low-density lipoprotein; LLCE, Liquid-liquid cartridge extraction; LLE,
Liquid-liquid extraction; LOD, Limit of detection; LOQ, Limit of quantitation; LOV, Lovastatin; LOVA, Lovastatin acid; M, mol/l; MeOH, Methanol;
Me- SV, Methyl-simvastatin; MEV, Mevastatin; MP, Mobile phase; MS, Mass spectrometry; MS/MS, Tandem MS; MTBE, Methyl-tert-butyl-ether;
ODS, Octadecylsilica, C18; o-OH-AT, o-hydroxyatorvastatin; p-OH-AT, p-hydroxyatorvastatin; PRA, Pravastatin; QC, Quality control; ROS,
Rosuvastatin; SDS, Sodium dodecyl-sulphate; SIM, Selected ion monitoring; SPE, Solid-phase extraction; SRM, Selective reaction monitoring; SV,
Simvastatin; SVA, Simvastatin acid; THF, Tetrahydrofuran; TIS, Turbo ionspray; ulv, ultra-low volume; UV, Ultraviolet
1. Introduction
Lucie Novakova*,
Dalibor Satnsky, Petr Solich
Department of Analytical
Chemistry, Faculty of
Pharmacy, Charles University,
Heyrovskeho 1203, 500 05,
Hradec Kralove, Czech
Republic
Corresponding author.
Tel.: +420 495067345;
Fax: +420 495067164;
E-mail: novakoval@faf.cuni.cz
352
Statins include natural (lovastatin), semisynthetic (simvastatin, and pravastatin)

and synthetic compounds (fluvastatin,
atorvastatin, cerivastatin, rosuvastatin
and pitavastatin) and are potent, specific
and competitive inhibitors of 3-hydroxy-3methlyglutaryl coenzyme A (HMG-CoA)
reductase. They are highly effective in
reducing total cholesterol and low-density
lipoprotein (LDL) cholesterol levels in the
human body.
HMG-CoA reductase is the key enzyme
that catalyzes the conversion of HMG-CoA
to mevalonate, which is an early ratelimiting step in the biosynthetic pathway
of cholesterol (Fig. 1). High-plasma LDL
cholesterol is a risk factor of cardiovascu-
lar diseases, such as atherosclerosis, which

is characterized by deposition of cholesterol on the arterial wall [13]. Atherosclerosis of the coronary and peripheral
vasculature is the leading cause of death
worldwide. Statistics show that 3842% of
deaths are related to cardiovascular diseases in Western and other developed
countries [4]. Lowering cholesterol levels
can arrest or reverse atherosclerosis in all
vascular beds and it can significantly decrease the morbidity and the mortality
associated with atherosclerosis. The main
methods of treating hyperlipidemia (or
hypercholesterolemia) are dietary and
lifestyle changes and the administration of
hypolipidemic drugs [5].
Statins are commonly used to treat
several
forms
of
hypercholesterol-
0165-9936/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2008.01.013
Trends
Figure 1. Biosynthesis of cholesterol. Cholesterol is synthesized from acetyl coenzyme A. The synthesis of mevalonate, mediated by HMG-CoA
reductase is the rate-limiting step, which regulates the cholesterol synthesis. It is the step where the statin molecule competitively effects the
synthesis of cholesterol.
emia.They have potent cholesterol-lowering effects and

they could reduce morbidity and mortality associated
with coronary heart disease significantly, as proved by
many clinical trials [2,68]. However, some statins
exhibit a number of adverse effects, such as myopathy or
rhabdomyolysis, so it is useful to monitor the levels of
statins in biological materials in order to establish an
appropriate dosage scheme, which would minimize
adverse effects and keep the cholesterol-lowering effect
[2].
Simvastatin and atorvastatin are the two most commonly occurring drugs in commercially available pharmaceutical formulations used for the clinical treatment of
hypercholesterolemia. Their structures can be seen in
Fig. 2.
2. Chemistry and pharmacokinetics

The first statin molecule mevastatin was discovered
by Endo et al. in 1976 as a fungal product extracted
from Penicillium citrinum [9]. Lovastatin, simvastatin and
pravastatin are also derivatives of fungal products.
Simvastatin and pravastatin are nowadays produced
semi-synthetically from lovastatin and mevastatin.
However, fluvastatin is a completely synthetic statin
with a very different structure from statins derived from
fungal products. It is a mevalonolactone derivative with
a fluorophenyl-substituted indole ring. Other synthetic
statins (cerivastatin, atorvastatin, rosuvastatin and
pitavastatin) have similar structures with fluorophenyl
groups. All of totally synthetic statins have open-ring
http://www.elsevier.com/locate/trac
353
Trends

OH
O
CH3
H3C
H3C
CH3
H3C
Simvastatin (lactone form)
HO
COOH
OH
CH3
F
N
CH3
H
N
O
Atorvastatin (acid form)
Figure 2. Chemical structures of simvastatin and atorvastatin.
acid forms [2,9]. All statins are absorbed rapidly following administration, reaching peak plasma concentration within 4 hours.
Statins exist in two forms, lactone and open-ring
hydroxy acid [10,11]. In vivo, the hydroxy acid forms
are the active drugs that lower plasma cholesterol while
the lactone forms are inactive (prodrug). The lactone
form of statin can be absorbed from the gastrointestinal
tract and transformed into the active drugs in liver and
non-hepatic tissues [11]. Depending upon chemical
structure, statins have different affinities for HMG-CoA
reductase, which determines their pharmacological
effects and different pharmacokinetic properties (e.g.,
tissue distribution, metabolic stability, enzymes and
transporters involved in their metabolism) [2]. We will
provide further information on simvastatin and atorvastatin, the most widely used statins in clinical treatment Fig. 2.
Simvastatin is a prodrug, which is administered as
an inactive lactone form. The lactone is absorbed from
gastrointestinal tract and hydrolyzed to the active
b-hydroxy acid form in the liver. Simvastatin and its
hydroxy acid are extensively (95%) bound to plasma
proteins. The substance undergoes extensive first-pass
metabolism in the liver and is mainly excreted in the bile.
About 85% of administered dose has been recovered
from the feces as metabolite and about 1015% from
urine, mainly as inactive forms [12,13].
Atorvastatin is administered in the open-ring hydroxy
acid form the active form. It is absorbed from the
354
gastrointestinal tract and it undergoes extensive firstpass metabolism in the liver. Liver metabolism produces
two active hydroxy metabolites ortho-hydroxyatorvastatin and para-hydroxyatorvastatin and three
inactive metabolites corresponding to the lactone form.
More than 98% is bound to plasma proteins. It is
excreted mainly in feces via the bile, with a smaller
proportion excreted in the urine. About 70% of the total
plasma HMG-CoA activity is attributed to active metabolites of atorvastatin, even if their concentrations are
very low [1214].
As apparent from the information above, the levels
of statins in biological fluids are very low, probably
because only about 5% of dosed statin reaches the
systemic circulation. For atorvastatin and simvastatin,
the plasma concentrations are typically at ng/ml levels.
The active metabolites of atorvastatin are present at
plasma concentration corresponding to pg/ml levels
[13], the typical concentration range being 0.120 ng/
ml.
3. Interconversion of lactone and hydroxy acid

