ORIGINAL PAPER

New UPLC–MS/MS method for simultaneous determinationof irbesartan and hydrochlorthiazide in human plasma

Seema Zargar • Tanveer Ahmad Wani

Received: 27 July 2013 / Accepted: 7 February 2014

� Iranian Chemical Society 2014

Abstract Ultra-performance liquid chromatography–tan-

dem mass spectrometry (UPLC–MS/MS) is a preeminent

analytical tool for rapid biomedical analysis with the

objective of reducing analysis time and maintaining good

efficiency. In this study a simple, rapid, sensitive and

specific ultra-performance liquid chromatography–tandem

mass spectrometry method was developed and validated

for quantification of the angiotensin II receptor antagonist,

irbesartan and hydrochlorthiazide in human plasma. After a

simple protein precipitation using methanol and acetoni-

trile, irbesartan, hydrochlorthiazide and internal standard

(IS) telmisartan were separated on Acquity UPLC BEHTM

C18 column (50 9 2.1 mm, i.d. 1.7 lm, Waters, USA)

using a mobile phase consisting of acetonitrile:10 mM

ammonium acetate:formic acid (85:15:0.1 % v/v/v)

pumped at a flow rate of 0.3 mL/min and detected by

tandem mass spectrometry with negative ion mode. The ion

transitions recorded in multiple reaction monitoring mode

were m/z 427.2 ? 193.08 for irbesartan, m/z 295.93 ?268.90 for hydrochlorthiazide and m/z 513.2 ? 287.14 for

IS. The assay exhibited a linear dynamic range of

30–500 ng/mL for irbesartan and 1–500 ng/mL in human

plasma with good correlation coefficient of (0.996) and

(0.997) and with a limit of quantitation of 30 and 1 ng/mL

for irbesartan and hydrochlorthiazide, respectively. The

intra- and inter-assay precisions were satisfactory; the

relative standard deviations did not exceed 10.13 % for

irbesartan and 11.14 % for hydrochlorthiazide. The pro-

posed UPLC–MS/MS method is simple, rapid and highly

sensitive, and hence it could be reliable for pharmaco-

kinetic and toxicokinetic study in both animals and

humans.

Keywords Irbesartan � Hydrochlorthiazide � Ultra-

performance liquid chromatography � Tandem mass

spectrometry � Pharmacokinetic � Toxicokinetic � High-

throughput analysis

Introduction

Most cardiovascular events are attributed to high blood

pressure. Hence, antihypertensive therapy is to reduce

considerably the risk of developing cardiovascular com-

plications that cause a high mortality rate in the patients

with hypertension. Hydrochlorothiazide (HCT) 6-chloro-

3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-

dioxide, is one of the oldest thiazide diuretics, often pre-

scribed in combination with other antihypertensive drugs

such as beta blockers, angiotensin-converting enzyme

inhibitors, or angiotensin II receptor blockers [1–3]. Irbe-

sartan (IBS), 2-butyl-3-[[20-(tetrazol-5-yl)biphenyl-4-yl]-

methyl]-1,3-diazaspiro[4.4]non-1-en-4-one, is a potent and

selective angiotensin II subtype 1 receptor antagonist

indicated for use in patients with hypertension, in addition

to those with type 2 diabetes mellitus and nephropathy [4].

Angiotensin II is regarded as the main effector of AT1

receptor in renin–angiotensin system. It causes vasocon-

striction, tachycardia, increase of aldosterone secretion

from the adrenal cortex and retention of sodium and body

fluid [4, 5]. The combination of IBS and HCT dosage form

S. Zargar

Department of Biochemistry, College of Science, King Saud

University, P.O. Box 22452, Riyadh 11211, Saudi Arabia

T. A. Wani (&)

Department of Pharmaceutical Chemistry, College of Pharmacy,

King Saud University, P.O. Box 2457, Riyadh 11451,

Saudi Arabia

e-mail: tanykash@yahoo.co.in

123

J IRAN CHEM SOC

DOI 10.1007/s13738-014-0429-3

has been indicated for treatment of edema and hypertension

and clinical studies conducted in this regard have shown

that the combination is clinically effective with a good

safety profile [6, 7]. IBS/HCT provided consistent blood

pressure lowering and tolerability regardless of age, obes-

ity, and prevalence type 2 diabetes and greater efficacy in

patients with high cardiovascular risk [8].

