atorva bioanalytical

CHAPTER 2. LITERATURE REVIEW

35

2. LITERATURE REVIEW

A number of analytical methods have been reported in various journals for the

determination of antihyperlipidemic drugs in pharmaceutical formulations and in

biological samples. Some of the work in this area of research is given below for

each drug.

2.1 Analytical Methods for Atorvastatin

Petkovska et al [170] developed and validated a Rapid Resolution Reversed Phase

High-Performance Liquid Chromatography method for the simultaneous

determination of atorvastatin and seven related compounds. Experimental design

was used during method optimization and robustness testing. Chromatography was

performed with mobile phase containing phosphate buffer pH 3.5 and a mixture of

10 % tetrahydrofuran in acetonitrile as organic modifier. A C18 Rapid Resolution

column was used. The developed method was able to determine atorvastatin

calcium purity and level of impurities in drug substances.

Khedr [171] developed a sensitive, selective, and validated stability-indicating

high-performance liquid chromatographic assay for atorvastatin in bulk drug and

tablet form. Atorvastatin was subjected to different stress conditions, including UV

light, oxidation, acid-base hydrolysis, and temperature. The analyte and the

degradation products were then analyzed on a C18 column using isocratic elution

with acetonitrile-0.02 M sodium acetate, pH 4.2 (45:55, v/v). The samples were

monitored with fluorescence detection at 282 nm (excitation)/400 nm (emission).

The method showed good resolution of atorvastatin from its decomposition

products. The linear range was 10-1200 ng/injection, and the limit of quantitation

(LOQ) was 2.0 ng/injection.

Sivakumar et al [172] applied statistical experimental design and Derringer's

desirability function to develop an improved RP-HPLC (Reverse Phase High


36

Performance Liquid Chromatography) method for the simultaneous analysis of

amlodipine and atorvastatin in pharmaceutical formulations. The predicted

optimum for the quality control samples was: methanol-acetonitrile-0.015 M

dipotassium hydrogen phosphate buffer (pH 5.33) (10:42.08:47.92, v/v/v) as the

mobile phase and 1.12 mL/min as the flow rate. The assay was validated according

to ICH guidelines.

Jamshidi et al [173] developed a two-step isocratic chromatography on silica gel

HPTLC layer and densitometric quantitation at λ = 280 nm for the separation of

atorvastatin from plasma constituencies and diclofenac sodium as peak-tracer. The

developed HPTLC method was validated in terms of LOD/LOQ (Limits of

detection/Limits of quantitation), linearity, recovery and repeatability. The method

was linear in the range 101–353.5 ng/zone. The LOD and LOQ were 30.3 ng/zone

and 101 ng/zone. The recovery and relative standard deviation (RSD) obtained

from between-days analysis were 97.5–103.0 and 1.7–3.4%.

Ma et al [174] developed a sensitive liquid chromatographic–electrospray

ionization–mass spectrometric method for direct concentration of atorvastatin in

human plasma. Plasma samples were extracted with ethyl acetate, and by a simple

reversed-phase chromatography. The LOQ was 0.25 ng/mL. The assay was linear

from 0.25–20 ng/mL. Intra-day and inter-day accuracy was better than 15 %.

Stanisz et al [175] developed and validated a rapid HPLC method for determination

of atorvastatin in pharmaceutical dosage forms. Separation of atorvastatin was

carried on a C-18 column using water-acetonitrile in the ratio of 48:52, adjusted to

pH 2.0 with 80% ortho-phosphoric acid. The wavelength was set as 245 nm. The

method was linear in the concentration range of 0.04 - 0.4 mg/mL. The RSD values

for intra and inter day precision were less than 1.00 and 0.90 %, respectively.


37

Nirogi et al [176] reported a review paper on published higher performance liquid

chromatographic-mass spectrometric methods for the quantification of presently

available seven statins, atorvastatin, simvastatin, lovastatin, pravastatin, fluvastatin,

rosuvastatin and pitavastatin. This review encompass that most of the methods used

for quantification of statins were in plasma and they were suitable for therapeutic

drug monitoring of these drugs.

Chaudhari et al [177] described the development and validation of a stability

indicating reverse-phase HPLC method for the simultaneous estimation of

atorvastatin and amlodipine from their combination drug product. The developed

RP-HPLC method used a C18 column, at ambient temperature. The mobile phase

was consisted of acetonitrile and 0.05 M potassium dihydrogen phosphate buffer

(60:40, v/v), adjusted to pH 3 ± 0.1 with 10 % phosphoric acid, at 1 mL/min, and

UV detection at 254 nm. The described method was linear over the range of 1-90

µg/mL and 1-80 µg/mL for atorvastatin and amlodipine respectively. The mean

recoveries were 99.76 % and 98.12 % for atorvastatin and amlodipine respectively.

The LOD for atorvastatin and amlodipine were found to be 0.4 µg/mL and 0.6

µg/mL, respectively and the LOQ was 1.0 µg/mL for both drugs.

Mohammadi et al [178] developed and validated a simple, rapid, precise and

accurate isocratic stability-indicating RP-HPLC method for the simultaneous

determination of atorvastatin and amlodipine in commercial tablets. The method

showed separation of amlodipine and atorvastatin from their associated main

impurities and their degradation products. Separation was achieved on an ODS-3,

column using a mobile phase consisting of acetonitrile-0.025 M sodium dihydrogen

phospahe buffer (pH 4.5) (55:45, v/v) at a flow rate of 1 mL/min and UV detection

at 237 nm. The linearity of the method was in the range of 2-30 µg/mL for

atorvastatin and 1-20 µg/mL for amlodipine. The LOD were 0.65 µg/mL and 0.35


38

µg/mL for atorvastatin and amlodipine respectively. The LOQ were 2 µg/mL and 1

µg/mL for atorvastatin and amlodipine respectively.

Borek-Dohalský et al [179] reported a validated, highly sensitive, and selective

isocratic HPLC method for quantitative determination of the atorvastatin and its

metabolite 2-hydroxyatorvastatin. Detection was performed with a mass

spectrometer equipped with an ESI interface in positive-ionization mode. The

method was linear in the concentration range 0.10-40.00 ng/mL for both

atorvastatin and 2-hydroxyatorvastatin. Inter-day and intra-day precision were less

than 8 % for both analytes. The LOQ was 0.02 ng/mL for atorvastatin and 0.07

ng/mL for 2-hydroxyatorvastatin.

Shen et al [180] developed a specific and accurate reversed-phase HPLC with UV

detection for the assay of atorvastatin in beagle dog plasma. After protein

precipitation; the extracts were separated on a C8 column with UV wavelength at

270 nm. The mobile phase consisted of acetonitrile: 0.1 M ammonium acetate

buffer (pH 4.0) (65:35, v/v) at a flow rate of 1 mL/min. Linearity was found to be

in the range of 0.05 µg/mL to 2.5 µg/mL. The LOQ was 25 ng/mL and the LOD

was 8 ng/mL. The total chromatographic analysis time was less than 9 min.