forms
A number of classes of drugs could potentially undergo
interconversion, which may occur during any of the
numerous steps of the bio-analytical method:
in the biological matrix before collecting aliquots of
samples for the analysis;
during extraction;
during evaporation to dryness or reconstitution;
in the solution in the injection vial; and,
in the case of MS, in the ion source as well.
There are several categories of drugs that could undergo interconversion. HMG-CoA reductase inhibitors
are typical examples of such a class, where the interconversion occurs between lactone and open-ring hydroxy acid [10,11]. Another category could be
conversion between samples that contain a carboxylic
acid and its acylglucuronide [15]. Samples that contain
a thiol group and its disulfide may also interconvert to
each other [16]. Minimizing the interconversion will
depend on the conditions during the bio-analytical procedure, pH being one of the most important.
HMG-CoA reductase inhibitor simvastatin is administered in its lactone form, although the active form is
open-ring hydroxy acid. Both forms are found in postdose samples.
On the other hand, atorvastatin is administered as
the open-ring hydroxy acid, but the post-dose samples contain both acid and lactonized forms
[10,11,17,18].
For the samples of hydroxy acid chemical structure
and the corresponding lactone forms, it is important to
maintain pH between 4 and 5 in order to minimize
interconversion. Increasing the pH above 6 facilitates the

conversion of lactone to acid (in the ionized form); by
contrast, lowering pH facilitates the conversion of acid to
lactone or lactone to acid (in the non-ionized form). Most
assays utilize pH around 4.5 (see Tables 1 and 2).
It is important to optimize the method in order to
minimize such interconversion. Unfortunately, even
optimal conditions may not totally prevent interconversion. It is thus essential to design the composition of
the calibration standards and quality control (QC) samples in terms of the ratio of the concentration of one
analyte to that of the other so that the accuracy and
precision obtained for QC samples realistically reflect
accuracy and precision that will be obtained for the expected samples. Ideally, the composition of the QC samples should be identical to that of real samples. This is
difficult to establish at the early stage of assay, where the
composition of real samples is not known and will
probably change from sample to sample [10].
It is recommended that the example of methoddevelopment design should include QC samples prepared
in real biological material containing only the lactone
form, only the hydroxy acid form, and both in varying
ratios of lactone to hydroxy acid (1:1; 1:10; 10:1; 1:3;
and, 3:1) in order to get good method accuracy and
precision. Such QC samples should be prepared at various concentration levels inside the range of calibration
curve [10]. In this study, the influence of pH (analytical
conditions at pH 4.2, 7.3 and 1.8) on interconversion of
analytes was described in detail. Under the conditions
that showed practically no interconversion, the results
for all QC samples gave excellent values for accuracy (up
to 8.6% RSD) and precision (up to 8.7% RSD) with no
influence of the ratio of both analytes.
However, in another experimental design, the results
for accuracy and precision were strongly influenced by
this ratio, except for 1:1, which was the same ratio as in
the calibration standards. Thus, under the conditions
that allow interconversion between analytes, a method
validated for the quantitation of the two analytes using
calibration standards with the ratio of analyte concentration of 1:1 and QC samples with the same 1:1 ratio
can be used for accurate measurements of the analytes
in real samples only if such samples also contain the two
analytes in the ratio 1:1. Therefore, to obtain an accurate indication of the performance of the method for all
real samples, it is necessary to use QC samples that cover
the entire spectrum of composition of real samples.
To summarize, in developing a method for the quantitation of two analytes that can undergo interconversion, the first step is to select the conditions that will
eliminate or minimize interconversion. The second step
is judicious selection of the composition of the QC
samples and the composition of calibration standards,
which should cover the spectrum of the composition of
real samples [10].
Trends
4. Analytical methods
The determination of drugs is a multi-disciplinary task.
During the manufacturing process of a drug substance
and drug formulation, there is a need for QC analytical
methods to include all known and unknown impurities.
Bio-analytical methods are necessary for clinical trials,
therapeutic drug monitoring and individual dosagescheme adjustment. Recently, there has also been great
interest in monitoring pharmaceutical residues in the
environment, so environmentally-focused analytical
methods are also necessary.
Determination of drugs in biological materials is an
important step in drug discovery and drug development, as it provides pharmacokinetic information, and
is necessary when the treatment-dose schedule and
safety margins need to be established. High-performance liquid chromatography (HPLC) together with
various types of detection UV (ultraviolet), FD (fluorescence detection) and MS has become the method of
choice for bio-analytical method development. Gas
chromatography (GC) is somewhat suppressed because
of the need for a derivatization step prior to analysis in
order to obtain volatile derivatives of the drug molecule,
which is often not volatile in the case of pharmaceuticals.
As expected from the different structures of simvastatin and atorvastatin, analytical methods for their quantitative determination were developed individually.
Because of the structural properties, there are not many
analytical methods that determine these two compounds
together in one analytical run or even in combination
with other statin molecules. This is also probably
because statins are not used with other statins simultaneously during treatment of hyperlipidemic patients.
There are only two works that describe the determination of a number of statin structures together in one
analytical run:
an environmental application where simvastatin,
atorvastatin, lovastatin and pravastatin (internal
standard (IS) = mevastatin) were determined in
aqueous samples [19]; and,
a pharmaceutical application where atorvastatin,
lovastatin, pravastatin, rosuvastatin and simvastatin
were determined together for QC of pharmaceutical
formulations [20]. Theophylline was employed as IS
for quantitation. The analytical run took about 40
min.
There have been two reviews on analytical methods
for the determination of HMG-CoA reductase inhibitors.
The first, published in 2003, referred to lovastatin,
simvastatin, pravastatin, fluvastatin and atorvastatin
[12]. HPLC and GC methods were discussed. Generally,
fluorescence and UV detection were applied together
with HPLC. Only two methods for the determination of
atorvastatin were available at that time. The second
355
Trends
356
Table 1. HPLC analytical methods for the determination of simvastatin

