Pharmacology & Pharmacy, 2011, 2, 341-346
doi:10.4236/pp.2011.24044 Published Online October 2011 (http://www.SciRP.org/journal/pp)
341
Validated LC-MS/MS Method for the
Determination of Rosuvastatin in Human
Plasma: Application to a Bioequivalence
Study in Chinese Volunteers
Dujuan Zhang1,2, Jing Zhang2, Xiaoyan Liu2, Chunmin Wei2, Rui Zhang2, Haojing Song2, Han Yao2,
Guiyan Yuan2, Benjie Wang2, Ruichen Guo2*
1School of Pharmacy, Shandong University, Jinan, China, 2Institute of Clinical Pharmacology, Qilu Hospital of Shandong University,
Jinan, China.
Received July 28th, 2011; revised August 26th, 2011; accepted September 20th, 2011.
ABSTRACT
A sensitive and selective liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) method
was developed and validated for the determination of rosuvastatin in human plasma using gliclazide as an internal
standard (IS). Rosuvastatin and gliclazide in plasma were extracted with ethyl acetate, separated on a C18 reversed
phase column, eluted with mobile phase of acetonitrile-methanoic acid (0.1%) (60:40, v/v), ionized by positive ion
pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor → product
ions of m/z 482.1 → 258.1 for rosuvastatin and m/z 324.2 → 127.2 for IS , respectively. The calibration curve was lin-
ear (r2 > 0.99, n = 5) over the concentration range of 0.1 - 60 ng/mL. The specificity, matrix effect, recovery, sensitivity,
linearity, accuracy, precision, and stabilities were validated for ro suvastatin in human plasma. In conclusion , the vali-
dation results showed that this method was sensitive, economical and less toxic and it can successfully fulfill the re-
quirement of bioequivalence study of rosuvastatin calcium tablets in Chinese healthy volunteers.
Keywords: Rosuvastatin, Bioequivalence, LC-MS/MS, Pharmacokinetics
1. Introduction
Rosuvastatin (Figure 1) is a hydroxyl-methyl-glutaryl
coenzyme A (HMG-CoA) reductase inhibitor used in the
treatment of patients with dyslipidemia [1]. Rosuvastatin
has been reported to reduce serum levels of low-density
lipoprotein cholesterol [2], accompanied by increases in
high-density lipoprotein cholesterol [3] and reductions in
triglycerides [4]. The population pharmacokinetic study
revealed that plasma exposure to rosuvastatin was sig-
nificantly higher in Asian subjects than in White subjects
living in the same environment [5].
In recent years, several methods have been reported
for the quantification of rosuvastatin in plasma. Kumar
TR et al published a HPLC-UV method for the determi-
nation of rosuvastatin in rat plasma [6]. Some LC-MS/
MS methods were developed [7-9], but they adopted ei-
ther ethyl ether as extraction solvent [7,8], which was
toxic and difficult to quantitate, or SPE [9], which was
relatively expensive, especially for the bioequivalence
study where a large number of samples were to be col-
lected and treated. Simultaneous quantitations of rosu-
vastatin and fenofibric acid [10] or other coadministrated
drugs [11,12] in biological samples were also proposed
by some literatures, using HPLC with ultraviolet detec-
tion [11,12] or LC-MS/MS [10], but the lowest detection
limit of these methods was 0.6 ng/mL, not sensitive
enough to meet the requirement for rosuvastation deter-
mination in plasma. It was therefore necessary to develop
a more optimized LC-MS/MS method for the quantita-
tion of rosuvastatin in plasma.
In this paper, a sensitive and selective LC-MS/MS
method has been developed and used to evaluate the bio-
equivalence of two rosuvastatin calcium tablets in healthy
Chinese volunteers.

Validated LC-MS/MS Method for the Determination of Rosuvastatin in Human Plasma: 342
Application to a Bioequivalence Study in Chinese Volunteers
2. Experimental
2.1. Chemicals and Instrumentation
Rosuvastatin (lot no. 091201, purity 98.6%) was ob-
tained from Shandong Dyne Marine Organism Pharma-
ceutical Co., Ltd. Gliclazide standard (Figure 1, lot no.
100269-9701) was obtained from National Institute for
the Control of Pharmaceutical and Biological Products.