Bahrami et al [181] developed and validated a rapid and sensitive high-

performance liquid chromatographic method for determination of atorvastatin in

human serum. After liquid-liquid extraction, chromatography was performed using

C18 column with a mobile phase consisting of sodium phosphate buffer (0.05 M,

pH 4.0) and methanol (33:67, v/v) at 247 nm. The average recovery of the drug was

95 %. The LOD and LOQ were 1 µg/mL and 4 ng/mL, respectively, and the

calibration curves were linear over a concentration range of 4-256 ng/mL.

Zarghi et al [182] developed a rapid and sensitive high-performance liquid

chromatographic method for the determination of atorvastatin in plasma. After


39

protein precipitation by acetonitrile, atorvastatin was separated on a C8 column

with mobile phase consisting of sodium dihydrogen phosphate buffer-acetonitrile

(60:40, v/v) adjusted to pH 5.5 at a flow rate of 1.5 mL/min and UV detection at

245 nm. The LOD for atorvastatin was 1 ng/mL. The method was linear over the

concentration range 20-800 ng/mL. The inter-day and intra-day assay precision was

found to be less than 7 %.

Pasha et al [183] developed and validated a specific, accurate, precise and

reproducible high-performance liquid chromatographic method for the

simultaneous quantitation of atorvastatin, lovastatin, pravastatin, rosuvastatin and

simvastatin, in pharmaceutical formulations and extended it to in vitro metabolism

studies of these drugs. Ternary gradient elution at a flow rate of 1 mL/min was

employed on an ODS 3V column at ambient temperature. The mobile phase

consisted of 0.01 M ammonium acetate (pH 5.0), acetonitrile and methanol, at a

wavelength of 237 nm. Drugs were found to be 89.6-105.6% of their label's claim

in the pharmaceutical formulations.

Hermann et al [184] developed a chromatographic method for the analysis of

atorvastatin, o- and p-hydroxyatorvastatin (acid and lactone forms) in human

plasma. After solid-phase extraction, analytes were separated on an HPLC system

with a linear gradient and a mobile phase consisting of acetonitrile, water and

formic acid. Detection was done by tandem mass spectrometry in electrospray

positive ion mode. Linearity was within the concentration range (0.2-30 ng/mL for

atorvastatin acid and p-hydroxyatorvastatin acid, and 0.5-30 ng/mL for o-

hydroxyatorvastatin acid). The LOD was 0.06 ng/mL for atorvastatin and p-

hydroxyatorvastatin, and 0.15 ng/mL for o-hydroxyatorvastatin.

Ertürk et al [185] developed a simple high-performance liquid chromatographic

method for the analysis of atorvastatin and its impurities in bulk drug and tablets

using gradient RP-HPLC assay with UV detection. Best resolution was determined


40

using a C18 column with acetonitrile-ammonium acetate buffer pH 4-

tetrahydrofuran (THF) as mobile phase. Samples were eluted gradiently with the

mobile phase at flow rate of 1 mL/min and detected at 248 nm.

Jemal et al [186] developed and validated a method for simultaneous quantitation

of both the acid and lactone forms of atorvastatin and both the acid and lactone

forms of its two biotransformation products, 2-hydroxyatorvastatin and 4-

hydroxyatorvastatin, in human serum by high-performance liquid chromatography

with electrospray tandem mass spectrometry. The acid compounds were stable in

human serum at room temperature but the lactone compounds in serum could be

stabilized by lowering the working temperature to 4 0C or lowering the serum pH to

6.0. The intra-day, inter-day precision and the deviations from the nominal

concentrations for all analytes were within 15 %. The required lower LOQ of 0.5

ng/mL was achieved for each analyte.

Bullen et al [187] developed and validated a liquid chromatographic/mass

spectrometric method to quantitate atorvastatin and its active metabolites ortho-

hydroxy and para-hydroxy atorvastatin in human, dog, and rat plasma.

Chromatographic separation of analytes was achieved by using a C-18 column with

a mobile phase consisting of acetonitrile-0.1 % acetic acid, (70:30, v/v). Analytes

were detected by tandem mass spectrometry. The method proved suitable for

routine quantitation of atorvastatin, o-hydroxyatorvastatin and p-

hydroxyatorvastatin over the concentration range of 0.250 ng/mL to 25.0 ng/mL.

Mean recoveries of atorvastatin, o-hydroxyatorvastatin and p-hydroxyatorvastatin

from plasma ranged 100 %-107 %, 70.6 %-104 %, and 47.6 %-85.6 %,

respectively. Mean recoveries of the [d5]-AT and [d5]-o-AT internal standards

ranged 98.0 %-99.9 % and 97.3 %, respectively. Inter assay precision for

atorvastatin, o-hydroxyatorvastatin and p-hydroxyatorvastatin, was < or = 7.19 %,

8.28 %, and 12.7 %, respectively. Inter assay accuracy for atorvastatin, o-


41

hydroxyatorvastatin and p-hydroxyatorvastatin was ± 10.6 %, 5.86 %, and 15.8 %,

respectively.

2.2 Analytical Methods for Simvastatin

Apostolou et al [188] developed a fully automated high-throughput liquid

chromatography/tandem mass spectrometry method for the simultaneous

quantification of simvastatin and simvastatin acid in human plasma. Plasma

samples were treated by acetonitrile for protein precipitation and subsequent two-

step liquid-liquid extraction in 96-deepwell plates, using methyl t-butyl ether as the

organic solvent. The method was very simple with chromatographic run time of

just 1.9 min.

Basavaiah et al [189] described two sensitive spectrophotometric methods for the

determination of simvastatin in bulk drug and in tablets. The methods were based

on the oxidation of simvastatin by cerium (IV) in acid medium followed by

determination of unreacted oxidant by two different reaction schemes. In one

procedure (method A), the residual cerium (IV) was reacted with a fixed

concentration of ferroin and the increase in absorbance was measured at 510 nm.

The second approach (method B) involved the reduction of the unreacted cerium

(IV) with a fixed quantity of iron (II), and the resulting iron (III) was complexed

with thiocyanate and the absorbance measured at 470 nm. In both methods, the

amount of cerium (IV) reacted corresponded to simvastatin concentration. The

systems obeyed Beer's law for 0.6-7.5 µg/mL and 0.5-5.0 µg/mL for method A and

method B, respectively.

