ISSN: 0970-020 X

ORIENTAL JOURNAL OF CHEMISTRY CODEN: OJCHEG

An International Open Free Access, Peer Reviewed Research Journal
2012, Vol. 28, No. (1):
Pg. 237-241
Est. 1984
www.orientjchem.org

A New HPLC Method for Determination of Losartan in

Human Plasma and its Application in Bioequivalence Studies

F. SHOKRANEH1, A. DABIRSIAGHI1* and N. ADIB2

¹Department of Pharmaceutics, Pharmaceutical Sciences Branch,

Islamic Azad University (IAU), Tehran, Iran.
²Food and Drug Lab Research Center, Ministry of Health, Tehran (Iran).
*Address For Correspondence: Islamic Azad University(IAU), DrShariati St. Gholhak,
Yakhchal St. Tehran, Iran.19395-6466
Email: [email protected]

(Received: February 04, 2012; Accepted: March 11, 2012)

ABSTRACT

A reliable, simple and sensitive reversed-phase high-performance liquid chromatographic

method was developed for determination of losartan in plasma .Separation is achieved by HPLC
after direct injection on a CN (250*4.6 mm) analytical column with a mobile phase composed of
sodium hydrogen phosphate buffer - acetonitrile - tetrahydrofurane - methanol and phosphoric
acid (0.1:5 :4 :21 :69.9 ) V/V% adjusted to pH= 9.9. Detection is by ultraviolent absorbance at
254nm. The flow rate was set at 0.6 ml/min. The lower limit of quantitation was 5 ng/ml. The intra and
inter-day precisions (CV %) of the quality control samples were 0.57-5.31% and 0.21 -4.52%
respectively. The recovery of method was 92.25± 2.19. The method was applied to a bioequivalence
study in human.

Key words: Losartan, Human plasma, HPLC, Bioequivalence

INTRODUCTION losartan as an AT1-receptor antagonist. The

metabolism of losartan to EXP 3174 and to inactive
The renin-angiotensin (RAS) is a major metabolites is mediated by CYP2C9 and CYP3A4.
regulator of blood pressure (BP), 1 Losartan Peak plasma levels of losartan and EXP 3174 occur
(COZAAR) is the prototype of a new class of orally 1–3 hours after oral administration, respectively,
active, non –peptide angiotensin II receptor and the plasma half-lives are 2.5 and 9 hours,
antagonists (ARBs) able to inhibit the renin- respectively. The plasma clearances of losartan and
angiotensin system specifically and selectively EXP 3174 (600 and 50 mL/min, respectively) are
without the agonistic effects of the peptide receptor due to renal clearance (75 and 25 mL/min,
antagonists.2Approximately 14% of an oral dose of respectively) and hepatic clearance (metabolism
losartan is converted to the 5-carboxylic acid and biliary excretion). The plasma clearance of
metabolite EXP 3174, which is more potent than losartan and EXP 3174 is affected by hepatic but
238 SHOKRANEH et al., Orient. J. Chem., Vol. 28(1), 237-241 (2012)

