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CHAPTER 5

METHODS FOR THE ANALYSIS OF VALSARTAN

5.1 Introduction

Angiotension II receptor blockers (ARBs) is a very potent chemical that causes

muscle surroundings blood vessels to contract, thereby contraction of blood vessels.

This contraction increases the pressure within the vessels and can cause high blood

pressure (hypertension), ARBs are medications that block the action of angiotension II

receptors on blood vessels, resulting in enlarge of blood vessels (dilate) and reduces

blood pressure. Reduced blood pressure makes it easier for the heart to pump blood

and can improve heart failure. These medicines also increase the release of water and

salt (sodium) to the urine, which in turn lowers blood pressure as well. ARBs also act

directly on the hormones that regulate sodium and water balance.

ARBs can be used to treat coronary artery disease or heart failure. In some

peoples who cannot tolerate angiotensin-converting-enzyme inhibitor (ACE) or who

have kidney disease from diabetes.

5.2 Drug profile and Literature review

Valsartan (VRT)

Valsartan is a new potent, highly selective and orally active anti hypertensive

drug belonging to the family of angiotension II type 1-receptor antagonists [1].

Valsartan has much greater affinity (about 20000 fold) for the angiotension II type 1

(AT1) receptor for the angiotension II type 2 (AT2) receptor, thereby relaxing blood

vessels and causing them to widen, which lowers blood pressure and improves blood

flow. Valsartan is rapidly absorbed after oral administration. Plasma levels peak 2-4 h

after oral and then decline with a terminal half-life reported in various studies in the

range of 6–9 h. Peak plasma concentration (Cmax) of 1.64 mg/L occurred after oral

administration of a single 80 mg dose of valsartan. A higher dose (200 mg) produced

a proportionately higher Cmax (3.46 mg/L) at a similar time post-dose.

141

Valsartan is chemically known as N-valeryl-N[[2-(1H-tetrazol-5- yl)biphenyl-

4- yl] methyl] valine . It has an empirical formula of C24H29N5O3 and a molecular

weight of 435.5 g/mol. Valsartan is available as a white, microcrystalline powder with

a melting range of 105-110 °C. It is freely soluble in methanol, ethanol sparingly

soluble in ethyl acetate, slightly soluble in dichloromethane and practically insoluble

in water.

Structure of valsartan

Telmisartan

It is an angiotension II receptor blocker. Used in the management of

hypertension [2]. This shows high affinity for the angiotension II receptor type1

(AT1), with a binding affinity 3000 times greater for AT1 than AT2.

Telmisartan is chemically known as 2-(4 -{[4methyl-6-(1-methyl-1H-1,3-

benzodiazol-2-yl)-2-propyl-1-H1,3-benzodiazol-1-yl]methyl}phenyl)benzoic acid.

The molecular formula of telmisartan is C33H30N4O2 . Structure of telmisartan is as

follows:

Telmisartan is used as an internal standard (IS) in this method.

142

Literature review

Chromatographic methods

Parambi et al. [3] have explained a HPLC method for the quantitation of

valsartan in tablet dosage form on a C18 column (250 x 4.6 mm) using a mobile phase

consisting of ammonium dihydrogen phosphate buffer: methanol (33.5:66.5) adjusted

to pH3 with formic acid at a flow rate of 1.0 mL/min and detection was performed at

265 nm. The retention time of valsartan was found to be at 11.9 min.

Chitlange et al. [4] have employed a RP-HPLC method for simultaneous

estimation of amlodipine besylate (AMLB) and valsartan (VAT) on RP C-18 column

(Kromasil, 250 x 4.6 mm) using acetonitrile: phosphate buffer (0.02M, pH 3.0),

(56:44 v/v) as mobile phase at a flow rate of 1.0 mL/min and the detection wavelength

was at 234 nm. The retention time for AMLB and VAT was found to be 3.07 and 6.20

min, respectively.

Prasad et al. [5] have developed stability indicating and validated RP-HPLC

method for the simultaneous estimation of valsartan (VAL) and amlodipine (AML) in

their combined dosage form. Water’s HPLC equipped with UV-visible and diode

array detectors, column used was XTerra® RP8, 5 μm, 100 mm × 4.6 mm i.d., at 30

°C. Mobile phase was consisting of 0.05M ammonium acetate and 0.5% TEA buffer

having pH 5.5 and in the ratio of 68:32 v/v at a flow rate of 1.0 mL/ min, and UV

detection was carried out at 238 nm and at 271 nm for AML and VAL, respectively.