Basavaiah et al [190] developed two simple and sensitive spectrophotometric

methods for the determination of simvastatin in pure form and in tablets using in

situ generated bromine, and p-phenylenediamine or o-dianisidine as reagents. The

methods were based on the bromination of simvastatin by in situ bromine in acid


42

medium followed by the determination of unreacted bromine by reacting with p-

phenylenediamine and measuring the resulting red colour at 510 nm (method A) or

reacting with o-dianisidine and measuring the absorbance at 470 nm (method B).

Beer’s law was obeyed over the concentration ranges 20-120 µg/mL and 2-12

µg/mL for method A and method B, respectively. The LOD and LOQ for method A

were found to be 2.96 µg/mL and 8.97 µg/mL, and the respective values for method

B were 0.14 µg/mL and 0.42 µg/mL. The assay precision was less than 5 % CV and

the accuracy was 97.38-103.4 %.

Nigovi et al [191] developed a cathodic square-wave stripping voltammetry method

for the determination of simvastatin at trace levels. The voltammetric response was

used to determine drug concentration in the range 1 × 10–8 mol/L to 7.5 × 10–7

mol/L, with LOD of 4.5 × 10–9 mol/L.

Arayne et al [192] developed a simple UV spectrophotometric method for the

determination of simvastatin in methanol and compared this with the existing

pharmacopoeial HPLC method. Analytical parameters such as stability, selectivity,

accuracy, and precision were established for the method in tablets and human

serum samples. The method was validated according to ICH and USP guidelines.

Jitender et al [193] developed and validated a sensitive HPLC assay for simvastatin

and its corresponding simvastatin hydroxyl acid for their simultaneous estimation

in solutions of various studies. HPLC separations were achieved on (i) C8 (ii) CN

and (iii) C18 columns. The eluents were monitored by diode array detector at 240

nm. Retention times were: simvastatin 8-9 min; and simvastatin hydroxy acid 5.5-6

min. The LOD of both on C-18 column was 0.05 µg/mL and on C8 and CN

columns was 0.1 µg/mL. Inter and intra assay precision were less than 6 %.

Malenović et al [194] developed a novel approach for the analysis of simvastatin

and its six impurities, applying micro emulsions as mobile phase. A micro


43

emulsion eluent containing 0.9 % w/w of di-isopropyl ether, 1.7 % w/w of sodium

dodecyl-sulphate, 7.0 % w/w of co-surfactant such as n-butanol and 90.4 % w/w of

aqueous 0.025 M di-sodium phosphate pH 7.0 was used for the analysis.

Separations were performed on a 3.5 µm X Terra 50 × 4.6 mm column at 30 0C.

Detection was performed at 238 nm and the flow rate of the mobile phase was set

to be 0.3 mL/min.

Coruh et al [195] studied the electrochemical behavior and determination of

simvastatin in aqueous alcohol medium at a stationary glassy carbon electrode.

Cyclic voltammetry showed one main oxidation peak between pH 2 and 8.

Differential pulse and square wave voltammetric techniques for the determination

of simvastatin in 0.1 M H2SO4 and a constant amount of methanol (20 %), allowed

quantitation over the 2 x 10-6-1 x 10-4 M range in supporting electrolyte with LOD

of 2.71 x 10-7 M and 5.50 x 10-7 M for differential pulse and square wave

voltammetric methods, respectively.

Abu-Nameh et al [196] proposed a simple and rapid HPLC method for the

determination of simvastatin using a C18 column and acetonitrile-phosphate buffer-

methanol (5: 3: 1, v/v/v) as a mobile phase with detection at 230 nm. The linear

range for simvastatin was up to 1.884 mg % and a regression coefficient of 0.9995.

Barrett et al [197] presented a validated, highly sensitive and selective isocratic

HPLC method for the quantitative determination of simvastatin and its metabolite

simvastatin hydroxy acid. Detection was done on triple quadrupole mass

spectrometer equipped with an ESI interface. The linearity was in the concentration

range of 0.10-16.00 ng/mL for simvastatin and 0.10-16.00 ng/mL for simvastatin

hydroxyl acid. Inter and intra-day precisions were lower than 7 % for all analytes.

The LOQ was 0.03 ng/mL for simvastatin and 0.02 ng/mL for simvastatin hydroxyl

acid.


44

Godoy et al [198] developed a simple HPLC method for the determination of

simvastatin in tablet dosage forms. The best results were obtained using

acetonitrile-0.03 M phosphate pH 4.5 buffer (70:30) at a flow rate of 3.0 mL/min.

Separation was achieved at room temperature on a C-18 monolithic column (100 x

4.6 mm), and the selected detection wavelength was 238 nm. The retention time

was 1.47 minutes.

Malenovic et al [199] used a novel and unique approach for retention modeling in

the separation of simvastatin and six impurities by liquid chromatography using a

micro emulsion as mobile phase. Optimal conditions for the separation of

simvastatin and its six impurities were obtained using an X Terra 50 x 4.6 mm,

column at 30 0C. The mobile phase consisted of 0.9 % w/w of diisopropyl ether, 2.2

% w/w of sodium dodecylsulphate , 7.0 % w/w of co-surfactant such as n-butanol,

and 89.9 % w/w of aqueous 0.025 M disodium phosphate pH 7.

Srinivasu et al [200] developed a micellar electrokinetic chromatographic method

for the quantification of lovastatin and simvastatin. Lovastatin and simvastatin were

separated using an electrolyte system consisting of 12 % acetonitrile (v/v) in 0.025

M sodium borate buffer pH 9.3 containing 0.025 M sodium dodecyl sulphate with

an extended light path capillary. Calibration curves were linear over the studied

ranges with correlation coefficients greater than 0.996. An LOD of 3.2 µg/mL and

LOQ of 10.6 µg/mL were estimated for both the drugs.

Tan et al [201] developed and validated a simple and sensitive reversed-phase

liquid chromatographic method for the analysis of simvastatin in human plasma.

After extraction with cyclohexane-dichloromethane (3.5:1, V/V), the drug was

measured by HPLC using a C18 column as stationary phase and an acetonitrile-

water (70:30, V/V) mixture as mobile phase. The flow rate was 1.2 mL/min and

with UV detection at 237 nm. The method was linear in the concentration range of


45

0.25-50.0 µg/L. Intra day and inter-day precision was less than 7.94 % and 8.58 %,

respectively. The recoveries of simvastatin were greater than 93.3 %.

Wang et al [202] developed a second derivative UV spectroscopic method for the

determination of simvastatin in the tablet dosage form. They carefully choose zero-

crossing technique of second derivative UV measurement at 243 nm. By using this

the selectivity and sensitivity of simvastatin was comparable to the previously

developed HPLC method.