not renal insufficiency. Losar tan should be concentration (2.5 to 500ng/ml) of Losartan curve.
administered orally once or twice daily for a total Data from HPLC in defined circumstances was
daily dose of 25–100 mg.3 Last studies showed that analyzed and evaluated.
several HPLC ,LC/MS/MS methods were used for
determination of losartan and its metabolite in Instrument and chromatographic conditions
human plasma.4-12The aim of this study was to Analyses were performed on younglin
develop a simple, rapid sensitive and reliable HPLC model ACME-900 pump equipped with Ultraviolet
method with Ultraviolent detection for quantization detector at wavelength of 254nm. Chromatography
of losartan in human plasma samples and to was performed at room temperature on a CN
compare the bioavailability of two losartan tablets column (250*4.6mm). The mobile phase consisted
(50 mg) formulations (losartan from Iranian of sodium hydrogen phosphate buffer - acetonitrile
company, as a test formulation and COZAR, MSD - tetrahydrofurane - methanol and phosphoric acid
,The Netherlands as a reference formulation) . The (0.1:5:4:21:69.9) V/V% adjusted to pH 9.9 using
method was validated according to procedures and phosphoric acid. The flow rate was set at 0.6ml/min
acceptance criteria based on FDA guideline and respectively.
recommendations of ICH, to provide enough
selectivity, sensitivity and reliability in Method validation
pharmacokinetic and bioequivalence studies. The method validation demonstrated the
specificity, lower limit of quantification (LOQ),
MATERIALS AND METHODS recovery, linearity, precision and accuracy of
measurements.13
Hydrochloric acid, methanol (HPLC grade)
,ammonia ,phosphoric acid, sodium hydrogen Specificity was investigated by analyzing
phosphate ,acetonitrile ,tetrahydrofurane , were six drug-free plasma samples for interference of
purchased from Merc. Losartan and Thioridazine endogenous compounds. For calibration curve five
were USP reference standard. different concentrations of losartan (2.5 to 500ng/
ml) in plasma were prepared by adding required
Sample and standard solutions preparation volume of working solutions to blank plasma. Plasma
Blood samples were collected from calibration curve was prepared by taking area ratio
individuals and centrifuged in order to separate the of analyte to internal standard as Y-axis and
plasma. (Separated plasma was stored at -20 °C.). concentration of analyte (ng/ml) as X-axis. Linearity
To a 0.5 ml aliquot of plasma, 1ml of internal standard of the standard curve was evaluated using least
(thioridazine) was added. 500 µl of 0.2 M HCL was squares linear regression analysis. The limit of
used to acidify the solution and then vortexed and quantification (LOQ) was taken in this work at the
transferred in to the cartridge (Bond ElutTM) CN which lowest concentration standard affording accuracy
was prepared by using pure methanol and water in and precision d”20%, using five plasma samples.
a proportion of 2:1. Initially 500µl distilled water and The intra and inter-day precisions (CV %)of the
10% methanol followed with 50µl of pure methanol assay procedure were determined by trice analysis
was put through the cartridge. The internal standard of quality control plasma samples (50 ,200 and 500
and drug was then collected. The pH of the solution ng/ml) at the someday and three different days.
was made alkaline (pH =8) using ammonia/ Recovery was determined by comparing the
methanol in a proportion of 1:99 respectively and response of three pre-treated quality control plasma
then dried under Nitrogen. The mobile phase was samples in three levels (25, 100 and 250 ng/ml)
then poured in to the product in order to reduce it with the absolute peak area of un-extracted samples
(The product was then reduced using the mobile containing the same concentration of the drug as
phase (100µl)). 80µl of this solution was injected in 100%.
to the HPLC with UV detector with the flow rate of
0.6ml/min and the column of CN (250*4.6mm). Stock Application
solution of losartan (25 to 5000ng/ml) was prepared The validated method was used in
and diluted by blank plasma to obtain the final bioequivalence study of losartan. It was an open,
 SHOKRANEH et al., Orient. J. Chem., Vol. 28(1), 237-241 (2012) 239