A number of methods have been employed for the

analysis of hydrochlorothiazide concentration alone or in

combination with other drugs in biological samples by

high-performance liquid chromatography (HPLC) with

ultraviolet or electrochemical detection [9–14], LC–MS

[15, 16] or with tandem LC–MS/MS [17–21]. However,

most of them were time consuming, insufficiently sensitive

and employed tedious liquid–liquid extraction. The liquid–

liquid extraction methods involved endogenous several

steps yielding poor separation from the plasma interfer-

ences and gave highly variable and relatively low recov-

eries. Several HPLC methods have also been reported for

the determination of IBS in human plasma which include;

HPLC coupled with UV [22], fluorescence [23, 24].

Although the simultaneous determination of IBS and HCT

has previously been suggested by other workers, who

employed HPLC coupled with UV detection [25, 26], or

with tandem LC–MS/MS detection [27] such methods

suffered from lack of sensitivity, demonstrated by the

lower limits of quantitation.

UPLC is a new category of separation science which

builds upon well-established principles of liquid chroma-

tography, using sub-2 lm porous particles. These particles

operate at elevated mobile phase linear velocities to pro-

duce significant reductions in separation time and solvent

consumption. Literature indicates that a UPLC system

allows approximately ninefold decreases in analysis time

as compared to the conventional high-performance (HP)

LC system using 5 lm particle size analytical columns, and

approximately threefold decrease in analysis time in

comparison with 3 lm particle size analytical columns

without compromise on overall separation [28–33]. Ac-

quity UPLC columns contain hybrid X-Terra sorbent,

which utilizes bridged ethyl siloxane/silica hybrid (BEH)

structure, ensures the column stability under the high

pressure and wide pH range (1–12) [33]. In all documented

references, no UPLC–MS/MS method has been used to

determinate IBS and HCT presence and concentration in

human plasma until now.

The present study describes the development and vali-

dation of a UPLC method coupled with tandem mass

spectrometry (UPLC–MS/MS) for the determination of

IBS and HCT in human plasma. The proposed method used

is more sensitive and relatively simple extraction procedure

using methanol and acetonitrile to directly precipitate

protein in combination with UPLC–MS/MS detection.

Experimental

Materials and methods

Hydrochlorthiazide was obtained from Sigma Chemical

Co. (St. Louis, USA). Telmisartan and irbesartan (IS) were

obtained from AK Scientific Inc. (CA, USA). Human

plasma was obtained from normal healthy volunteers at

King Khalid University Hospital (Riyadh, Saudi Arabia),

and they were kept frozen at -20 �C until analysis. HPLC-

grade acetonitrile, methanol and ammonium acetate were

obtained from Winlab Laboratory, UK. Formic acid was

obtained from BDH Laboratory, UK. All other reagents

were of analytical grade unless stated otherwise. All

aqueous solutions was prepared using water that was

purified using Milli-QR Gradient A10R (Millipore, Mosc-

heim Cedex, France) having pore size 0.22 lm.

Apparatus and operating condition

Liquid chromatography

The UPLC system included quaternary solvent manager, a

binary pump, degasser, autosampler with an injection loop

of 10 lL and a column heater-cooler. The separation was

performed on Acquity UPLC BEHTM C18 column

(50 9 2.1 mm, i.d., 1.7 lm, Waters, USA) maintained at

40 �C. The mobile phase was composed of acetoni-

trile:10 mM ammonium acetate:formic acid (85:15:0.1 %

v/v/v)) pumped at a flow rate of 0.3 mL/min The injection

volume was 5 lL in partial loop mode and the temperature

of the autosampler was kept at 4 �C.

Mass spectrometric conditions

Waters Acquity liquid chromatography system coupled

with a Waters TQD triple quadrupole mass spectrometer

was used (Waters, Milford, USA). Mass spectrometric

detection was carried out using an electrospray interface

(ESI) operated in the negative ionization mode with

multiple reaction monitoring (MRM) for all IBS, HCT and

IS. Nitrogen was used as a desolvating gas at a flow rate

of 550 L/h. The desolvating temperature was set at 350 �C

and the source temperature was set at 150 �C. The colli-

sion gas (argon) flow was set at 0.1 mL/min. The capillary

voltage was set at 3.2 kV. Decrease in capillary voltage

had minimal effect on peak intensity, however, decreasing

the capillary voltage reduced the peak intensity drasti-

cally. The MS analyzer parameters were as follows: LM1

and HM1 resolution 15.0 and 15.0; ion energy 1, 0.8 V;

LM2 and HM2 resolution 12.0 and 14.0, respectively, ion

energy 2, 0.1 V, dwell time, 0.146 s. The cone voltage

and collision energy were optimized in case of each

J IRAN CHEM SOC

123

analyte so as to maximize the signal corresponding to the

major transition observed in the MS/MS spectra, follow-

ing the fragmentation of the [M?H]? ions corresponding

to the selected compounds. The Mass Lynx software

(Version 4.1, SCN 805) was used to control the UPLC–

MS/MS system as well as for data acquisition and

processing.