Matrix sample
preparation
Stationary phase
analytical column
Mobile phase
Detection
Precursor ions notes
Time [min]
Validation data
LOQ/LOD
Ref.
SV, SVA
LOV, LOVA
PRA
Pu-Ehr tea SPE
Luna C18
(4.6 250 mm, 5 lm)
ACN, water, AcAc (70:30:0.5)
ESI+
MS-MS
SRM
Not stated
15
Not given stability

and interconversion study
[11]
SV, AT
LOV
PRA
IS = MEV
Aqueous samples
SPE
Genesis C18
(2.1 50 mm, 3 lm)
Gradient elution A:
ACN + 2 mM MeA + 0.1%
AcAc B: water + 2 mM
MeA + 0.1% AcAc
ESI+
MS-MS
SRM
[M + CH3NH3]+
[M + H]+
[M + CH3NH3]+
[M + CH3NH3]+
5.5
LOD = 0.11.0 ng/l

LOD = 0.11.2 ng/l
LOD = 0.11.2 ng/l
LOD = 1.015.4 ng/l
[19]
SV
AT, LOV
PRA, ROS
IS = theophylline
Pharmaceutical
formulation
metabolism study
in vitro
extraction MeOH
Interstil ODS 3V
(4.6 250 mm, 5 lm)
0.01 M AmAc pH 5.0

ACN
MeOH
UV 237 nm
40
LOQ = 0.1 lg/ml
[20]
SV, SVA
IS = LOV, LOVA
Human plasma
SPE
Capcell Pak C18

(4.6 150 mm, 5 lm)
ACN-water (80:20)
FD
360 nm
430 nm
Derivatization by
1-bromoacetylpyrene
35
LOQ = 0.1 ng/ml
[21]
SV, SVA
No IS
Human plasma
LLE
ODS Hypersil
(4.6 250 mm, 5 lm)
0.025 M sodium
dihydrogenphosphate pH4.5
ACN (35:65)
UV
238 nm
10
LOD = 15 ng/ml
[22]
SV
IS = LOV
Human plasma
LLE
Capcell Pak C18 UG

120U (1.5 250
mm, 5 lm)
ACN 20 mM potassium
phosphate
buffer pH 5.6 (65:35)
30
LOQ = 0.5 ng/l
[23]
SV and impurities
(SVA, LOV
Me-SV, dimmer
acetate ester
anhydro-SV
IS = propyphenazon
Tablets extraction MP
X-Terra (4.6
50 mm, 3.5 lm)
Microemulsion: 0.9%
diisopropylether
1.7% SDS 7.0%
co-surfactant 90% 25
mM di-sodium
phosphate pH 7.0
25
LOD = 5 ng/ml
LOQ = 10 ng/ml
(SV and SVA)
[24]
UV
238 nm
UV
238 nm
Determined
substances
Table 1 (continued)
Matrix sample
preparation
Stationary phase
analytical column
Mobile phase
Detection
Precursor
ions notes
Time
[min]
Validation
data LOQ /LOD
Ref.
SV, LOV
Standard solutions
No LC
No LC
ESI+MSMS SRM
[M+H]+its
fragmentations
using MS
Not stated
fragmentation study
[25]
SV, SVA
Standard solutions
C18 (3.9 50 mm,

5 lm)
ACN, 3.0 mM FAc (70:25)
ESI+
MS-MS
SRM
[M + H]+
[M + NH4]+
2.5
Chromatographic and
mass resolution study
[26]
SV + impurities
No IS
SV substance tablets
Extraction ACN/FAc
Zorbax C8
(4.6 150 mm,
3.5 lm)
Gradient elution
A: 0.085 phosphoric acid
B:ACN
A: 0.001% FAc in water
B: 0.001% FAc in CAN
DAD
ESI+
MS-MS
45
Identification of
impurities
[27]
SV, SVA
IS = LOV, LOVA
Human plasma
SPE
Discovery C18
(4.6 50 mm, 5 lm)
ACN, MeOH, AmAc 0.1 M

(62:10:28)
ESI+/
MS-MS
SRM
[M + CH3CN + Na]+
[M H]
4.5
LOQ = 0.03 ng/ml SV

LOQ = 0.02 ng/ml SVA
[28]
SV, SVA
IS = LOV, LOVA
Human
plasma on-line
SPE
C18 (3.9 50 mm,