one-centre, cross-over ,randomized, three way and RESULTS AND DISCUSSION

double-blind study to asses relative bioavailability
of losartan in twenty-four healthy volunteers Some HPLC and LC-MS-MS methods
following single dose administration of losartan as have been developed for determination of losartan.
50 mg tablet (All subject gave informed consent to The proposed method is suitable for losartan
the work). The reference (COZAR, MSD, The quantification in plasma samples. The based on a
Netherlands) and test (manufactured by Iranian simple liquid – liquid extraction (LLE) followed by
company) product were used in this study. The high – performance liquid chromatography with UV
blood collecting times were 0 ,0.25 ,0.5 ,0.75 ,1 ,1.5 detector, using thioridazine as an internal standard,
,2 ,3 ,4 ,5 ,6 ,8 ,12 ,24 ,36 ,48 hour after oral respectively. Blood samples were collected at
administration of 50mg losartan reference and test specified time intervals, and the plasma separated
to fasting volunteers. The plasma samples were and analyzed for losartan. Blank plasma and spiked
analyzed by the described method. The plasma with losartan and internal standard are
pharmacokinetic parameters like area under the shown in Fig. 1. Retention time for the losartan and
plasma-concentration curve from zero to the last internal standard were 3.5 min and 8 min. The
measurable losartan sample time and to infinity chromatograms also confirmed the complete
(AUC o-t and AUC o-inf),maximum concentration (C separation. The calibration cur ve could be
max
) ,time to maximum concentration (T max) were described by the equation:
determined for the period of 0-48 hour.
Conc = 77area% + (-6.1), r2 =0.9921
Statistical analysis
The analysis of variance was performed The assay exhibited a linear dynamic
on data for differences between and within the range of 2.5 – 500 ng/ml, a run time of 20 min for
subspecies using the ANOVA (SPSS ver.10). mean each sample made it possible to analyze more than
separation were determined by least significant 70 samples per day. The limit of quantitation (LOQ )
difference (LSD) at P≤0.05% . was 5 ng/ml. Intra and inter-day precisions (CV%)

Volt (mV)
131.03

100.57

Losartan Thioridazine
70.10

39.64

9.17

Fig.1. Chromatograms of (1) Blank human plasma; (2) Plasma spiked with thioridazine (rt=3.5min)
and losartan (rt=8.0 min); (3) Human plasma after administration of losartan tablet
240 SHOKRANEH et al., Orient. J. Chem., Vol. 28(1), 237-241 (2012)

Table 1: Precision and Recovery of Losartan Assay in Human Plasma (n=3)

Spiked Intra-day Inter-day Spiked Recovery

(ng/ml) (ng/ml) %
Found SD CV% Found SD CV%

50 50.30 6.02 11.51 55.66 2.51 4.52 25 89.99

200 207.30 11.01 5.31 205.66 2.51 1.22 100 94.37
500 501.10 2.85 0.57 501.46 1.05 0.21 250 92.38
Losartan Conc (ng/ml)

Sampling Time (hour)

Fig. 2: Mean plasma concentration – time curve for test and reference preparation following single
oral administration of losartan 50 mg tablet in 24 healthy volunteers

of the quality control samples were 0.57 – 5.31% were observed for C max, T max, AUC o-infand other
and 0.21 – 4.52% ,the accuracy of this bioanalytical pharmacokinetic parametersand the test
method was 102.85±2.33 respectively (Table 1). The formulation is bioequivalent to the reference
overall intra and inter assay variations were within formulation for losartan.
acceptance limits according to FDA guidelines.
The paper describes a rapid and
The plasma samples from 24 health reproducible HPLC method which enables the
human volunteers were assayed with the validated determination of losartan in plasma.
method described above. The mean concentration
– time curve is shown in figure 2. The main advantage of this method is the
use of precipitation for purification, which is easily
Maximum plasma concentration (C max) and fast in comparison with other purification and
ranged from 104.53 to 176.44 ng/ml for test product extraction methods. This HPLC method is reliable,
and 121.03 to 192.60 ng/ml for reference product at reproducible and sensitive with respect to validation
1.5 to 4 hour (T max). Also the mean value of area parameters. It can be used as an assay method in
under the concentration time curve (AUC o-t) the study of losartan pharmacokinetics as well as
obtained was 1485.60 ng h/ml, AUC o-inf was found bioavailability/ bioequivalence studies.
to be 2397.45 ng h/ml . No statistical differences
 SHOKRANEH et al., Orient. J. Chem., Vol. 28(1), 237-241 (2012) 241

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