Ochiai et al [203] developed a highly sensitive and selective high performance

liquid chromatographic method for the determination of simvastatin (I) and its

active hydrolyzed metabolite (II) in human plasma. Compounds were separately

extracted from plasma into two fractions. Compound I in first fraction was

hydrolyzed to II. A fluorescent derivative was then prepared by esterification with

1-bromoacetylpyrene in the presence of 18-crown-6. The pyrenacyl ester of II thus

obtained was purified on a phenyl boronic acid solid-phase extraction column, and

was measured by column-switching HPLC with fluorescence detection. The

calibration curves were linear in the concentration range of 0.1-10 ng/mL. The

intra-day precision was less than 11.0 %, and the accuracies were between 91.7 %

and 117 %. The LOQ for both analytes were 0.1 ng/mL.

Carlucci et al [204] developed and validated a fast, simple and accurate method for

determining simvastatin and simvastatin acid concentrations in human plasma. This

method involved an extraction procedure using a mixture of acetonitrile-water and

reversed-phase high-performance liquid chromatography with UV detection. The

method was linear from 20 ng/mL to 1000 ng/mL for simvastatin and from 25

ng/mL to 1000 ng/mL for simvastatin acid, respectively. Relative standard

deviations less than 2.3 % and relative errors of less than 5.2 % were obtained from

human plasma controls containing simvastatin at identical concentrations.


46

2.3 Analytical Methods for Lovastatin

Wang et al [205] developed a fast and sensitive ultra performance liquid

chromatography tandem mass spectrometry method for the determination of

lovastatin in human plasma. Sample pretreatment involved one-step extraction with

n-hexane-methylene dichloride-isopropanol (20:10:1, v/v/v) of 0.5 mL plasma.

Chromatographic separation was carried out on a C 18 column with mobile phase

consisting of acetonitrile-water (containing 0.005 M ammonium acetate; 85:15,

v/v) at a flow-rate of 0.35 mL/min. The detection was performed on a triple-

quadrupole tandem mass spectrometer by multiple reactions monitoring via

electrospray ionization source with positive mode. The analysis time was shorter

than 1.7 min per sample. The method was linear in the concentration range of

0.025-50.0 ng/mL with LOQ of 0.025 ng/mL. The intra and inter-day precision

values were below 11 % and the accuracy (relative error) was within 6.0 % at three

quality control levels.

Yuan et al [206] developed a selective, rapid and sensitive ultra performance liquid

chromatography–tandem mass spectrometry method for the quantitative

determination of lovastatin in human plasma. Sample pretreatment involved a one-

step extraction with tert-butyl methyl ether. The analysis was carried out on a C-18

column with flow rate of 0.35 mL/min. The mobile phase was water and

acetonitrile 80: 20 (v/v). The detection was performed on a triple-quadrupole

tandem mass spectrometer by multiple reaction monitoring mode via electrospray

ionization (ESI). Method was linear in the concentration range of 0.08–

24.50 ng/mL, with LOQ of 0.08 ng/mL. The intra and inter-day precision values

were below 15 %.

Yu et al [207] developed and validated a sensitive and selective liquid

chromatographic tandem mass spectrometric method for analysis of lovastatin in

human plasma. Ethyl acetate extraction was used for plasma sample preparation.

Chromatographic separation was achieved on a C18 column by isocratic elution


47

with 83:17:0.1 (v/v) methanol–0.002 M aqueous sodium acetate–formic acid as

mobile phase, at a flow rate of 1.0 mL/min. MS–MS detection was performed using

positive electrospray ionization and multiple-reaction monitoring. Method was

linear in the concentration range of 0.05 ng/mL to 20 ng/mL with LOQ of 0.05

ng/mL. Intra and inter-day precision were ranged from 0.4 % to 11.4 % with the

deviation always less than 15 %. Extraction recoveries were from 86.8 % to 94.1 %

for lovastatin.

Zhang et al [208] developed and validated a simple HPLC method for the

determination of lovastatin in rat tissues. Samples were prepared by a simple

protein precipitation. Separation was carried out on a C-18 column with a mobile

phase of acetonitrile: 0.05 M ammonium acetate at a flow rate of 1.0 mL/min and

detection at 238 nm. The method was linear from 0.0175 µg/mL to 7.0 µg/mL with

LOD of 0.006 µg/mL.

Li et al [209] developed a simple and sensitive method for lovastatin in urine based

on capillary electrophoresis. The following optimal conditions were determined for

stacking and separation: electrophoretic buffer of 0.1 M Gly- NaOH (pH 11.52),

sample buffer of 0.02 M Gly-HCl (pH 4.93), fused-silica capillary of 76 cm×75-µm

i.d (67 cm from detector), and sample injection at 14 mbar for 3 min. A 21- to 26-

fold increase in peak height was achieved for detection of lovastatin in urine under

the optimal conditions compared with normal capillary zone electrophoresis. The

LOD and LOQ for lovastatin in urine were decreased to 8.8 ng/mL and 29.2

ng/mL, respectively. The intra day and inter-day precision values were 2.23–3.61

% and 4.03–5.05 %, respectively. The recoveries of the analyte ranged from 82.65

% to 100.49 %.

Alvarez et al [210] described an HPLC stability-indicating method to study the

hydrolytic behaviour of lovastatin in different simulated fluids. The selected

chromatographic conditions were a C-18 column, acetonitrile/methanol/phosphate


48

buffer solution pH 4 (32/33/35) as mobile phase, 45 ºC temperature column, flow

rate of 1.5 mL/min and UV detection at 238 nm. Lovastatin exhibited a pH-

dependent degradation with an instantaneous hydrolysis in alkaline media at room

temperature. One or two degradation products were observed when lovastatin was

hydrolyzed in alkaline or acid medium, respectively.

Orkoula et al [211] developed FT-Raman spectroscopy and HPLC methods for

monitoring the stability of lovastatin in the solid state in the presence of gallic acid,

a natural antioxidant. A Raman calibration curve was constructed using the area of

the strong but overlapping vibration mode of lovastatin at 1645 cm-1 and of the

gallic acid at 1595 cm-1. Mixtures of the active ingredient with the antioxidant were

heated in the presence of atmospheric air up to 120 0C. The molar ratios of

lovastatin and gallic acid in the artificially oxidized mixtures were determined from

their Raman spectra using the calibration curve. The HPLC analysis was based on a

reserved-phase C 18 column, using a gradient elution program by varying the

proportion of solvent A acetonitrile 100% to solvent B 0.1% v/v phosphoric acid,

and a programmable diode array detection at 225 nm.

Sharma et al [212] developed a simple validated HPLC method utilizing an

isocratic mobile phase with short retention times for cyclosporine A and lovastatin.