5 lm)
ACN, 3.0 mM FAc (70:25)
ESI+
MS-MS
SRM
[M + H]+
2.5
LOQ = 0.5 ng/ml SV
[29]
SV
IS = LOV
Human plasma LLE
Shim-pack ODS
(4.6 150 mm,
5 lm)
MeOHwater (9:1)
ESI+
MS
SV
[M + Na]+
LOD = 0.05 ng/ml
[30]
SV, SVA
IS = deuterium labeled
Eects of
mobile phase
on ionization/
fragmentation
plasma
LLCE
Kromasil C18
(2.0 50 mm, 4 lm)
ACN-buffer 2 mM pH 4.5
(70:30) buffers: AmAc,
hydrazine acetate, alkylammonium acetates
ESI+
MS-MS
SRM
[M H] SVA various SV: [M + H]+

[M + Na]+ [M + K]+
[M + NH4]+
[M + RNH3]+
R = alkyl
3.5
LOQ = 50 pg/ml
[31]
SV, SVA IS = stable

isotope labeled
Human plasma
LLCE
Kromasil C18
(2.0 50 mm, 5 lm)
ACN AmAc pH 4.5 by FAc

(75:25)
TIS
+
MS-MS
SRM
[M + H] SV
[M H] + SVA
3.5
LOQ = 50 pg/ml
[32]
Determined
substances
(continued on next page)
Trends
357
358
[34]
LOQ = 0.05 ng/ml
Kromasil C18
(2.0 50 mm, 5 lm)
Human plasma LLE
SV, SVA
IS = stable isotope
labeled
ACN 1 mM methyl ammonium

acetate pH 4.5 (75:25)
TIS
+
MS-MS
SRM
[M + H] SV
[M H] + SVA
3.5
[33]
LOQ = 0.05 ng/ml
3.0
[M H] SVA
[M + CH3NH3]+ SV
TIS+
MS-MS SRM
Synergie Max RP
(2.0 50 mm, 4 lm)
Human plasma ulv-SPE
ACN 1 mM methyl ammonium

acetate pH 4.5 (80:20)
Precursor
ions notes
SV, SVA IS = deuterium

labeled
Table 1 (continued)
Stationary phase
analytical column
Matrix sample
preparation
Determined
substances
Mobile phase
Detection
Time
[min]
Ref.
Validation
data LOQ /LOD
Trends
work [13] referred to some MS bio-analytical methods

for the determination of statins.
Fig. 3 shows the trend in developing new methods for
the determination of simvastatin and atorvastatin. Simvastatin has been monitored since the early 1990s,
while, due to its later synthesis, atorvastatin has been
monitored since 1999. Since 2003, the number of newly
developed methods has been increasing for both analytes.
4.1. Simvastatin
LC-MS/MS (LC coupled to tandem MS) is nowadays the
method of choice for the determination of simvastatin
and its hydroxy acid (Table 1). While, in 2003, there
was the same number of GC methods (3), HPLC-UV or
FD (3) and LC-MS (3) [12], newly developed methods
employed MS/MS. Only simvastatin acid (open-acid
form, which arises from interconversion during analytical procedure or in the human body as an active form of
a prodrug) was monitored together with simvastatin; no
other metabolites were described.
Whereas the HPLC-FD method [21] using 1-bromoacetylpyrene for the derivatization could be very
convenient for bio-analytical purposes with its LOQ of
0.1 ng/ml, the HPLC-UV assay has borderline sensitivity
for the quantitation of simvastatin in biological materials
because of the poor UV-absorption properties of simvastatin molecule, giving an LOD of 15 ng/ml [22]. A more
sensitive HPLC-UV assay for the determination of simvastatin in human plasma was developed later with an
LOQ of 0.5 ng/ml [23]. However, lower sensitivity is no
problem for quantitation in pharmaceutical preparations. Such a method was developed by Malenovic et al.
[24] utilizing a microemulsion mobile phase for quantitation of simvastatin and its six impurities (simvastatin
acid, lovastatin, methyl-simvastatin, dimmer of simvastatin, acetate ester of simvastatin and anhydro-simvastatin) in tablets or the one previously cited [20].
HPLC-UV and FD analytical methods utilized C18
analytical columns for the retention/separation of simvastatin/simvastatin acid together with mobile phase
containing acetonitrile in combination with phosphate
buffer (to maintain pH around 45) or with water in an
older method [21]. X-terra hybrid stationary phase was
employed in one case together with application of a
microemulsion mobile phase [24]. Isocratic elution was
applied in most analyses. Detection was performed at
238 nm in all cases [2224]. A fluorescence method
utilized 360 nm and 430 nm [21]. Analytical run time
was usually very long, starting from 10 min [22] going
up to 35 min using FD [21]. Two bio-analytical methods
also determined simvastatin acid next to the lactone
form [21,22], as did a pharmaceutical QC method,
where simvastatin acid was determined among six
impurities using IS propyphenazon [24]. Lovastatin was
often employed as an IS because of its unique structural
Determined substances
Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor
ions (notes)
Time
[min]
Validation data
LOQ /LOD
Ref.
AT, SV
LOV, PRA
IS = MEV
Aqueous samples SPE
Genesis C18 (2.1

50 mm, 3 lm)
Gradient elution
A: ACN + 2 mM
MeA + 0.1% AcAc
B: water + 2 mM
MeA + 0.1% AcAc
ESI
+
MS-MS
SRM
[M + CH3NH3]+
[M + H]+
[M + CH3NH3]+
[M + CH3NH3]+
5.5
LOD = 0.11.0 ng/l

LOD = 0.11.2 ng/l
LOD = 0.11.2 ng/l
LOD = 1.015.4 ng/l
[19]
AT, LOV
PRA
ROS, SV
IS = theophylline
Pharmaceutical
formulation
metabolism study
in vitro extraction:
ACN:buffer
Interstil ODS 3V
(4.6 250 mm, 5 lm)
0.01 M AmAc pH 5.0

ACN
MeOH
UV
237 nm
40
LOQ = 0.1 lg/ml
[20]
AT impurities = DSAT,
DFAT, no IS
Bulk drug tablets

extraction MeOH
C18 Luna
(4.6 250 mm, 5 lm)
Gradient elution
ACN-ammonium acetate
buffer pH 4.0 THF
UV
248 nm
32
LOQ = 0.13 lg/ml

