Validated LC-MS/MS Method for the Determination of Rosuvastatin in Human Plasma: Application to a Bioequivalence Study in Chinese Volunteers

doi:10.4236/pp.2011.24044

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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.

Email: *[email protected]

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)

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

(%)

(%) Mean ± SD RSD

(%)

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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