Drugs were analysed by a reversed-phase HPLC method using a C18 column. An

isocratic mobile phase containing acetonitrile and water in the proportions 70:30

and 80:20 was used for the HPLC analysis of cyclosporine A and lovastatin,

respectively. The flow-rate was 1 mL/min and detection was done at 238 nm at 25 0C. The LOD were 250 ng/mL and 10 ng/mL and LOQ were 400 ng/mL and 30

ng/mL for cyclosporine A and lovastatin respectively. The method was linear in

concentration range of 0.5-6 µg/mL for cyclosporine A and 0.05-0.4 µg/mL for

lovastatin.


49

Ye et al [213] developed a simple, rapid HPLC assay with ultraviolet detection for

the analytical determination of lovastatin and its acid in human plasma. Sample

clean up involved the use of C10 solid-phase extraction cartridges. LOQ was 100

ng/mL. Standard curves were linear from 100 ng/mL to 5,000 ng/mL. The assay

was able to measure steady-state lovastatin concentration at the initial dose level in

a phase I trial of lovastatin as a modulator of apoptosis.

Strode et al [214] developed a reliable supercritical fluid chromatography method

for the analysis of lovastatin. Methanol-modified carbon dioxide was used to elute

the drug, and it’s dehydro lovastatin and hydroxy acid lovastatin degradation

products from a silica column. The hydroxy acid lovastatin was tailed in this

mobile phase. This was eliminated by the addition of trifluoroacetic acid to the

mobile phase which permitted the drug and its two main degradation products to

elute from the silica column in under 6 min with symmetrical peak shape.

Mazzo et al [215] developed a flow injection method to determine simultaneously

lovastatin, and butylated hydroxyanisole in tablets. The system involved ultraviolet

absorbance detection for the drug and oxidative amperometric electrochemical

detection for butylated hydroxyanisole. The method was found to be reproducible

for routine determinations with accuracy of ± 1 % for lovastatin and ± 4 % for

butylated hydroxyanisole. Precision for both analytes was approximately ± 1 %.

The method with UV detection was specific for the drug in the presence of

potential autoxidation products as well as butylated hydroxyanisole and its

oxidation products.

Chaudhari et al [216] developed a simple and reproducible HPTLC method for the

separation and quantitation of simvastatin, pravastatin sodium and rosuvastatin

calcium, in pharmaceutical dosage forms. The stationary phase used was precoated

silica gel. The mobile phase was a mixture of chloroform, methanol and toluene


50

(6:2:2, v/v/v). All the drugs were extracted from the respective tablets using

methanol. The percentage recoveries ranged from 100 % to 101 % for simvastatin,

98 % to 101 % for pravastatin sodium and 98 % to 102 % for rosuvastatin calcium.

The LOD for simvastatin, pravastatin sodium and rosuvastatin calcium were found

to be 15 ng/spot, 9 ng/spot and 8 ng/spot, respectively and LOQ were 200 ng/spot

for simvastatin and 100 ng/spot for pravastatin sodium and rosuvastatin calcium.

2.4 Analytical Methods for Rosuvastatin

Suslu et al [217] developed and validated a capillary zone electrophoretic method

with diode array detection for the determination of rosuvastatin calcium in

pharmaceutical formulations. Optimum results were obtained with 0.05 M borate

buffer at pH 9.5, capillary temperature 30 0C and applied voltage 25 kV. The

samples were injected hydrodynamically for 5 s at 50 mbar. Detection wavelength

was set at 243 nm. The migration times of rosuvastatin calcium and diflunisal were

3.20 ± 0.01 minutes and 4.20 ± 0.02 minutes. The total time of analysis was less

than 6 minutes.

Uyar et al [218] developed a simple, rapid and reliable spectrophotometric method

for the determination of rosuvastatin calcium in pharmaceutical preparations. The

solutions of standard and pharmaceutical samples were prepared in methanol at 243

nm. The developed method was validated with respect to linearity, range, LOD and

LOQ, accuracy, precision, specificity and ruggedness. The linearity range of the

method was 1.0–60.0 µg/mL and LOD was 0.33 µg/mL.

Gao et al [219] developed and validated a sensitive liquid chromatography/tandem

mass spectrometric method for the determination of rosuvastatin in human plasma.

Chromatographic separation was accomplished on a C18 column. The mobile

phase consisted of methanol-water (75:25, v/v, adjusted to pH 6 by aqueous

ammonia). Detection was achieved by ESI MS/MS in the negative ion mode. The


51

LOQ was 0.02 ng/mL. The linear range of the method was from 0.020 to 60.0

ng/mL. The intra and inter-day precisions were lower than 8.5 % and the accuracy

was within -0.3 to 1.9% in terms of relative error (RE).

Lan et al [220] developed and validated a simple and sensitive liquid

chromatography/tandem mass spectrometry method for the quantification of

rosuvastatin in human plasma. The analyte was extracted by simple one-step liquid-

liquid extraction. The chromatographic separation was performed on a C18 column

with a mobile phase consisting of 2 % formic acid/methanol (20:90, v/v) at a flow

rate of 1.00 mL/min. The retention time of rosuvastatin was 2.3. Triple-quadrupole

MS/MS detection was operated in positive mode by monitoring the transition of

m/z 482-->258 for rosuvastatin. The LOQ was 0.1ng/mL and the assay was linear

from 0.1-20 ng/mL. Inaccuracy was less than 8.4 % and imprecision less than 12.8

% at all tested concentration levels.

Vittal et al [221] described a simple, sensitive and specific high-performance liquid

chromatography method for simultaneous determination of rosuvastatin (RST) and

gemfibrozil (GFZ) in human plasma. Following separation, the residue was

reconstituted in the mobile phase and injected onto a C18 column. The

chromatographic run time was less than 20 min using flow gradient (0.0-1.60

mL/min) with a mobile phase consisting of 0.01 M ammonium acetate, acetonitrile

and methanol (50:40:10, v/v/v) and UV detection at 275 nm. Nominal retention

times of RST, GFZ and IS were 6.7 min, 13.9 min and 16.4 min, respectively. The

LOQ of RST and GFZ was 0.03 µg/mL and 0.30 µg/mL, respectively. Linearity

was in the 0.03-10 µg/mL and 0.3-100 µg/mL ranges for RST and GFZ,

respectively. The inter and intra-day precisions were in the range 2.37-9.78 % and

0.92-10.08 %, respectively.


52

Kumar et al [222] developed a specific, accurate, precise and reproducible high-

performance liquid chromatography method for the estimation of rosuvastatin in rat

plasma. The assay procedure involved simple liquid-liquid extraction. After

separation, rosuvastatin was reconstituted in the mobile phase and injected onto a

C18 column. Mobile phase consisting of 0.05 M formic acid and acetonitrile

(55:45, v/v) was used at a flow rate of 1.0 mL/min. The detection of the analyte

peak was achieved at 240 nm. The standard curve for RST was linear in the

concentration range of 0.02-10 µg/mL. Absolute recovery of RST was 85-110. The

LOQ was 0.02 µg/mL. The inter and intra-day precisions were in the range of 7.24-

12.43 % and 2.28-10.23 % respectively. Accuracy was in the range of 93.05-112.17

%.