VAL, AML and in their combined dosage form were exposed to thermal, photolytic,

oxidative and acid-base hydrolytic stress conditions; the stressed samples were

analyzed by the proposed method. The retention time of valsartan and amlodipine

were 1.98 and 4.03 min, respectively. The method was found linear over the range of

1-20 μg/mL for amlodipine and 1.6-240 μg/mL for valsartan.

Jothieswari et al. [6] have described a reverse phase high performance liquid

chromatographic method for the simultaneous estimation of valsartan and

hydrochlorothiazide in bulk and in pharmaceutical formulations using RP-C18

column. The mobile phase (acetonitrile: methanol: 50 mM phosphate buffer adjusted

to pH 3 with orthophosphoric acid) was pumped at a flow rate of 1.0 mL/ min in the

ratio of 20: 50: 30%v/v and the eluents were monitored at 250 nm. Linearity was

143

obtained in the concentration range of 4-40 mg/mL for valsartan and 1–10 mg/mL for

hydrochlorothiazide.

Afshin Zarghi et al. [7] have developed a high-performance liquid

chromatographic (HPLC) method using amonolithic column for determination of

valsartan in human plasma. The assay is based on protein precipitation using

acetonitrile and fluorescence detection. The assay enables the measurement of

valsartan for therapeutic drug monitoring with a minimum quantification limit of 20

ng/mL. The method involves, one-step extraction procedure, and analytical recovery

was nearly complete. Isocratic mobile phase consisting of 0.01 M disodium hydrogen

phosphate buffer-acetonitrile (60:40 v/v) adjusted to pH 3.5 with diluted phosphoric

acid was used. The excitation and emission wavelengths were set at 230 and at 295

nm, respectively. The calibration curve was linear over the concentration range 20-

2000 ng/mL.

Zong-Zhu Piao et al. [8] have established a method of determining valsartan

concentration in human plasma samples using high performance liquid

chromatography (HPLC) combined with ultraviolet (UV) detection. Methanol

appeared to be the best with a high recovery efficiency compared to other solvents

such as acetonitrile, ethylacetate and methyl-tert-butyl ether. After a simple protein

precipitation using methanol, the analytes were separated on a Phenomenex® Luna

C18 column using 42% acetonitrile with 15 mM potassium dihydrogen phosphate in

water (pH- 2.0 adjusted with phosphoric acid) as the mobile phase at a flow rate of 1.2

mL/min. The standard calibration curve was constructed in the concentration range of

50-2000 ng/mL. Spironolactone was used as an internal standard (IS). Valsartan and

IS were eluted at 10.25 and 12.17 min, respectively.

Vinzuda et al. [9] have developed a reverse phase isocratic RP-HPLC method

for the determination of valsartan in tablet dosage form. The method was carried out

using thermo-hypersil ODS column (150 mm × 4.6 mm i.d., 5 μm particle size) with

mobile phase comprised of water: acetonitrile: glacial acetic acid (500:500:01). The

flow rate was set at 1.0 mL/min and effluent was detected at 273 nm. The retention

time of valsartan was found to be 4.6 min. LOD and LOQ were found to be 2.72

μg/mL and 8.25 μg/mL, respectively. The calibration curve was linear in the

concentration range of 40-140 μg/mL.

144

Ramakrishna Yadav et al. [10] have reported a high performance liquid

chromatography (RP-HPLC) method for the simultaneous estimation of valsartan and

hydrochlorothiazide in tablets. The separation was achieved on Hypersil C18 column

(250 cm length, 4.6 mm internal diameter and 5 μm particle size) with a mobile phase

having a fixed composition of acetonitrile: 0.1M-ammonium acetate buffer in the ratio

of 55:45 v/v and at a flow rate was 1.0 mL/min. The effluent was monitored with UV

detector at 254 nm. The retention times for valsartan and hydrochlorothiazide were

found to be 2.38 and 2.97 min., respectively. The linearity range for valsartan and

hydrochlorothiazide were 32-160 and 5-25 μg/mL, respectively.