Calibration standards and quality control samples

A standard stock solution of IBS, HCT and telmisartan (IS)

were prepared by dissolving the compounds in methanol, to

give a final concentrations of 1 mg/mL. The 1 mg/mL

stock solution of IBS and HCT was serially diluted to

prepare working solutions in the required concentration

range with diluent methanol–water (50:50, v/v). The cali-

bration standards and quality control (QC) samples were

prepared by spiking with working solutions yielding eight

standard solutions ranging from 30 to 500 ng/mL for IBS

and 1–500 ng/mL for HCT. QC stock solutions were pre-

pared separately in methanol–water (50:50, v/v). QC

samples at three different concentrations levels: 60, 250,

and 400 ng/mL for IBS and 3, 250 and 400 ng/mL for

HCT. Spiked plasma calibration standards and quality

control samples were kept at -80 �C until assayed or used

for validating the assay procedures. The IS working solu-

tion (1 lg/mL) for routine use was prepared by diluting the

telmisartan stock solution in methanol and kept in refrig-

erator for storage.

Plasma blank: 190 lL of plasma was spiked with 10 lL

of methanol–water (50:50, v/v).

Plasma blank with internal standard: 190 lL of plasma

was spiked with 10 lL of 1 lg/mL IS.

Sample preparation

A simple protein precipitation method was used to extract

IBS, HCT and IS. Plasma samples stored at around -80 �C

were thawed, left for 1 h and vortexed for 30 s on room

temperature before extraction to ensure homogeneity. To

190 lL of plasma sample, 10 lL (0.6 lg/mL) of IS was

added. The samples were vortex mixed for about 30 s and

then 100 lL of methanol was added to it and vortex mixed

again for another 30 s. After vortex mixing, further 500 lL

of acetonitrile was added to the sample. The samples were

again vortex mixed gently for 1.0 min and the supernatant

was separated after centrifugation at 15,000g for 10 min

and evaporated to dryness under a gentle stream of nitrogen

at 40 �C. The residue was reconstituted with 190 lL of

methanol–water (50:50, v/v) and transferred to UPLC vials.

5 lL volumes (in partial loop with needle over fill mode)

of the sample were subjected to the analysis by UPLC–MS/

MS.

Bioanalytical method validation

A full method validation was performed according to

guidelines set by the United States Food and Drug

Administration (US-FDA) and European Medicines

Agency (EMEA) guidelines. [34, 35] The validation of this

procedure was performed in human plasma in order to

evaluate the method in terms of selectivity, linearity of

response, accuracy, precision, recovery, dilution integrity

and stability of analytes during both short-term sample

processing and long-term storage. Selectivity, linearity,

accuracy and precision exercise was also performed in

human plasma.

Selectivity and specificity

The selectivity of the method towards endogenous plasma

matrix components, metabolites and component medica-

tions was assessed in human blank plasma. Among the

analyzed plasma batches, plasma batch showing no or

minimal interference at the retention time of analytes and

internal standards was selected. They were processed and

analyzed using the proposed extraction protocol spiked

with standard IBS and HCT at lower limit of quantification

(LLOQ) level (30 and 1 ng/mL, respectively, for IBS and

HCT) and IS 30 ng/mL.

Carry over

Carryover effect was evaluated to ensure that the rinsing

solution used to clean the injection needle and port was

able to avoid any carry-forward of injected sample in

subsequent runs. The design of the experiment comprised

blank plasma, LLOQ and upper limit of quantitation

(ULOQ) followed by blank plasma to check for any pos-

sible interference due to carryover.