LOD = 0.013 lg/ml
[36]
AT, amolodipine
+ impurities
no IS
Tablets
MeOH extraction
Perfectsil Target ODS-3

(4.6 250 mm, 5 lm)
ACN 0.025 M NaH2HPO4

buffer pH 4.5 (55:45)
UV
237 nm
18
LOD = 0.35 lg/ml

LOQ = 2 lg/ml
[37]
AT amlodipine
No IS
Drug products
MeOH extraction
Lichrosphere 100 C18

(4.6 250 mm, 5 lm)
50 mM potassium dihydrogen
phosphate buffer pH 3.0
ACN (40:60)
UV
254 nm
LOD = 0.4 lg /ml
[38]
AT
IS = DCF
Human serum
LLE
Shim-pack CLC-ODS
(4.6 150 mm, 5 lm)
0.05 M sodium phosphate

buffer, pH 4.0 by
phosphoric acid
MeOH (33:67)
UV
247 nm
LOQ 4 ng/ml
LOD = 1 ng/ml
[39]
AT
IS = ibuprofen
Bulk drug tablets

human plasma
MeOH extraction
RP Supelcosil C18
(4.6 150 mm, 5 lm)
ACN:MeOH:water
(45:45:10)
UV
240 nm
LOD = 0.008 lg /ml

LOQ = 0. 18 lg/ml
[40]
Table 2. HPLC analytical methods for the determination of atorvastatin
(continued on next page)
Trends
359
Trends
360
Table 2 (continued)
Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor
ions (notes)
Time
[min]
Validation data
LOQ /LOD
Ref.
AT novobicin
roxytromycin
Aqueous samples
SPE
YMC ODS-AQ (1.0 100 mm,

3 lm)
Gradient elution
A: ACN
B: 10 mM AmAc
ESI+
MS-MS
SRM
[M + H]+
5.0
ILOQ = 1 pg
[41]
AT, ATA
2-OH-AT/L
4-OH-AT/L
Human serum LLE
YMC Basic (2.0 50 mm, 5 lm)
Gradient elution
A: water, MeOH, FAc 88%
B: ACN, MeOH, FAc 88%
(950 ml:50 ml:43 ll)
ESI+
MS-MS
SRM
[M + H]+
3.5
LOQ = 0.5 ng/ml
[42]
AT o-OH-AT, p-OH-AT
Human, dog and rat

plasma LLE
YMC JSphere H 80 (C18)

(3.0 150 mm, 4 lm)
ACN, AcAc 0.1% (70:30)
ESI+
MS-MS
SRM
[M + H]+
LOQ = 0.250 ng/ml
[43]
AT, ATA
p-OH AT/L
o-OH AT/L
IS = metachalon
Human plasma SPE
Omnisphere C18
(2.0 30 mm, 3 lm)
Gradient elution
A: ACN, water, FAc 1mM
(30:70)
B: ACN, water, FAc 1mM
(60:40)
ESI+
MS-MS
SRM
[M + H]+
21
LOD = 0.06 ng/ml

LOD = 0.16 ng/ml o-AT
[18]
AT
p-OH AT
o-OH AT
IS = ROS
Human plasma LLE
Waters Symmetry C18

(4.6 100 mm, 5 lm)
0.03%
FAc ACN (30:70)
TSI+
MS-MS
SRM
[M + H]+
2.5
LOQ = 100 pg/ml
[44]
AT
2-OH-AT
IS = clindamycin
Human plasma LLE
Atlantis dC18 (4.0 100 mm,

3 lm)
ACN, AcAc (70:30)
ESI+
MS-MS
SRM
[M + H]+
4.0
LOD = 0.02 ng/ml

LOQ = 0.1 ng/ml
[45]
AT, AT-L
p-OH AT/L
o-OH AT/L
Human plasma SPE
4 analytical columns in parallel

Genesis C18 (2.2 50 mm, 4 lm)
Gradient elution
A: 0.1% AcAc in water
B: ACN
ESI+
MS-MS
SRM
[M + H]+
1.65
[46]
Determined Substances
Trends
Analytical methods for simvastatin and atorvastatin
4
simvastatin
atorvastatin
0
1992
1997
1999
2000
2001
2002
2003
2004
2005
2006
2007
Figure 3. Analytical methods for the determination of simvastatin and atorvastatin first analytical methods for the determination of simvastatin
were published in the early 1990s, and the first for atorvastatin in 1999. The increasing trend for new methods to be developed for both statins
can be observed since 2003.
similarity with the molecule of simvastatin (Fig. 4). The

difference in the structure is only one methyl group, so
such a molecule could also be used as convenient IS in
MS detection, since it will have not just the same
retention and extraction properties, but also similar
ionization in the ion source.
Since 2000, there has been a growing number of
newly developed methods for the determination of simvastatin, where most of methods employed MS/MS
detection (Fig. 3). In the case of simvastatin, there were
not only methods for its quantitation also other methods
that studied detailed fragmentation [25], chromatographic and mass resolution necessary for analysis of
simvastatin and simvastatin acid [26], identification of
unknown impurities in simvastatin substance and tablets [27], and the influence of various media (used for
simvastatin standard solutions) for the interconversion
between simvastatin and simvastatin acid [11].
Quantitative LC-MS methods for simvastatin included,
above all, bio-analytical assays [2834] determining
simvastatin in human plasma, one pharmaceutical QC
application [27] and one environmental application
[19]. They employed MS/MS using a specific SRM (selected reaction monitoring) experiment except for one
work, where a single quadrupole was utilized for quantitation enabling only an SIM (selected ion monitoring)
experiment for quantitation [30].
Simvastatin in its lactone form was always determined
in electrospray positive ion mode (ESI+) or in positive
turbo ionspray (TIS+) respectively, while its hydroxy acid
form was determined in electrospray negative ion mode
(ESI ), negative turbo ionspray (TIS ) and ESI+ or TIS+.