Manoranjani et al. [11] have described a HPLC method for the assay of

valsartan in tablet dosage form. Isocratic elution at a flow rate of 1mL/min was

employed on asymmetry C18 column at ambient temperature. The mobile phase

consisted of methanol: water: THF 60:35:05(v/v/v). The uv detection wavelength was

at 269 nm. Linearity was observed in concentration range of 10-35 ppm. The retention

time for valsartan was 4.6 min.

A validated and stability indicating RP-HPLC method [12] was reported in

literature for the simultaneous estimation of valsartan (VAL) and amlodipine (AML)

in their combined dosage form. Column used was XTerra® RP18, mobile phase was

consisted a mixture of solution A (1000 mL water + 0.2 mL trifluoro acetic acid) and

solution B (water:acetonitrile: trifluoro acetic acid, 400:600:1, v/v/v) with flow rate of

1.5 mL/ min and UV detection was carried out at 237 and at 265 nm for AML and

VAL, respectively. The described method was linear over the range of 1.6-240 μg/mL

and 1-30 μg/mL for VAL and AML, respectively.

UV spectrophotometric methods

First-derivative ultraviolet spectrophotometric and high-performance liquid

chromatographic (HPLC) methods [13] were used to determine valsartan and

hydrochlorothiazide simultaneously in a combined pharmaceutical dosage forms. The

derivative procedure was based on the linear relationship between the drug

concentration and the first derivative amplitudes sat 270.6 and 335 nm for valsartan

and hydrochlorothiazide, respectively. The calibration graphs were linear in the range

of 12.0–36.1g/mL for valsartan and 4.0–12.1 g/mL for hydrochlorothiazide.

145

Furthermore, a high performance liquid chromatographic procedure with ultraviolet

detection at 225 nm was developed for a comparison method. For the HPLC

procedure, a reversed phase column with a mobile phase of 0.02 M phosphate buffer

(pH 3.2)-acetonitrile (55: 45; v/v) was used to separate for valsartan and

hydrochlorothiazide. The plot of peak area ratio of each drug to the internal standard

versus the respective concentrations of valsartan and hydrochlorothiazide were found

to be linear in the range of 0.06–1.8 and 0.07–0.5 g/mL, respectively.

Sevgi Tatar et al. [14] have described UV- and second derivative-

spectrophotometric and high-performance liquid chromatographic methods for the

determination of valsartan in pharmaceutical formulation. For the first method, UV-

spectrophotometry, standard solutions were measured at 205.6 nm. The linearity range

was found to be 2.0–10.0g/mL in ethanol with a correlation coefficient (r=0.9997).

For the second method, the distances between two extremum values (peak-to-peak

amplitudes) at 221.6 and 231.2 nm were measured in the second order derivative-

spectra of standard solutions. The third method was based on high-performance liquid

chromatography on C18 column using acetonitrile, phosphate buffer as a mobile

phase and losartan was used as an internal standard. Detection was carried out at 265

nm with a UV-detector. The assay was linear over the concentration range at 1.0–5.0

µg/mL.

Meyyanathan and coworkers [15] have explained two UV spectrophotometric

methods, simultaneous equation method and Q-value analysis method for the

simultaneous estimation of nebivolol hydrochloride and valsartan used as

cardiovascular drugs available in capsule dosage form and nebivolol hydrochloride

with hydrochlorothiazide used as antihistaminic H blocker available in tablet dosage

form. The methods are based on the measurement of the absorbance of nebivolol

hydrochloride and hydrochlorothiazide at 270.4, 280.2 and 270 nm for nebivolol HCl

and valsartan at 246.6, 280.2 and 275 nm, respectively. These methods were obeyed

Beer's law in the concentration range of 0.5 - 2.5 μg/mL for nebivolol HCl, 1.0–20

μg/mL for valsartan 1.0–3.0 μg/mL for hydrochlorothiazide.

Gupta et al. [16] have described two UV spectrophotometric methods for the

estimation of valsartan (VRT) in bulk and in tablet dosage form. Calibration graphs

146

were found to be linear (r=0.999) over the concentration range of 10-50 μg/mL. Both

the methods can be adopted in its routine analysis.

Deshpande et al. [17] have developed two UV spectrophotometric methods for

the estimation of valsartan and hydrochlorothiazide in pharmaceutical formulation.