Linearity and standard curve

The linearity of the method was determined by analysis of

standard plots associated with eight-point standard cali-

bration curve recorded individually for IBS and HCT. Each

was plotted against corresponding concentration level in

the ranges 30–500 ng/mL for IBS, and 1–500 ng/mL for

HCT. Calibration curves from accepted three precision and

accuracy batches were used to establish linearity. Curves

were best fitted using a least-square linear regression model

y = mx ? b, weighted by 1/x2, in which y is the peak area

ratio, m is slope of the calibration curve, b is the y axis

intercept of the calibration curve and x is the analyte (HCT

or IBS) concentration. Back-calculations were made from

these curves to determine the concentration of IBS and

HCT in each calibration standards and the resulting

J IRAN CHEM SOC

123

calculated parameters were used to determine concentra-

tions of analyte in quality control samples. The determi-

nation coefficient r2 [ 0.98 was desirable for all the

calibration curves. The lowest standard on the calibration

curve was to be accepted as the lower limit of quantifica-

tion (LLOQ), if the analyte response was at least ten times

more than that of drug-free (blank) extracted plasma. In

addition, the analyte peak of LLOQ sample should be

identifiable, discrete, and reproducible with accuracy

within ±20 % and a precision B20 %. The deviation of

standards other than LLOQ from the nominal concentration

should not be more than ±15.0 %.

Precision and accuracy

Intra- and inter-day accuracies expressed as a percentage of

deviation from the respective nominal value. The precision

of the assay was measured by the percent coefficient of

variation (% CV) at four concentrations in human plasma.

Intra-day precision and accuracy were assessed by analyzing

six replicates of the quality control samples at three levels

(quality control) during a single analytical run. The inter-day

precision and accuracy were assessed by analyzing 18 rep-

licates of the quality control samples at each level through

three precision and accuracy batches runs on three consec-

utive validation days. The deviation at each concentration

level from the nominal concentration was expected to be

within ±15.0 % except LLOQ, for which it should not be

more than 20.0 %. Similarly, the mean accuracy should not

deviate by ±15.0 % except for the LLOQ where it can be

±20.0 % of the nominal concentration.

Extraction recovery and matrix effect

To investigate extraction recovery, a set of samples (n = 6

at each low, medium, and high concentration levels in

unique lots of plasma) was prepared by spiking IBS and

HCT into plasma at 60, 250, and 400 ng/mL for IBS and 3,

250 and 400 ng/mL for HCT, respectively. Each of the

samples were processed as per the procedure described

previously. A second set of plasma samples was processed

and spiked post-extraction with the same concentrations of

IBS, HCT and IS that actually existed in the pre-extraction

spiked samples. Extraction recovery for each analyte was

determined by calculating the ratios of the raw peak areas

of the pre-extraction spiked samples to those of the samples

spiked after extraction. The matrix effect was evaluated by

analyzing MQC sample.

Stability and dilution integrity evaluation

Stability of IBS and HCT in plasma was assessed by

analyzing six replicates of QC samples at low and high

concentrations under a variety of storage and processing

conditions. Six aliquots of each low and high concentration

quality control samples were taken to evaluate the bench-

top stability (short-term stability), freeze–thaw stability,

auto sampler storage stability and long-term stability.

Bench-top stability was assessed after exposure of the

plasma samples to room temperature for *6 h, which

exceeds the residence time of the sample processing pro-

cedures. The freeze–thaw stability was evaluated after

undergoing three freeze (at around -80 �C)–thaw (room

temperature) cycles. The autosampler storage stability was

determined by storing the reconstituted QC samples for

*48 h under autosampler condition (maintained at 8 �C)

before being analyzed. Long-term stability was assessed

after storage of the test samples at around -80 �C for

60 days. The working solutions and stock solutions of IBS,

HCT and IS were also evaluated for stability at room

temperature for 24 h and at refrigerator temperature (below

10 �C) for 30 days. All stability exercises were performed

against freshly spiked calibration standards. The samples

were considered stable in plasma at each concentration if

the deviation from the mean calculated concentration of

stability quality control samples was within ±15 %.

The dilution integrity experiment was intended to vali-

date the dilution test to be carried out on higher analyte

concentrations (above ULOQ), which may be encountered

during real subject samples analysis. It was performed at

1.6 times the ULOQ concentration. Six replicates samples

of half and quarter concentration were prepared and their

concentrations were calculated by applying the dilution

factor of 2 and 4, respectively, against the freshly prepared

calibration curve. The integrity of the samples was con-

sidered to be maintained if % nominal is within ±15 % of

nominal values and % CVs B 15 % at both diluted levels.