The selection of precursor ion for the determination of
simvastatin lactone was not remotely unequivocal see
Tables 1 and 3. Simvastatin forms various adducts
influenced by mobile-phase composition, and, unfortunately, such adducts sometimes give higher intensity
than [M+H]+, which would be an ideal precursor ion for
SRM transition and was utilized in some studies
[26,29,32,34]. However, other authors found that simvastatin was not sensitive enough and they observed
[M+Na]+, [M+K]+, [M+NH4]+ in the spectra of simvastatin next to the [M+H]+, especially [M+Na]+ and
[M+NH4]+, which gave very high intensities [30,31].
They were therefore used as precursor ions for quantitation in some works [28,30].
Favorable experimental conditions could achieve high
sensitivity for [M+Na]+ over major molecular ions, as
published in earlier LC-MS/MS studies. Nevertheless, the
sodium content was found not to be totally controllable
during sample analysis. It caused variations in the
abundance of the sodium-adduct ion. Sources of sodium
(potassium respectively) are believed to originate from
the biological matrix or glass containers used during
sample preparation and analysis. Sodium acetate could
be added to the mobile phase as the way of maintaining
better consistency in the abundance of the sodium-adduct ion. Unfortunately, the non-volatile nature of the
sodium buffer makes it preferable not to use it during LCMS/MS assays. The factors affecting the relative abundance of [M+Na]+ and [M+H]+ include not only general
experimental conditions, such as mobile-phase-buffer
361
Trends
OH
H3C
H3C
R=
Lovastatin
R
O
O
O
CH3
CH3
H3C
H3C
R=
H3C
Simvastatin
O
CH3
H3C
R=
D313C
[13CD3] Simvastatin
Figure 4. Similarity of simvastatin and lovastatin structures, and stable-isotope labeling of simvastatin.
content and pH, solvents and mass spectrometer

parameters, but also the materials of the sample and
solution containers. Generally, to reduce sodium content
to favor the formation of [M+H]+ ions, containers made
of glass and solutions or solvents containing an alcohol,
such as methanol, should be avoided throughout sample
preparation and analysis [31,35].
There were some attempts to favor the occurrence of
[M+NH4]+ by addition of ammonium ions into the
mobile phase [31]; however in this extensive study
methylammonium adduct [M+CH3NH3]+ was found to
be more convenient than other alkylammonium and
ammonium adducts tested. It was therefore chosen as a
precursor ion in this study and in one other work [33].
Monitored SRM transitions differed among the analytical
methods, and the details can be seen in Table 3.
The choice of precursor ion is important as well as
chromatographic and mass resolution [26]. In the case
of simvastatin, where mass unit 18 corresponds the
difference between lactone and hydroxy acid form and it
is, by coincidence, different by only one mass unit from
the 17 mass unit, which corresponds to the difference
between [M + H]+ and [M + NH4]+ ions of the lactone or
the hydroxy acid form arising in positive ionization
mode. The specificity of selected SRM must be verified or
chromatographic resolution of the acid and lactone form
is required [26].
Most LC-MS quantitative methods determined simvastatin hydroxy acid together with simvastatin, except for
one method, where only simvastatin was determined
[30]. Simvastatin and its hydroxy acid were separated on
C18 analytical column in all cases. Acetonitrile was
usually present in mobile phase at more than 70%.
Volatile additives including ammonium acetate, formic
362
acid and methyl-ammonium acetate were added in order

to enhance ionization and to get better sensitivity of the
method. Ammonium acetate buffer often had pH of 4.5
in order to minimize the interconversion between simvastatin and simvastatin hydroxy acid, because it is
much stronger than in the case of atorvastatin. Analytical runs were very short, ranging from 2.5 min up to
5 min. Isocratic elution was applied in most cases.
The best ISs for precise and accurate quantitation in
MS are stable-isotope-labeled standards. In the case of
simvastatin labeling usually occurs at one of the methyl
group, [13CD3]-simvastatin is obtained [31,32], see
Fig. 4. Four works employed deuterium-labeled ISs [31
34]. All other works utilized lovastatin and lovastatin
acid ISs, as its high stated above, because of its high
similarity with the structure of simvastatin Fig. 4.
Method sensitivity expressed as LOQ was typically about
0.05 ng/ml in almost all methods.
4.2. Atorvastatin
Analytical methods, even those newly developed, for
the determination of atorvastatin and its five possible
metabolites, including the lactone form of atorvastatin,
p-hydroxyatorvastatin and o-hydroxyatorvastatin and
their lactone forms (overview in Table 2), still employ
HPLC-UV detection in spite of its lower sensitivity.
However, such methods could be successfully applied
for the analysis of atorvastatin in drug substances or
drug formulations together with impurities, as reported by Ertuk et al. [36], Mohammadi et al. [37]
(where amlodipine was also determined in combined
tablet formulation), as it was in the work of Chaudhari et al. [38] but without the determination of
impurities.
Trends
Table 3. Specific SRM transition for the determination of simvastatin, its various precursor ions, simvastatin hydroxy acid and their stable-isotope
labeled forms
Compound determined
Simvastatin
Molecular weight
Precursor ion
+
418.27
[M + H]
[M + Na]+
[M + CH3NH3]+
[M + CH3CN + Na]+
[13CD3]-simvastatin
[M + H]+
422.27
[M + CH3NH3]+
Simvastatin acid
436.27
[M H]
[13CD3]-simvastatin acid
440.27
[M + H]+
[M H]
Two HPLC-UV assays for the determination of atorvastatin in biological materials have been published.
Determination of atorvastatin in human serum using
diclofenac as the IS was published by Bahrami et al. [39]
with an LOQ of 4 ng/ml. Altuntas et al. developed a
method for the determination of atorvastatin in human
serum, bulk drug and tablets using ibuprofen as the IS
with an LOQ of 18 ng/ml [40].
HPLC-UV methods generally utilize the C18 stationary
phase. In most cases, a combination of acetonitrile and
buffer (ammonium acetate or phosphate) with the aim of
keeping pH between 4 and 5. Both isocratic and gradient
elution has been utilized. Detection has usually been
performed at 237 nm [20,37], 247 nm [36,39] or 254
nm [38]. Analytical run times have been very variable,
the shortest about 34 min [39,41], the longest about
30 min [36]. None of HPLC-UV methods also determined
the active metabolites of atorvastatin or its lactone form.
Specific SRM transition (SIM)
Ref.
419.1 285.1
419.1 199.1
441 (SIM)
450.3 285.1
481.2 440.9
[29,32]
[34]
[30]
[31,33]
[28]
423.2 285.1
423.2 199.1
454.3 285.1
[32]
[34]
[31,33]
435.3 114.0
435.3 319.1
437.3 303
439.2 319.1
[28]
[3134]
[29]
[3134]
Fluorescence detection has not been employed in the