Mehta et al [223] applied a forced degradation study for the development of a

stability-indicating assay for the determination of rosuvastatin in the presence of its

degradation products. Degradation of the drug was done at various pH values.

Moreover, the drug was degraded under oxidative, photolytic, and thermal stress

conditions. The proposed method was able to resolve all of the possible degradation

products formed during the stress study.

Hull et al [224] developed a selective, accurate and precise assay for the

quantification of the N-desmethyl metabolite of rosuvastatin in human plasma. The

method employed automated solid phase extraction followed by HPLC with

positive ion electrospray tandem MS. The standard curve range for N-desmethyl

rosuvastatin in human plasma was 0.5-30 ng/mL with 0.5 ng/mL being the value of

LOQ.

2.5 Analytical Methods for Gemfibrozil

Prabu et al [225] developed a simple, precise and rapid RP-HPLC method for the

determination of racecadotril in a pharmaceutical formulation using gemfibrozil as


53

internal standard. Ratio of the peak area of analyte to internal standard was used for

quantification. The chromatographic separation was carried out by using a Reverse

Phase C18 column. The mobile phase consisting of a mixture of 0.02 M phosphate

buffer (pH 3.5) and acetonitrile in the ratio of (40:60) with detection at 230 nm at a

flow rate of 1 mL/min was employed. The method was statistically validated for

linearity, accuracy and precision.

Kim et al [226] developed a sensitive and simple high performance liquid

chromatography for the determination of gemfibrozil in a small plasma sample.

The analysis of gemfibrozil in the plasma sample was carried out using a reverse

phase C18 column with fluorescence detection (a maximum excitation at 242 nm

and a minimum emission at 300 nm). A mixture of acetonitrile–0.4 % phosphoric

acid solution (53:47, v/v) was used as a mobile phase. The detection limit of this

method was 10 ng/mL. The method was linear over a range of 0.05 mg/mL –15

mg/mL. The inter- and intra-day precision did not exceed 15 %.

Ulu et al [227] developed and validated a simple, selective, precise and accurate

reversed phase-HPLC assay for analysis of gemfibrozil in tablets. Separation and

quantification were achieved on a C-18 column under isocratic conditions using a

mobile phase (methanol: water, 80:20, v/v) maintained at 1.1 mL/min. UV-

detection was at 280 nm. The method was linear over the range of 0.5 µg/mL –3.0

µg/mL. The LOD and LOQ were 0.20 µg/mL and 0.51 µg/mL respectively. The

intra-day and inter-day precision were below 1.74 and 1.83 %, respectively.

Roadcap et al [228] developed and validated a sensitive LC–MS/MS assay for the

quantitative determination of gemfibrozil in dog plasma. The assay involved the

extraction of the analyte from dog plasma using Chem Elut cartridges and methyl

tert.-butyl ether. Chromatography was performed on a Metasil basic column (50×2

mm I.D., 3 µm) using a mobile phase consisting of 70:30 acetonitrile–ammonium


54

acetate (0.001 M, pH 5.0) with a flow-rate of 0.2 mL/min. The method showed

inter and intra-assay precision of less than 8.9%, with inter and intra-assay accuracy

between 99 % and 101 %.

González-Peñas et al [229] developed a sensitive high-performance liquid

chromatographic assay for the quantitative determination of gemfibrozil. The assay

involved a single cyclohexane extraction and LC analysis with fluorescence

detection. Chromatography was performed at 40 0C on an ODS column. The

mobile phase was a mixture of a solution of phosphoric acid 0.4% and acetonitrile

(45:55). The detection limit was 0.025 µg/mL. The method was linear from 0.05 to

0.5 µg/mL. Intra and inter-day precision was less than 15 %. Mean recovery was

90.15 % for gemfibrozil.

Nakagawa et al [230] described sensitive and specific methods for the simultaneous

determination of gemfibrozil and its metabolites in plasma and urine. The methods

were based on a fully automated high performance liquid chromatographic system

with fluorescence detection. Urine samples, diluted with acetonitrile, were directly

analysed by HPLC using a flow and eluent programming method. In the case of

plasma, gemfibrozil and its main metabolites were extracted from acidified samples

and the resulting extracts injected into the chromatographic system. The sensitivity

was approximately 100 ng/mL for gemfibrozil and its four metabolites.

Hengy et al [231] described a sensitive and specific method for the determination

of gemfibrozil at therapeutic concentrations in plasma. The method was based on

high performance liquid chromatography. Gemfibrozil and the internal standard

ibuprofen were extracted from acidified plasma into cyclohexane and the resulting

residue was analyzed on a commercial reversed phase column with

acetonitrile/water 1:1 and 0.2 % phosphoric acid as mobile phase. The eluted peaks

were detected by UV-absorption at 225 nm. The sensitivity was approx. 50 ng/mL.


55

2.6 Analytical Methods for Fenofibrate

Kadav et al [232] developed and validated a stability indicating UPLC method for

the simultaneous determination of atorvastatin, fenofibrate and their impurities in

tablets. The chromatographic separation was performed on C18 column (1.7 µm,

2.1 mm × 100 mm) using gradient elution of acetonitrile and ammonium acetate

buffer (pH 4.7; 0.01 M) at flow rate of 0.5 mL/min. UV detection was performed at

247 nm. Total run time was 3 min within which main compounds and six other

known and major unknown impurities were separated. The method was validated

for accuracy, repeatability, reproducibility and robustness. Linearity, LOD and

LOQ.

Nakarani et al [233] developed two simple and accurate methods to determine

atorvastatin and fenofibrate in combined dosage using second-derivative

spectrophotometry and reversed-phase liquid chromatography. Atorvastatin and

fenofibrate in combined preparations were quantitated using the second-derivative

responses at 245.64 nm for atorvastatin and 289.56 nm for fenofibrate in spectra of

their solution in methanol. The method was linear in the concentration range of 3–

15 µg/mL for atorvastatin and fenofibrate. In the HPLC method, analysis was

performed on a C-18 column in the isocratic mode using the mobile phase

methanol-water (90 + 10, v/v), adjusted to pH 5.5 with orthophosphoric acid, at a

flow rate of 1 mL/min. Measurement was made at a wavelength of 246.72 nm. The

method was linear in the concentration range of 3–15 µg/mL for atorvastatin and

fenofibrate.