Result and discussion

Optimization and validation of assay

Optimization of chromatographic condition

Initial feasibility experiments of various mixture(s) of

organic solvents such as acetonitrile and methanol along

with Millipore water; both having 0.1 % formic acid, also

these organic solvents along with different concentration of

ammonium acetate (2–15 mM) with altered flow-rates (in

the range of 0.20–0.50 mL/min) were performed to optimize

an effective chromatographic conditions of IBS, HCT and IS

(chemical structures given in Fig. 1). The best conditions

were achieved with mobile phase comprising acetonitrile:

10 mM ammonium acetate:formic acid (85:15:0.1 % v/v/v))

pumped at a flow rate of 0.3 mL/min, on Acquity UPLC

J IRAN CHEM SOC

123

BEH� C18 column (50 9 2.1 mm, i.d. 1.7 lm. The selected

conditions were found to be suitable for the determination of

electrospray response for IBS, HCT and IS.

UPLC–MS/MS operation parameters were carefully

optimized for the determination of IBS. Analytes were

detected by tandem mass spectrometry using MRM of

precursor–product ion transitions with 0.146 s dwell time,

at m/z 427.2 ? 193.08 for IBS, 295.93 ? 268.90 for HTZ

and m/z 513.2 ? 287.14 for IS. A standard solution

(100 ng/mL) of IBS, HCT and the IS were directly infused

along with the mobile phase into the mass spectrometer

with ESI as the ionization source. The mass spectrometer

was tuned initially in the negative ionization modes for

IBS, HCT and IS. Parameters, such as capillary and cone

voltage, desolvation temperature, ESI source temperature

and flow rate of desolvation gas and cone gas, were opti-

mized to obtain the optimum intensity of protonated mol-

ecules of IBS, HCT and IS for quantification. Among the

parameters, capillary and cone voltage, especially cone

voltage, were important parameters. The cone voltage was

optimized using cone ramp (2–100) V. The precursor ion

intensities increased significantly when cone voltage was

raised gradually. Lastly, analytes produced the strongest

ion signals when cone voltage was set up at 42 V for IBS

and at 46 V for HCT. Decrease in the cone voltage reduced

the ion signal and increase in the cone voltage had minimal

effect on the ion signal. The collision energy was investi-

gated from 2 to 80 eV to optimize the response of product

ion, and the best values were found to be 28 eV for the

product ions m/z 193.08 for IBS and 18 eV for the product

ions m/z 268.9 for HCT. For IS, m/z 287.14 spectra were

produced at cone voltage of 48 eV optimum collision

energy of 34 eV.

Optimization of sample processing

Protein precipitation was used for sample preparation in this

study. Protein precipitation can be helpful in producing a clean

sample and avoiding endogenous substances in plasma with

the analytes and IS onto the column and MS system. Clean

samples are essential for minimizing ion suppression and

matrix effect in UPLC–MS/MS analysis. Two organic

Fig. 1 Chemical structure of

IBS (a), HCT (b) and

telmisartan (IS) (c)

Fig. 2 a–c Represent chromatograms obtained from blank plasma

showing no interference at the retention time of IBS, HCT and IS,

respectively. d–f Represent chromatogram of LLOQ for IBS, HCT

and IS, respectively. g–i Representative chromatogram HQC for IBS,

HCT and IS, respectively

J IRAN CHEM SOC

123

solvents, acetonitrile and methanol, were used for precipita-

tion of these proteins. Finally a combination of methanol and

acetonitrile was found to be optimal, which can produce a

clean chromatogram for a blank plasma sample and yield the

highest recovery for the analytes from the plasma.

Selectivity

Selectivity of the method was assessed by comparing the

chromatogram of blank plasma with the corresponding

spiked LLOQ sample. Six different batches of blank human

plasma were tested to identify the peaks due to the possible

biogenic plasma components. Thus the method looks to be

selective enough for determination of IBS, HCT and IS in

plasma. Representative chromatograms obtained from

blank plasma showing no interference at the retention time

of IBS, HCT and IS are shown in Fig. 2a–c, respectively.

Representative chromatogram of LLOQ for IBS, HCT and

IS is shown in Fig. 2d–f, respectively; whereas, represen-

tative chromatogram HQC for IBS, HCT and IS are shown

in Fig. 2g–i, respectively.

Linearity and sensitivity

The linearity of the method was determined by a weighted

least-square regression analysis of standard plot associated

with an eight-point standard curve for both IBS and HCT.

The calibration curves were generated by plotting area ratio

(IBS/IS) as a function of IBS concentration and was found to

be linear from 30 to 500 ng/mL for IBS in human plasma and

(HCT/IS) as a function of HCT concentration and was found

to be linear from 1 to 500 ng/mL for HCT in human plasma.