Methanol, acetonitrile and ethyl acetate were all of
HPLC grade, from J. T. BAKER. Methanoic acid and
hydrochloric acid were analytical grade, from Sinopharm
Chemical Reagent Co., Ltd. Blank human plasma was
provided by Shandong blood center (P. R. of China). The
test formulation (lot no. 100102) was manufactured by
Shandong Dyne Marine Organism Pharmaceutical Co.,
Ltd. The reference formulation (lot no. GF550) was do-
nated by IPR Pharmaceuticals, Inc.
Determination of rosuvastatin in human plasma was
performed with an Agilent 1200 series HPLC and an
Agilent 6410 Triple Quadrupole mass spectrometer equip-
ped with an electrospray ionization source (Agilent Tech-
nologies, USA).
2.2. LC-MS/MS Conditions
The chromatographic separation was achieved on a Dia-
monsil C18 column (150 mm × 4.6 mm, 5 μm) at 30˚C
maintained with a thermostated column oven. The mo-
bile phase consisting of acetonitrile-methanoic acid
(0.1%) (60:40, v/v) was employed at a flow rate of 0.8
mL/min. The injection volume was 20 μL.
Mass spectrometric analysis was performed in the
positive ion MRM mode by monitoring ion transitions at
m/z 482.1 → 258.1 for rosuvastatin and m/z 324.2 →
127.2 for IS (Figure 2), with spray gas pressure of 350
Pa, protective air of nitrogen gas at a flow rate of 9 L/min,
dwell time of 200 ms, capillary voltage of 4000 V, frag-
ment electric voltage of 140 V for rosuvastatin and 100
V for IS, and collision energy of 40 eV for rosuvastatin
and 20 eV for IS. All data were acquired employing
Agilent 6410 Quantitative Analysis version analyst data
processing software.
Figure 1. The chemical structure of rosuvastatin and gli-
clazide.
Figure 2. Product ion spectrum of rosuvastatin and gli-
clazide (IS).
2.3. Preparation of Standard Solutions,
Calibration and Quality Control Samples
Rosuvastatin standard was accurately weighted and dis-
solved in methanol achieving concentration of 0.1 mg/ mL
stock solution, and further diluted with mobile phase ob-
taining 1, 10, 100, 1000 ng/mL working solutions, respec-
tively. The stock solution of IS of 1 mg/mL was diluted
with mobile phase to obtain the working solution of 10
ng/mL. All were stored in the refrigerator (4˚C), and equi-
librated to room temperature before use (approximately 15
minutes).
Rosuvastatin calibration of 0.1, 0.5, 2, 5, 10, 20 and 60
ng/mL and quality control (QC) of 0.2, 5 and 50 ng/mL
in blank human plasma were prepared by spiking with
rosuvastatin and IS working solutions and used for
method validation, standard curve or quality control dur-
ing run of analysis, and all were disposed with the estab-
lished procedure.
2.4. Plasma Disposition
30 µL of the IS (10 ng/mL gliclazide in mobile phase)

Validated LC-MS/MS Method for the Determination of Rosuvastatin in Human Plasma:
Application to a Bioequivalence Study in Chinese Volunteers
343
was mixed with 0.5 mL plasma sample, then 100 µL hy-
drochloric acid (0.1 mol/L in water) and 4 mL ethyl ace-
tate were added, vortex-mixed for 2 min, and centrifuged
at 5000 rpm for 5 min. The organic phase was transferred
to a clean tube and evaporated to dryness under gentle
stream of nitrogen gas at 40˚C. The residue was recon-
stituted with 100 µL mobile phase, and 20 µL was in-
jected onto the LC-MS/MS for analysis.
2.5. Method Validation
The method was validated for specificity, matrix effect,
recovery, sensitivity, linearity, precision, accuracy, and
stability according to the US Food and Drug Administra-
tion (FDA) guidelines for the validation of bioanalytical
methods [13].
The specificity of the method was evaluated by com-
paring chromatograms of blank plasma, rosuvastain and
IS standard, blank plasma spiked with rosuvastain and IS,
plasma sample from a volunteer after administration of
rosuvastatin calcium tablet.