Straka et al [234] determined steady-state fenofibric acid serum concentrations

using anion-exchange solid-phase extraction in combination with reverse-phase

HPLC. Chromatographic separation under isocratic conditions, with use of

ultraviolet detection at 285 nm, provided clean baseline and sharp peaks for

clofibric acid, 1-napthyl acetic acid (internal standards), and fenofibric acid. The


56

assay was employed to quantify fenofibric acid in more than 800 human subject

specimens. Fenofibric acid analysis was found to be linear over the range of 0.5

mg/L to 40 mg/L. Accuracies ranged from 98.65 % to 102.4 %, whereas the within-

and between-day precisions ranged from 1.0 % to 2.2 % and 2.0 % to 6.2 %,

respectively.

El-Gindy et al [235] presented several spectrophotometric and HPLC methods for

the determination of fenofibrate, vinpocetine and their hydrolysis products. The

resolution of either fenofibrate or vinpocetine and their hydrolysis products were

accomplished by using numerical spectrophotometric methods as partial least

squares (PLS-1) and principal component regression (PCR) applied to UV spectra;

and graphical spectrophotometric methods as first derivative of ratio spectra (1DD)

or first (1D) and second (2D) derivative spectrophotometry for vinpocetine and

fenofibrate, respectively. In addition HPLC methods were developed using ODS

column with mobile phase consisting of acetonitrile-water (80:20, v/v, pH 4) with

UV detection at 287 nm for fenofibrate and a mobile phase consisting of

acetonitrile-0.001 M KH2PO4, containing 0.1 % diethylamine (60:40, v/v, pH 4.6)

with UV detection at 270 nm for vinpocetine. The proposed methods were

successfully applied for the determination of each drug and its hydrolysis product

in laboratory-prepared mixture and pharmaceutical preparation.

Yardimci et al [236] investigated the electrochemical reduction of fenofibrate at a

hanging mercury drop electrode by cyclic voltammetry, square-wave voltammetry,

and chronoamperometry. The best analytical signals was found in borate buffer

(pH 9.0)–tetra butyl ammonium iodide mixture containing 12.5 % (v/v) methanol at

–1.2 V (versus Ag/AgCl). According to cyclic voltammetric studies, the reduction

was irreversible and diffusion controlled. The diffusion coefficient was 2.38×10–

6 cm2 s–1 as determined by chronoamperometry. Under optimized conditions of

square-wave voltammetry, a linear relationship was obtained between 0.146–


57

4.96 µg/mL of fenofibrate with LOD of 0.025 µg/mL. Validation parameters such

as sensitivity, accuracy, precision, and recovery were evaluated.

Hernando et al [237] described a multi residue method for the extraction and

determination of two therapeutic groups of pharmaceuticals, lipid-regulating agents

(clofibric acid, bezafibrate, gemfibrocil, fenofibrate) and beta-blockers (atenolol,

sotalol, metoprolol, betaxolol) in waters by solid-phase extraction followed by

liquid chromatography-electrospray ionization tandem mass spectrometry.

Recoveries obtained from spiked HPLC water, as well as, from spiked real samples

were all above 60 %, with the exception of betaxolol with a 52 % recovery. The

quantitative MS analysis was performed using a multiple reaction monitoring. The

LC-MS-MS method gave detection limits ranging from 0.017 µg/L to 1.25 µg/L in

spiked effluent. Precision of the method ranged from 3.7 % to 18.5 %.

Lossner et al [238] described a sensitive HPLC method for the determination of

fenofibric acid (FA) in serum. FA from human serum samples was isolated by an

easy one step extraction procedure with a mixture of n-hexane and ethyl acetate

(90:10, v/v). The recovery was 84.8 % of the total FA in serum. The compound was

separated isocratically on a reversed phase column with acetonitrile and 0.02 M

phosphoric acid (55:45, v/v) at a flow-rate of 1 mL/min. Absorbance at 287 nm was

recorded for quantification. The LOD was 0.03 µg/mL and the LOQ was 0.1

µg/mL.

Streel et al [239] developed a new fully automated method for the determination of

fenofibric acid in plasma which involved the solid-phase extraction (SPE) of the

analyte from plasma on disposable extraction cartridges (DECs) and reversed-phase

HPLC with UV detection. After extraction, 100 µL of the extract was directly

introduced into the HPLC system. The liquid chromatographic separation of the

analytes was achieved on a RP-8 stationary phase. The mobile phase consisted of a

mixture of methanol and 0.04 M phosphoric acid (60:40, v/v). The analyte was

monitored photometrically at 288 nm. The absolute recovery was close to 100 %


58

and a linear calibration curve was obtained in the concentration range from 0.25

µg/mL to 20 µg/mL. The mean RSD values for repeatability and intermediate

precision were 1.7 and 3.9 % respectively.

Lacroix et al [240] developed HPLC methods for drug content and HPLC and

NMR methods for related compounds in fenofibrate raw materials. The HPLC

methods resolved 11 known and six unknown impurities from the drug. The HPLC

system was comprised of ODS column, a mobile phase consisting of acetonitrile,

water, trifluoroacetic acid in the ratio of 700/300/l (v/v/v) at a flow rate of 1

mL/min and a UV detector set at 280 nm. Minimum quantifiable amounts were

about 0.1 % for three of the compounds and less than 0.05 % for the other eight.

Individual impurities in 14 raw materials ranged from trace levels to 0.25 %, and

total impurities from 0.04 to 0.53 % (w/w). Six unknown impurities were detected

by HPLC, all at levels below 0.10 %. An NMR method for related compounds was

also developed and it was suitable for 12 known and several unknown impurities.

The results for related compounds by the two techniques were consistent. The main

differences stem from the low sensitivity of the HPLC method for some of the

related compounds at 280 nm, or from the higher limits of quantitation by the NMR

method for several other impurities using the conditions specified. Results for the

assay of 15 raw materials by HPLC were within the range 98.5-101.5%.

Abe et al [241] developed a reliable HPLC method for the determination of

fenofibric acid and reduced fenofibric acid in the biological samples. After addition

of the internal standard solution and 0.5 M HCl to the biological sample, fenofibric

acid, reduced fenofibric acid and the internal standard were extracted with a mixed

solvent of n-hexane and ethyl acetate (90:10) from the mixture. The acids were

back-extracted from the organic phase with 0.1 M Na2HPO4 and then re-extracted

from the aqueous phase with a mixed solution of n-hexane and ethyl acetate (95:5)

after addition of 0.5 M HCl. The organic phase was evaporated to dryness under


59

the vacuum. The residue was dissolved in methanol and diluted with distilled

water. An aliquot of the resulting solution was injected on the HPLC.