determination of atorvastatin, probably due to the difficulties with the derivatization step and the repeatability
of the procedure.
Methods about 10100 times more sensitive have
been developed using MS/MS detection, most of which
were for bio-analytical applications, determining atorvastatin in human plasma. One method referred to the
determination of atorvastatin together with other drugs
(novobiocin and roxytromycin in aqueous samples [41]
for the environmental applications).
Bio-analytical methods [18,4246] have, in all cases,
employed MS/MS using specific SRM conditions with
electrospray ionization in positive-ion mode (ESI+). This
may be surprising, taking into account the carboxylic
acid group present in the structure of atorvastatin.
However, several works confirmed that no higher
sensitivity was obtained with electrospray ionization in
Table 4. Specific SRM transition for the determination of atorvastatin, its metabolites and their stable-isotope-labeled forms
Compound determined
Molecular weight
Precursor ion
+
Specific SRM transition
Ref.
559.2 440.2
564.2 440.2
564.2 445.2
541.2 448.2
546.2 448.2
546.2 453.2
[18,4246]
[42]
[43,46]
[18,42,46]
[42]
[46]
Atorvastatin
[d5]-atorvastatin
558.25
563.25
[M + H]
[M + H]+
Atorvastatin lactone
[d5]-atorvastatin lactone
540.25
545.25
[M + H]+
[M + H]+
p-hydroxy atorvastatin
[d5]-p-hydroxyatorvastatin
p-hydroxyatorvastatin lactone
[d5]-p-hydroxyatorvastatin lactone
574.25
579.25
556.25
561.25
[M + H]+
[M + H]+
[M + H]+
[M + H]+
575.2 440.2
580.2 445.2
557.2 448.2
562.2 453.2
[18,4246]
[42,43,46]
[18,42,46]
[42,46]
o-hydroxyatorvastatin
574.25
[M + H]+
[d5]-o-hydroxyatorvastatin
o-hydroxyatorvastatin lactone
[d5]-o-hydroxyatorvastatin lactone
579.25
556.25
561.25
[M + H]+
[M + H]+
[M + H]+
575.2 440.2
575.2 466.2
580.2 445.2
557.2 448.2
562.2 453.2
[4246]
[18,44]
[42,43,46]
[18,42,46]
[42,46]
363
Trends
364
Table 5. Extraction procedures utilized for sample preparation during simvastatin analysis
Matrix
Extraction
procedure
Stationary phase
LLCE cartridge
SPE eluent/LLE
extraction agent
Stabilization/
pre-extraction
treatment
Interconversion
data
Validation data:
recovery
Ref.
SV, SVA
LOV, LOVA, PRA
Pu-Ehr tea
SPE
1. Extraction in water at 100C

2. Supelco DSC C18
90% MeOH
None
Given at various
conditions
recSV = 95.098.9%
[11]
SV, AT
LOV, PRA
Aqueous samples
SPE
1. Filtration
2. Oasis HLB
MeOH
pH 4.5 by 3.0 M H2SO4
1%
recSV = 6984%
[19]
SV, SVA
Human plasma
SPE
C8
1. MeOH, water
6:4 2. ACN
None
Not significant
recSV = 40%
recSVA = 40%
[21]
SV, SVA
Human plasma
LLE
ACN:water
(60:40) ACN
None
Not given
recSV = 95.296.3%
recSVA = 92.895.1%
[22]
SV
Human plasma
LLE
diethylether
10 M HCl
Not given
recSV = 86.990.7%
[23]
SV, SVA
Human plasma
SPE
Oasis HLB
ACN 0.1 M AmAc

pH 4.5 (75:25)
None
Not given
recSV = 88.8%
recSVA = 85.6%
[28]
SV, SVA
Human plasma
on-line SPE
Oasis HLB
ACN 3.0 mM
FAc (10:90)
0.1 M sodium acetate pH 4.2
<1%
recSV P 75%
recSVA P 38%
[29]
SV
Human plasma
LLE
Et-Ac
None
Not given
rec = 101.4%
[30]
SV, SVA
Plasma
LLCE
ChemElut cartridge
MTBE
100 mM AmAc pH 4.5
<0.07%
recSV = 78%
recSVA = 87%
[31]
SV, SVA
Human plasma
LLCE
ChemElut cartridge
MTBE
100 mM AmAc pH 4.5
<0.07%
recSV = 66%
recSVA = 73%
[32]
SV, SVA
Human plasma
ulv-SPE
Oasis HLB l-elution