The determination coefficients (r2) were consistently greater

than 0.995 during the course of validation. The lower limit of

quantification for this assay was 30 and 1 ng/mL for IBS and

HCT in plasma, respectively. Representative LLOQ is sen-

sitive enough to investigate the pharmacokinetic behavior of

IBS and HCT in human plasma.

Precision and accuracy

Tables 1 and 2 summarize the inter- and intra-day preci-

sion and accuracy values for QC samples. The coefficient

of variation values of both intra- and inter-day results of

plasma were 2.54–10.13 and 1.14–2.29 %, respectively, for

IBS and intra- and inter-day results of plasma were

2.21–11.14 and 0.62–1.06 %, respectively, for HCT. These

results indicate that the method has good precision and

accuracy and are within the acceptance limit of\15 % and

±\15 % for precision and accuracy, respectively.

Recovery

At three QC concentration levels 60, 250, and 400 ng/mL

for IBS and 3, 250 and 400 ng/mL for HCT the percent

extraction recoveries (mean ± SD) of IBS and HCT

obtained are given in Table 3. The mean extraction

recovery for IBS was 82.04 ± 1.80 % and for HCT was

85.85 ± 1.43 %. The mean recovery for the IS telmisartan

at the concentration employed was 88.62 ± 9.68 %. This

result indicates that the extraction efficiency for IBS and

HCT using protein precipitation method was satisfactory,

consistent and concentration independent.

Table 1 Intra- and inter-day precision and accuracy of IBS in human

plasma

Spiked

conc.

(ng/mL)

Run Measured conc.

(ng/mL ± SD)

Precision

(CV, %)

Accuracy

(recovery, %)

Intra-day variation (six replicates at each concentration)

60 1 58.88 ± 5.96 10.13 98.13

2 61.51 ± 5.12 8.32 102.53

3 59.49 ± 5.23 8.80 99.16

250 1 248.61 ± 15.33 6.16 99.44

2 256.11 ± 12.73 4.96 102.68

3 248.19 ± 17.04 6.86 99.27

400 1 402.38 ± 17.04 4.23 100.59

2 406.37 ± 10.33 2.54 101.60

3 404.80 ± 15.50 3.83 101.20

Inter-day variation (18 replicates at each concentration)

60 59.96 ± 1.37 2.29 96.38

250 251.17 ± 4.80 1.91 100.91

400 406.19 ± 4.66 1.14 101.54

Table 2 Intra- and inter-day precision and accuracy of HCT in

human plasma

Spiked

conc.

(ng/mL)

Run Measured conc.

(ng/mL ± SD)

Precision

(CV, %)

Accuracy

(recovery, %)

Intra-day variation (six replicates at each concentration)

3 1 3.03 ± 0.32 10.87 101.11

2 3.05 ± 0.34 11.14 101.72

3 3.04 ± 0.23 7.60 101.61

250 1 250.28 ± 10.74 4.29 100.11

2 248.38 ± 14.16 5.70 99.35

3 247.19 ± 18.35 7.42 98.87

400 1 393.83 ± 8.71 2.21 98.45

2 400.61 ± 11.81 2.94 100.15

3 401.63 ± 15.65 3.89 100.40

Inter-day variation (18 replicates at each concentration)

3 3.08 ± 0.03 1.07 96.38

250 248.61 ± 1.55 0.62 100.91

400 398.69 ± 4.23 1.06 101.54

J IRAN CHEM SOC

123

Matrix effect and other recoveries

In this study, the matrix effect was evaluated by analyzing

MQC sample. The matrix effect was calculated via the

formula:

Matrix effect (%) = X2/X1 9 100 (%)

where X1 = response of neat concentrations and X2 is

response of post-spiked concentrations

From the calculations, it was observed that IBS and

HCT showed an average (n = 6) matrix factor of 101.98 %

with a CV of 3.72 % for IBS and 101.72 % with a CV of

5.21 % for HCT at MQC level

Stability and dilution integrity

The stabilities of IBS and HCT were investigated at two

concentrations of QC samples (low and high concentra-

tions) to cover expected conditions during analysis, storage

and processing of all samples, which include the stability

data from various stability exercises like in-injector, bench

top, freeze/thaw and long-term stability tests. The stability

results summarized in Table 4 showed that IBS and HCT

spiked into human plasma was stable for at least 6 h at

room temperature, for at least 48 h in final extract at 8 �C

under autosampler storage condition, for 30 days at around

-80 �C, and during three freeze–thaw cycles when stored

at around -80 �C and thawed to room temperature. The

stock solutions and working standard of IBS, HCT and IS

were stable for 30 days at refrigerator temperature (below

10 �C) and at least for 24 h at room temperature.