Blank biological samples were extracted and then
spiked with rosuvastatin at three concentration levels (0.2
ng/mL as low, 5 ng/mL as medium and 50 ng/mL as high)
and IS in five replicates, respectively, to evaluate the
matrix effects of plasma. The corresponding peak areas
were then compared to those of standard solutions, and
peak area ratio is defined as the matrix effect.
The extraction recoveries were evaluated by compar-
ing peak areas of analytes in spiked biological samples
with those of samples to which the analytes had been
added after extraction. Five replicates of each QC level
were disposed with the established extraction procedure.
The lower limit of quantization (LLOQ) was evaluated
by analyzing five replicates of spiked plasma samples at
the concentration of 0.1 ng/mL.
The calibration curve was prepared by analyzing spiked
calibration samples at 7 different concentration levels on
each day of analysis, typically described by equation y =
ax + b, where y corresponds to the peak-area ratio and x
to the concentration ratio of rosuvastatin to IS. The line-
arity of calibration curve was assessed by linear regres-
sion with a weighting factor of the reciprocal of the con-
centration squared (1/x2).
Accuracy and precision were assessed by the deter-
mining of QC samples with five replicates for each con-
centration level on the same day or on three consecutive
days. Precision was expressed by coefficient of variation
(RSD) and accuracy by relative error (RE).
The stabilities of rosuvastatin in plasma samples at
different concentrations were examined under different
study conditions; i.e. storing at –20˚C for 45 days. Freeze/
thaw stability was determined after freezing (–20˚C) and
thawing (25˚C) QC samples for two cycles. Stability of
post-extracted samples in the HPLC auto-sampler at
room temperature for 7 hours was also observed.
2.6. Bioequivalence Study Design
The study was approved by the Ethics Committee of Qilu
Hospital, Shandong University according to Declaration
of Helsinki. Twenty healthy young male Chinese volun-
teers with age of (24.6 ± 1.4) years, weight of (64.9 ± 7.5)
kg, height of (173.7 ± 4.9) cm and body mass index
(BMI) of (21.5 ± 1.9) kg/m2 were screened to participate
and signed Informed Consent Form. 10 mg rosuvastatin
tablet was orally administered with 200 mL of warm wa-
ter after an overnight fast (10 hours). Standard meals or a
given amount of water were provided 4 hours after ad-
ministration in the study, and no coffee, smoke, choco-
late or other food was allowed. All volunteers were under
supervision of physicians for any possible adverse
events.
Blood samples were collected before and 0.5, 1.0, 2.0,
3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0, 24.0, 36.0, 48.0, 72.0
hours after administration. Blood was centrifuged imme-
diately at 5000 rpm for 5 min, and plasma was trans-
ported into two EP tubes equally, clearly labeled and
stored at –20˚C pending analysis. All plasma samples of
20 volunteers were determined and the pharmacokinetic
parameters including the maximum plasma concentration
(Cmax), time to maximum plasma concentration (Tmax),
half-life of drug elimination during the terminal phase
(t1/2), area under the plasma concentration-time curve
from 0 to last measurable time (AUC0-t) or from 0 to in-
finity (AUC0-∞) were estimated using non-compart-
mental model method. Cmax and Tmax were obtained di-
rectly from the measured data. T1/2, AUC0-t and AUC0-∞
were calculated using Drug and Statistical Software-
Version 2.0 (DAS 2.0, P. R. of China).
3. Results
3.1. Method Validation
3.1.1. Specificity
Typical MRM chromatograms of blank plasma, rosuvas-
tatin and IS standard, blank plasma spiked with rosuvas-
tatin and IS, plasma from a volunteer after administration
of rosuvastatin calcium tablet spiked with IS were shown
in Figure 3. Retention times of rosuvastatin and IS were
3.0 and 5.1 min, respectively, and overall run time was
within 6 min. No significant interfering peak was ob-
served around the rosuvastatin and IS during analysis.
3.1.2. Matrix Effect and Extraction Recovery
The mean matrix effect values of rosuvastatin at three
concentrations of 0.2 ng/mL, 5 ng/mL and 50 ng/mL and

Validated LC-MS/MS Method for the Determination of Rosuvastatin in Human Plasma: 344
Application to a Bioequivalence Study in Chinese Volunteers
(a) (b)
(c) (d)
Figure 3. Typical chromatograms of rosuvastatin and IS in human plasma: blank plasma sample (a); rosuvastatin and IS
standard (b); a blank plasma sample spiked with rosuvastatin (3 ng/mL) and IS (c); a plasma sample of a volunteer at 0.5
hours after administration of 10 mg rosuvastatin calcium tablet (d).