96-well SPE plate
ACN:water (95:5)
100 mM AmAc pH 4.5
<0.08%
recSV = 66%
recSVA = 73%
[33]
SV, SVA
Human plasma
LLE
96-well LLE plate
MTBE
100 mM AmAc pH 5.0
<0.06%
lactonization
<0.07%
hydrolysis
recSV = 87.1%
recSVA = 72.7%
[34]
Substances
isolated
Substances isolated
Matrix
Extraction
procedure
Stationary phase
SPE eluent LLE extraction agent
Stabilization /preextraction treatment
Interconversion
data
Validation data:
recovery
Ref.
AT, SV
LOV, PRA
Aqueous samples
SPE
1. Filtration - Soxhlet
2. Oasis HLB
MeOH
pH 4.5 by 3.0 M H2SO4
10%
recAT = 6486%
[19]
AT
Human serum
LLE
Et-Ac
0.1 M phosphate
buffer pH 7.0
Not given
recAT = 95%
[39]
AT novobiocin
roxytromycin
Aqueous samples
SPE
1. Filtration - Soxhlet
2. HLB
MeOH
pH 4.0 by
3.5 M H2SO4
Not given
recAT = 70.572.2%
[41]
AT, ATA
2-OH-AT/L
4-OH-AT/L
Human serum
LLE
MTBE
0.1 M sodium
acetate buffer pH 5.0
Given at various
conditions
rec = 60100%
all analytes
[42]
AT o-OH-AT,
p-OH-AT
Human, dog
and rat plasma
LLE
Diethyl ether
0.1 M NaOH
Given at various
conditions
recAT = 100107%
[43]
AT, ATA
p-OH AT/L
o-OH AT/L
Human plasma
SPE
Varian C18
ACN 0.1 M AmAc (95:5)
1 M sodium
formate pH 3
<4%
rec = 5378%
all analytes
[18]
AT
p-OH AT
o-OH AT
Human plasma
LLE
Diethyl ether
dichloromethane (7:3)
10% o-phosphoric acid
Not given
recAT = 54.2%
[44]
AT
2-OH-AT
Human plasma
LLE
Diethyl ether
dichloromethane (60:40)
0.01 M sodium
acetate pH 6.0
Not given
recAT = 89.692.5%
[45]
AT, AT-L
p-OH AT/L
o-OH AT/L
Human plasma
SPE
Isolute C-18
95% MeOH
100 mM AmAc
pH 4.6
Not given
Not given
[46]
Table 6. Extraction procedures utilized for sample preparation during atorvastatin analysis
Trends
365
Trends
negative-ion mode (ESI ), probably because this compound contained two nitrogen groups [42,43]. The
precursor ion chosen for quantitation was [M+H]+ in all
methods developed (see Table 2). The monitored SRM
transition for atorvastatin therefore utilized 559 440
(which was also the most intensive transition in all
methods).
Compared to HPLC-UV methods, all LC-MS/MS
methods determined atorvastatin together with its phydroxy and o-hydroxy metabolites, except [45] where
only the o-hydroxy metabolite was determined. All
metabolites including hydroxy acid and lactone forms
were determined by Jemal et al. [42], Hermann et al.
[18] and Van Pelt et al. [46]. Atorvastatin metabolites
were all also determined in ESI+ using [M + H]+ as a
precursor ion; the specific transition for each metabolite
and their stable isotope-labeled forms can be seen in
Table 4.
Atorvastatin and its metabolites have been separated
on the C18 analytical column using acetonitrile usually about 70% (to shorten retention times of analytes;
containing methanol only they would be eluted much
later) as part of the mobile phase, together with acetic
or formic acid as additives to enhance ionization. Both
isocratic and gradient elution have been utilized. Analytical run times were, in most cases, about 6 min or less,
except for one method [18].
The best ISs for precise and accurate quantitation in
MS are stable-isotope-labeled standards. In the case of
atorvastatin and its metabolites, [d5] labeling usually
occurs on the phenyl ring, which does not contain
fluorine [42,43]. Only three works employed deuteriumlabeled ISs [42,43,46] because they were very expensive
and sometimes they were not easily available. Other
works utilized ISs of various structures, including
metachalon [18], rosuvastatin [44] or clindamycin [46].
Method sensitivity expressed as LOQ was quite similar for
all methods, typically in the range 0.5 ng/ml [42] to 0.1
ng/ml [45]. [Please check this range?]
5. Sample preparation
A convenient sample-preparation procedure should isolate the analytes from the complex matrix while
removing endogenous interfering substances. This is
often the most time-consuming, critical step of analysis.
The analytes should be pre-concentrated in order to increase the sensitivity and the selectivity of the method.
The procedures for sample preparation of simvastatin
and atorvastatin have in most cases included LLE (liquidliquid extraction), and SPE (solid-phase extraction).
However, simple extraction into the organic solvent has
also been used in pharmaceutical applications
[20,24,27,3638,40] see Tables 1 and 2. There have
also been specific approaches to sample preparation (e.g.,
366
LLCE (liquid-liquid cartridge extraction) [31,32] or online SPE utilizing a column-switching technique).
Tables 5 and 6 give overviews of LLE and SPE samplepreparation techniques utilized for the isolation of simvastatin and atorvastatin, focusing on data recovery,
interconversion during sample preparation (details given
in the work of Herman et al. [18], Zhao et al. [32] and
Zhang et al. [34]) and stabilization data.
Statins have mostly been isolated using SPE on the
Oasis HLB cartridge, employing methanol for elution, or
using LLE, where MTBE, ethyl acetate or diethyl ether
have generally been applied as extraction agents.
6. Conclusions
Analysis of statins is a current problem, because these
drugs are widely used for the treatment of hypercholesterolemia.
This review has presented HPLC methods for the
determination of simvastatin and atorvastatin in various
fields of application (pharmacology, clinical medicine
and environmental). We divided the methods according
to the type of detection. We discussed interconversion
between acid and lactone form of statins.
LC-MS/MS methods have undoubtedly become the
method of choice. In order to get high selectivity and
sensitivity, MS/MS techniques employ specific SRM
conditions, which are convenient, especially in bioanalytical applications. There is no problem in determining the metabolites, even if they are not also sufficiently separated chromatographically.
Chromatographic conditions must be carefully arranged, as must selection of a precursor ion for a specific
SRM transition in order not retain the sensitivity and the
selectivity of the method.
Acknowledgement
The authors gratefully acknowledge the financial support of IGA MZ CR No. 1A/8689-4.
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HPLC Methods For The Determination of Simvastatin and Atorvastatin

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HPLC Methods For The Determination of Simvastatin and Atorvastatin

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Trends

Trends in Analytical Chemistry, Vol. 27, No. 4, 2008