Method I-Absorption ratio method (Q-analysis) using two wavelengths, 265 nm (iso

bestic point at which both the drugs exhibit absorbance) 249 nm (λmax of valsartan)

and Method II- area under curve method. For the second method area under the curve

in the range of 249 -259 nm and 261-281 nm was selected for the analysis of valsartan

and hydrochlorothiazide respectively. Linearity for detector response was observed in

the concentration range of 2-24 µg/mL and 2-14 µg/mL for valsartan and

hydrochlorothiazide, respectively.

An extraction-free spectrophotometric method for the simultaneous estimation

of valsartan and ezetimibe in pharmaceuticals using acidic dyes, namely,

bromophenol blue (BPB) and bromocresol green (BCG) was reported [18]. The

method was based on selective ion-pair formation between valsartan and the acidic

dyes. The yellow coloured ion-pair induces a bathochromic shift in the spectrum with

maximum absorbance at 425 and 428 nm for BPB and BCG, respectively. With BPB,

the ion-pair was formed obeyed Beer`s law in the ranges 5-40 and 1–50 μg/mL for

valsartan and ezetimibe, respectively.

Nevin Erk [19] has developed two methods using two different techniques for

the simultaneous determination of valsartan and hydrochlorothiazide in

pharmaceutical dosage forms. The first method, based on compensation technique is

presented for the derivative spectrophotometric determination of binary mixtures with

overlapping spectra. By using ratios of the derivative maxima or the derivative

minimum, the exact compensation of either component in the mixture can be

achieved, followed by its determination. The second method, differential derivative

spectrophotometry, comprised of measurement of the difference absorptivities

derivatized in the first order (_D1) of a solution in methanol at wavelengths of 227.8

and 276.5 nm, respectively.

Erdal Dinc et al. [20] have explained a new signal processing approach based

on continuous wavelength transform (CWT) was proposed for the simultaneous

147

spectrophotometric determination of valsartan and amlodipine in tablets without

making use of any chemical separation procedure. After the CWT method was applied

to the analyzed spectra, the results plotted in wavelength domain. Several CWT

families were tested and Daubechie10 (db10-CWT) and D Meyer (dmey-CWT) were

found suitable for the determination of VRT and AML. The procedure of the CWT

family was based on the recovery results of signal analysis. Linear calibration ranges

were considered as 2-42 g/mL for VRT and 2-32 g/mL for AML, respectively. The

validation treatments for the proposed CWT methods were performed by analyzing

various synthetic mixtures and using the standard addition technique.

Nashwah [21] has developed a spectrophotometric method for simultaneous

determination of amlodipine (AML) and valsartan (VRT) without previous separation.

In this method, amlodipine in methanolic solution was determined using zero order

UV spectrophotometry by measuring its absorbance at 360.5 nm without any

interference from valsartan. Valsartan spectrum in zero order is totally overlapped

with that of amlodipine. First, second and third derivative could not resolve the

overlapped peaks. The first derivative of the ratio spectra technique was applied for

the measurement of valsartan. The ratio spectrum was obtained by dividing the

absorption spectrum of the mixture by that of amlodipine, so that the concentration of

valsartan could be determined from the first derivative of the ratio spectrum at 290

nm. Quantification limits of amlodipine and valsartan were 10–80 g/mL and 20–180

g/mL, respectively.

Gaikwad et al. [22] have explained a high-performance thin-layer

chromatographic (HPTLC) method for the simultaneous determination of ramipril and

valsartan in capsules. Identification and determination were performed on 10 cm × 10

cm aluminum-backed TLC plates, coated with 0.2 mm layers of silica gel 60F254,

previously washed with methanol using ethyl acetate : chloroform: glacial acetic acid,

(8:2:0.2, v/v) as mobile phase. Detection was carried out densitometrically using UV

detector at 220 nm. The Rf values were 0.15 for ramipril and 0.49 for valsartan. The

linear response for ramipril and valsartan was observed over 800–4000 ng/spot (r =

0.9933) and 800–4000 ng/spot (r = 0.9906), respectively.

Kadam et al. [23] have developed a high-performance thin-layer

chromatographic (HPTLC) method for simultaneous analysis of valsartan and

148

hydrochlorothiazide in tablet formulations. Standard and sample solutions of valsartan

and hydrochlorothiazide were applied to pre coated silica gel G 60 F254 HPTLC

plates and the plates were developed with chloroform–ethyl acetate–acetic acid,

5:5:0.2 (v/v), as mobile phase. UV detection was performed densitometrically at 248

nm. The retention factors of valsartan and hydrochlorothiazide were 0.27 and 0.56,

respectively. The linear range was 800–5600 ng per spot for valsartan and 125–875 ng

per spot for hydrochlorothiazide.