IS at 3 ng/mL were (96.41 ± 6.14)%, (99.75 ± 1.43)%,
(98.35 ± 3.01)%, and (88.93 ± 3.39)%, respectively. It
demonstrated no matrix significantly affected the deter-
mination of rosuvastatin in human plasma.
Mean extraction recoveries of rosuvastatin at three QC
levels and IS were (85.73 ± 6.62)%, (83.99 ± 1.52)%,
(79.67 ± 4.13)%, and (75.98 ± 1.34)%, respectively. The
results were shown to be consistent, precise and repro-
ducible.
3.1.3. Calibration Curve and LLOQ
The calibration curves showed a good linearity in the
concentration range of 0.1 - 60 ng/mL with correlation
coefficient (r2 > 0.99) and the LLOQ for rosuvastatin was
proved to be 0.1 ng/mL with RSD and RE were 4.5% and
–5.6%, respectively.
3.1.4. Accuracy and Precision
The results of intraday and interday precision and accu-
racy were shown in Table 1. Both precision values (RSD)
were less than 7.7%. Intraday and interday accuracy (RE)
ranged from –10.1% to –4.3% and –9.5% to –2.8%, re-
spectively.
3.1.5. Stability
The results of stability of rosuvastatin were shown in
Table 2. The data demonstrated that rosuvastain was
stable under the indicated conditions for the bioequiva-
lence study. The stock solutions of rosuvastatin and IS
were comparable to the freshly prepared ones after stor-
age at −20˚C for days and two freeze-thaw cycles.
3.2. Application to Bioequivalence Study
The validated method was applied to the bioequivalence
study of rosuvastatin in human plasma after a single oral
administration of 10 mg rosuvastatin calcium tablet to 20
healthy male volunteers. The mean plasma concentra-
tion-time profile and main pharmacokinetic parameters

Validated LC-MS/MS Method for the Determination of Rosuvastatin in Human Plasma: 345
Application to a Bioequivalence Study in Chinese Volunteers
Table 1. Interday and intraday precision and accuracy for
the determination of rosuvastatin in human plasma (n = 5).
Intraday Interday
Nominal
Conc.
(ng/mL) Mean ± SD RSD
(%)
RE
(%) Mean ± SD RSD (%)
RE
(%)
0.2 0.19 ± 0.01 7.7 –4.30.19 ± 0.01 6.3 –2.8
5 4.50 ± 0.16 3.6 –10.14.52 ± 0.18 4.1 –9.5
50 46.35 ± 0.99 2.1 –7.347.05 ± 3.34 7.1 –5.9
Table 2. Stability of rosuvastatin at various conditions (n = 5).
Condition Nominal Conc.
(ng/mL) Mean ± SD RSD (%)RE (%)
0.2 0.21 ± 0.01 7.0 3.4
5 4.76 ± 0.29 6.2 –4.7 Fresh samples
50 48.84 ± 3.39 6.9 –2.3
0.2 0.19 ± 0.01 6.4 –3.6
5 5.28 ± 0.27 5.2 5.6 –20˚C, 45 days
50 45.81 ± 3.28 7.2 –8.4
0.2 0.19 ± 0.02 10.4 –5.8
5 5.06 ± 0.17 3.4 1.2
Two
freeze-thaw
cycles
50 52.52 ± 3.88 7.4 5.0
0.2 0.21 ± 0.01 4.6 5.1
5 4.59 ± 0.19 4.2 –8.2
Post-extracted
samples, 25˚C,
7 h
50 48.26 ± 1.24 2.6 –3.5
were shown in Figure 4 and Table 3, respectively.
Bioequivalence of test and reference was evaluated by
calculating 90% confidence intervals (90% CI) for the
test/reference ratio of logarithmic transformed Cmax,
AUC0-t and AUC0-∞. The 90% confidence intervals (90%
CI) for the test/reference ratio of logarithmic transformed
Cmax, AUC0-t and AUC0-∞ were shown in Table 4, and all
were within the bioequivalence acceptance range 80% -
125% adopted by US-FDA [14]. The two rosuvastatin
calcium tablets are bioequivalent.