In dilution integrity study, the % accuracy of two and

four times diluted sample are indicated in Table 4 and

showed %CV of less than 15 %. These results conclude

that the dilution of the concentrated plasma sample up to

four times maintains legibility and integrity of IBS and

HCT concentration.

Table 3 Recovery data of irbesartan, hydrochlorthiazide (three QC

samples) and telmisartan in human plasma

Compound Spiked conc. (ng/mL) Recovery (% ± SD)

IBS (analyte) 60 80.30 ± 7.41

250 81.91 ± 5.67

400 83.91 ± 4.11

Mean ± SD 82.04 ± 1.80

HCT (analyte) 3 84.38 ± 6.43

250 85.91 ± 6.34

400 87.25 ± 5.79

Mean ± SD 85.85 ± 1.43

Telmisartan (IS) 30 88.62 ± 9.68

Table 4 Stability and dilution integrity data of irbesartan and hydrochlorthiazide in human plasma

Stability Drug Spiked conc.

(ng/mL)

Measured conc.

(ng/mL ± SD)

Precision

(CV, %)

Accuracy

(recovery, %)

Bench top (6 h) Irbesartan 60 59.75 ± 2.75 4.61 99.58

400 399.24 ± 16.17 4.05 98.88

Hydrochlorthiazide 3 3.01 ± 0.34 11.28 100.5

400 396.00 ± 14.70 3.71 99.00

Freeze thaw (3 cycle) Irbesartan 60 61.41 ± 4.26 6.94 102.36

400 402.57 ± 11.80 2.93 100.64

Hydrochlorthiazide 3 3.15 ± 0.31 10.52 100.50

400 402.67 ± 15.77 3.91 100.66

Auto sampler (48 h) Irbesartan 60 59.41 ± 5.98 10.07 99.03

400 397.52 ± 19.40 4.88 99.38

Hydrochlorthiazide 3 2.94 ± 0.28 9.50 98.27

400 399.33 ± 13.03 3.26 99.83

30 days at -80 �C Irbesartan 60 61.25 ± 6.65 10.85 102.08

400 400.91 ± 16.32 4.07 100.22

Hydrochlorthiazide 3 2.94 ± 0.28 9.50 98.27

400 401.00 ± 11.36 2.83 100.25

Dilution integrity Irbesartan 160 157.95 ± 9.29 5.88 98.72

320 316.44 ± 8.61 2.72 98.88

Hydrochlorthiazide 160 159.62 ± 11.04 6.92 99.76

320 324.61 ± 13.16 4.05 101.44

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Advantages of the proposed method over the reported

methods

This study represents the first report describing the deter-

mination of IBS and HCT in human plasma by UPLC–MS/

MS method. The proposed method is superior to the pre-

viously reported LC–MS methods in terms of the sensi-

tivity, simplicity as the method described herein is based

on simple one-step protein precipitation for sample prep-

aration. The run time was only 2 min which is suitable for

high-throughput analysis and reduction in the use of

organic solvents as flow rate of 0.3 mL/min was used for

just 2 min for each sample run.

Conclusions

A novel simple, economical high-throughput and highly

sensitive UPLC–MS/MS method was successfully devel-

oped and validated for the determination of IBS and HCT

in human plasma. The method involved simple one-step

protein precipitation method for plasma sample preparation

for analysis and short runtime (2.0 min). The proposed

method could be practical and reliable for pharmacokinetic

and toxicokinetic study for IBS and HCT in humans.

Acknowledgements This research project was supported by a grant

from the ‘Research Center of the Center for Female Scientific and

Medical Colleges’, Deanship of Scientific Research, King Saud

University.

References

1. A. Lant, Drugs 29, 162 (1985)

2. A.S. Manalan, H.R. Besch Jr, A.M. Watanabe, Circ. Res. 49, 326

(1981)

3. K. Wellington, D.M. Faulds, Drugs 62, 1983 (2002)

4. K.F. Croom, M.P. Curran, K.L. Goa, C.M. Perry, Drugs 64, 999

(2004)

5. J.K. Kirk, Am. Fam. Physician 59, 3140 (1999)

6. J.M. Neutel, E. Saunders, G.L. Bakris, W.C. Cushman, K.C.

Ferdinand, E.O. Ofili, J.R. Sowers, M.A. Weber, J. Clin. Hy-

pertens. 7, 578 (2005)