Masnatta et al [242] developed a selective high-performance liquid

chromatographic method to assess either bezafibrate, ciprofibrate or fenofibric acid

plasma levels. Drugs were extracted with diethyl ether, after acidification with

HCL. An isocratic acetonitrile-0.02 M H3PO4 (55:45) mobile phase, a C18 column

and UV detection were used. The LOQ was 0.25 µg/mL for the three fibrates. Intra-

and inter-assay accuracy ranged were 90-107 % and 82-111 %: 96-115 % and 94-

107 %: 94-114 % and 94-126 % for bezafibrate, ciprofibrate and fenofibric acid,

respectively. Intra- and inter-assay precision were 1.72-3.06 % and 2.66-7.67 %:

1.88-4.64 % and 0.62-2.99 %: 1.26-4.69 % and 3.56-7.17 % for the three fibrates

studied.

2.7 Analytical Methods for Ezetimibe

Doshi et al [243] developed and validated a simple, precise, and accurate HPLC

method for the assay of ezetimibe in tablets and for determination of content

uniformity. Reversed-phase liquid chromatographic separation was achieved by use

of phosphoric acid (0.1 %, v/v)–acetonitrile 50:50 (v/v) as mobile phase. The

method was validated for specificity, linearity, precision, accuracy, robustness, and

solution stability. Method was linear in the concentration range of 20–80 µg/mL.

Accuracy was between 100.8 % and 102.7 %.

Dixit et al [244] established a simple, selective and stability-indicating HPTLC

method for the analysis of simvastatin and ezetimibe. The method used aluminum-

backed silica gel 60F254 TLC plates as stationary phase with n-hexane–acetone 6:4

(v/v) as mobile phase. Densitometric analysis of both drugs was carried out in

absorbance mode at 234 nm. Method was linear in the range of 200–1,600 ng/band.

The LOD and LOQ were 25 ng/band and 150 ng/band, respectively. Simvastatin


60

and ezetimibe were subjected degradation by acid, pH 6.8 phosphate buffer,

oxidation, dry heat, and wet heat. The degradation products were well resolved

from the pure drug with significantly different R F values.

Sharma et al [245] developed, UV, first, second and third derivative

spectrophotometric methods for the determination of ezetimibe in pharmaceutical

formulation. For the first method, based on UV spectrophotometry, the quantitative

determination of the drug was carried out at 233 nm and the linearity range was

found to be 6-16 µg/mL. For the first, second and third derivative

spectrophotometric methods the drug was determined at 259.5 nm, 269 nm and 248

nm with the linearity ranges 4-14 µg/mL, 4-14 µg/mL and 4-16 µg/mL.

Basha et al [246] accomplished simultaneous separation and quantification of

ezetimibe (EZM) and its phase-I metabolite i.e., ezetimibe ketone (EZM-K) and

phase-II metabolite i.e., ezetimibe glucuronide (EZM-G) in various matrices by

gradient HPLC with UV detection. The assay involved deproteinization of 500 µL

of either incubation or bile sample containing analytes and internal standard (IS,

theophylline) with 75 µL acetonitrile containing 25 % perchloric acid. An aliquot

of 100 µL supernatant was injected onto a C-18 column. The chromatographic

separation was achieved by gradient elution consisting of 0.05 M formic acid:

acetonitrile: methanol: water at a flow rate of 1 mL/min. The detection of analyte

peaks were achieved at 250 nm. Average extraction efficiencies of EZM, EZM-G

and IS was greater than 75-80 % and for EZM-K was greater than 50 % from all

the matrices tested. LOQ for EZM, EZM-K and EZM-G was 0.02 µg/mL.

Rajput et al [247] developed a simple, accurate and precise spectroscopic method

for the simultaneous estimation of ezetimibe and simvastatin in tablets using first

order derivative zero-crossing method. Ezetimibe showed zero crossing point at

245.4 nm while simvastatin showed zero crossing point at 265.2 nm. The method

was linear in the range of 5-40 µg/mL for ezetimibe at 265.20 nm. The linear


61

correlation was obtained in the range of 5-80 µg/mL for simvastatin at 245.4 nm.

The limit of detection was 0.39 µg/mL and 0.12 µg/mL for ezetimibe and

simvastatin, respectively. The LOQ was 1.10 µg/mL and 0.4 µg/mL for ezetimibe

and simvastatin, respectively.

Singh et al [248] developed a stability-indicating HPLC method for the analysis of

Ezetimibe in the presence of the degradation products. Ezetimibe was subjected to

different ICH prescribed stress conditions. It involved a C-8 column and a mobile

phase composed of ammonium acetate buffer (0.02 M, pH adjusted to 7.0 with

ammonium hydroxide) and acetonitrile, which was pushed through the column in a

gradient mode. The detection was carried out at 250 nm. The method was validated

for linearity, range, precision, accuracy, specificity, selectivity and intermediate

precision.

Oliveira et al [249] developed and validated an analytical method based on liquid

chromatography-tandem mass spectrometry for the determination of ezetimibe in

human plasma. Ezetimibe and etoricoxib (internal standard) were extracted from

the plasma by liquid-liquid extraction and separated on a C-18 analytical column

with acetonitrile: water (85:15, v/v) as mobile phase. Detection was carried out by

positive electrospray ionization (ESI+) in multiple reactions monitoring (MRM)

mode. The chromatographic separation was obtained within 2.0 min and the

method was linear in the concentration range of 0.25–20 ng/mL for free ezetimibe

and of 1–300 ng/mL for total ezetimibe. The mean extraction recoveries for free

and total ezetimibe from plasma were 96.14 % and 64.11 %, respectively.

Oswald et al [250] developed a selective assay to measure serum concentration–

time profiles, renal and fecal elimination of ezetimibe in pharmacokinetic studies.

Ezetimibe was measured after extraction with methyl tert-butyl ether using 4-

hydroxychalcone as internal standard and liquid chromatography coupled with

tandem mass spectrometry (LC–MS/MS) for detection. The chromatography was


62

done isocratically with acetonitrile/water (60/40, v/v; flow rate 200 µl/min) using

C-18 Column. The MS/MS analysis was performed in the negative ion mode. The

validation ranges for ezetimibe and total ezetimibe were as follows: serum 0.0001–

0.015 µg/mL and 0.001–0.2 µg/mL; urine and fecal homogenate 0.025–10 µg/mL

and 0.1–20 µg/mL, respectively.

Sistla et al [251] developed a rapid, specific reversed-phase HPLC method for

assaying ezetimibe in pharmaceutical dosage forms. The assay involved an

isocratic elution of ezetimibe on a C18 column using a mobile phase composition

of water (pH 6.8, 0.05%, w/v 1-heptane sulfonic acid) and acetonitrile (30:70, v/v).

The flow rate was 0.5 mL/min and the analyte monitored at 232 nm. The assay was

linear from 0.5 to 50 µg/mL. All the validation parameters were within the

acceptance range.

atorva bioanalytical

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