4. Discussion
Mass spectrometric detection was carried out on an
Agilent 6410 triple quadrupole instrument equipped with
an ESI source operated in the positive ion mode. During
optimization of the mass spectrometric parameters,
strong and stable signals of rosuvastatin and IS can be
observed in the form of their [M + H]+ molecular ions
with mass to charge ratios of m/z 482.1 and m/z 324.2,
respectively. Each of the precursor ions was subjected to
Table 3. Main pharmacokinetic parameters of rosuvastatin
after a single dose of 10 mg rosuvastatin calcium tablet test
and reference (n = 20, mean ± SD).
Parameter Test Reference
t1/2z (h) 18.30 ± 11.34 16.74 ± 12.70
Tmax (h) 2.55 ± 0.51 2.50 ± 0.61
AUC0-t (ng·h/mL) 95.15± 46.68 91.89± 44.34
AUC0-∞ ( ng·h/mL)99.10 ± 48.24 95.24 ± 45.65
Cmax (ng/mL) 17.52 ± 8.04 16.53 ± 7.20
Table 4. The 90% Confidence Interval (90% CI) for the
test/reference ratio of Cmax, AUC0-t and AUC0-∞.
Parameter Mean Ratio (%) 90% CI (%) P-Value
Cmax 105.1 99.4 - 111.1 0.137
AUC0-t 103.2 96.2 - 110.8 0.441
AUC0-∞ 103.8 96.8 - 111.4 0.368
Figure 4. Mean (±SD) plasma concentration-time curves of
rosuvastatin calcium after a single oral administration of 10
mg rosuvastatin calcium tablet test and reference (n = 20).
collision-induced dissociation to determine the resulting
product ion. Different collision energy (CE) values (20,
30, 40 eV) for rosuvastatin were tested, and at higher CE
(40 eV), a major fragment ion at m/z 258.1 was formed,
giving a considerably better response and a higher signal.
Fragmentation of IS produced the most abundant product
ion at m/z 127.2. Therefore, the ion transitions m/z 482.1
→ 258.1 and 324.2 → 127.2 were selected for MRM of
the rosuvastatin and the IS, respectively.
An ideal IS used to guarantee high accuracy of LC-
MS/MS assay should track the analyte during the extrac-
tion with almost the same recovery of the analyte. It was
also eluted close to the analyte on the column and com-
pensated for potential inconsistent response for matrix
effects. Several compounds were investigated such as
pravastatin sodium, fluvastatin sodium, lorazepam, losar-

Validated LC-MS/MS Method for the Determination of Rosuvastatin in Human Plasma:
Application to a Bioequivalence Study in Chinese Volunteers
346
tan potassium, and gliclazide. Significant interfering peaks
were observed for pravastatin sodium, fluvastatin sodium
and lorazepam during analysis. Different responds were
detected for losartan potassium when spiked with pur-
chased blank plasma and plasma samples from volunteers.
Gliclazide, with suitable retention time, acceptable matrix
effects, and extraction recoveries was selected as internal
standard.
Protein precipitation (PPT), liquid-liquid extraction
(LLE) and solid-phase extraction (SPE) were tried to
obtain a simple and excellent plasma preparation proce-
dure. PPT was easy to dilute the sample and failed to
sufficiently remove endogenous interference. The SPE
column for solid-phase extraction is relatively expensive
for a great quantity of samples. LLE method with various
extraction solvents, including chloroform, ethyl acetate,
n-hexane, dichloromethane, n-hexane-dichloromethane-
isopropanol (20:10:1, v/v/v) was investigated and evalu-
ated for acceptable extraction recoveries and matrix effect,
and ethyl acetate with no-concentration-dependent extrac-
tion recovery and acceptable matrix effect was adopted.
In conclusion, the developed method was simple, spe-
cific, sensitive, and successfully applied to bioequiva-
lence study of rosuvastatin calcium tablets. The two ro-
suvastatin calcium tablets were bioequivalent.
5. Acknowledgements
The authors appreciated Shandong Dyne Marine Organ-
ism Pharmaceutical Co., Ltd, providing financial support
and test and reference products.
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