7. J.M. Neutel, S.S. Franklin, P. Lapuerta, A. Bhaumik, A. Ptas-

zynska, J. Hum. Hypertens. 22, 266 (2008)

8. M.R. Weir, J.M. Neutel, A. Bhaumik, M.E.D. Obaldia, P.

Lapuerta, J. Clin. Hypertens. 9(S 12), 23 (2007)

9. R.B. Miller, C. Amestoy, J. Pharm. Biomed. Anal. 10, 541 (1992)

10. I. Niopas, A. Daftsios, J. Liq. Chromatogr. Relat. Technol. 25,

487 (2002)

11. D. Zendelovska, T. Stafilov, P. Milosevski, Biomed. Chromatogr.

18, 71 (2004)

12. A. Medvedovici, C. Mircioiu, V. David, D.S. Miron, Eur. J. Drug

Metab. Pharmacokinet. 25, 91 (2000)

13. K. Richter, R. Oertel, W. Kirch, J. Chromatogr. A 729, 293

(1996)

14. B.S. Kuo, A. Mandagere, D.R. Osborne, K.K. Hwang, Pharm.

Res. 7, 1257 (1990)

15. L. Li, J. Sun, P. Yang, Z. He, Anal. Lett. 39, 2797 (2006)

16. A. Vonaparti, M. Kazanis, I. Panderi, J. Mass Spectrom. 41, 593

(2006)

17. M.F. Tutunji, H.M. Ibrahim, M.H. Khabbas, L.F. Tutunji, J

Chromatogr. B 877, 1689 (2009)

18. M. Song, T. Hang, H. Zhao, L. Wang, P. Ge, P. Ma, Rapid

Commun. Mass Spectrom. 21, 3427 (2007)

19. H. Li, Y. Wang, Y. Jiang, Y. Tang, J. Wang, L. Zhao, J. Gu, J.

Chromatogr. B 852, 436–442 (2007)

20. N.V.S. Ramakrishna, K.N. Vishwottam, S. Manoj, M. Koteshw-

ara, S. Wishu, D.P. Varma, Biomed. Chromatogr. 19, 751 (2005)

21. W. Fang, W. Xie, J.Y.K. Hsieh, B.K. Matuszewski, J. Liq.

Chromatogr. Relat. Technol. 28, 2681 (2005)

22. R.T. Sane, M. Francis, S. Pawar, Indian Drugs 40, 104 (2003)

23. A.K. Shakya, Y.M. Al-Hiari, O.M.O. Alhamami, J. Chromatogr.

B 848, 245 (2007)

24. W.L. Wang, Q. Liu, Zhongguo Yaofang 18, 2512 (2007)

25. N. Erk, J. Chromatogr. B 784, 195 (2003)

26. S. Najma, A.M. Saeed, A.S. Shahid, S. Shahnawaz, Chin.

J. Chromatogr. 26, 544 (2008)

27. L.F. Tutunji, M.F. Tutunji, M.I. Alzoubi, M.H. Khabbas, A.I.

Arida, J. Pharm. Biomed. Anal. 51, 985 (2010)

28. R. Russo, D. Guillarme, D.T.T. Nguyen, C. Bicchi, S. Rudaz,

J.L. Veuthey, J. Chromatogr. Sci. 46, 199 (2008)

29. D.T. Nguyen, D. Guillarme, S. Rudaz, J.L. Veuthey, J. Sep. Sci.

29, 1836 (2006)

30. J.R. Mazzeo, U.V. Neue, M. Kele, R.S. Plumb, Anal. Chem. 77,

460 (2005)

31. A. de Villiers, F. Lestremau, R. Szucs, S. Gelebart, F. David, P.

Sandra, J. Chromatogr. A 1127, 60 (2006)

32. S.A. Wren, P. Tchelitcheff, J. Chromatogr. A 1119, 140 (2006)

33. L. Novakova, L. Matysova, P. Solich, Talanta 68, 908 (2006)

34. US Food and Drug Administration, Center for Drug Evaluation

and Research (CDER). Guidance for industry, bioanalytical

method validation. 2001. http://www.fda.gov/downloads/Drugs/%

e2%80%a6/Guidances/ucm070107.pdf

35. European Medicines Agency. Guideline on bioanalytical method

validation. 2011. http://www.ema.europa.eu/docs/en_GB/docu

ment_library/Scientific_guideline/2011/08/WC500109686.pdf

J IRAN CHEM SOC

123