Hiten Janardan shah et al. [24] have proposed LC-MS/MS method for

simultaneous analysis of valsartan (VAL) and hydrochlorothiazide (HCTZ) in human

plasma. VAL and HCTZ were chromatographed on a C8 column with 75:15:10

actonitrile-methanol-0.001% aqueous ammonia as mobile phase. The samples were

extracted from solid phase extraction.

Ramani et al. [25] have described the simultaneous determination of three

drugs namely simvastatin, amlodipine and valsartan in 500 µL plasma. The total run

time involved 2.8 min. This was achieved with a mobile phase consisting of 0.02M

ammonium formate (pH4.5):acetonitrile (20:80) at a flow rate of 0.5 mL/min. on an

X-terra C18 column. The range of concentration validated of valsartan was 0.5-50

ng/mL.

Senthamil Selvan et al. [26] have described the fixed dose combination of

nebivolol and valsartan in human plasma by LC-MS/MS. These two drugs were

extracted from plasma using acetonitrile and separated on a C18 column. The mobile

phase consisting of acetonitrile and 0.05 mM formic acid (50:50v/v, pH3.5) was

delivered at a flow rate of 0.25 mL/min. The linearity was obtained over the

concentration range of 1.0-2000 ng/mL for valsartan.

A LC-MS/MS method [27] was described by Xiaoling Hu et al. for the

simultaneous quantification of benazepril, gliclazide and valsartan in human plasma.

Chromatographic separation was performed on a Shim-pack VP-ODS C18 column

(250 x2.0mm i.d., 5µm) using methanol-0.05% formic acid (90:10,v/v) as mobile

phase. Lower limit of quantification of valsartan was 20 ng/mL.

Nozomu Koseki et al. [28] have developed an LC-MS/MS method for

valsartan using solid phase extraction in an Empore high performance extraction disk

149

plate, universal resin 96-well formate. Calibration range of the method was 2-2000

ng/mL.

Arbinda Patnaik et al. [29] have developed an HPLC method for valsartan in

tablet dosage forms, Microbondapak column was used and mobile phase was

consisted of methanol and phosphate buffer pH-3 (65:35). Total run time was found to

be 20 min and linearity of the concentration range was 10-100µg/mL.

From the above literature survey, it is found that, only few LC-MS/MS and

spectrophotometric methods are available for the determination of valsartan. The

reported spectrophotometric and chromatographic methods are less sensitive and also

having some deficiencies in their methods. In order to overcome these short comings,

the author has presented a simple spectrophotometric method for the determination of

valsartan using p-chloranilic acid as a π-acceptor in the Section 5A, and a sensitive

LC-MS/MS method for its determination in human plasma is described in Section 5B.

150

Section 5A

Spectrophotometric determination of valsartan using p-chloranilic acid as π-

acceptor in pure and in dosage forms

5A.1. Reagent profile

p-Chloranilic acid (PCA)

p-Chloranilic acid is chemically known as 2,5-dichloro-3,6-dihydroxy-p-

benzoquinone with a molecular formula of C6H2Cl2O4. It is widely used in

Spectrophotometric studies of isoniazid [30]. It has the following molecular structure.

5A.2. Experimental

5A.2.1 Apparatus

Absorbance measurements for spectrophotometric analysis were performed

using a Systronics Model 166 digital spectrophotometer provided with 1-cm matched

quartz cells.

5A.2.2 Material and reagents

Analytical reagent grade chemicals and reagents were used.

p-Chloranilic acid (0.05 %, w/v) (p-CA). It was freshly prepared by dissolving 0.05

g p-chloranilic acid (Rolex, Mumbai, India) in 100 mL acetone.

Working standard solution. The pure grade VRT, certified to be 99.99% pure was

received from Cipla India Ltd., Mumbai, India, as a gift sample and was used as

received. A stock standard solution equivalent to 100 µg/mL of VRT was prepared by

dissolving 10 mg of the pure drug in 100 mL methanol.

151

Pharmaceutical formulations of VRT such as Valzaar [Torrent], Diovan

[Novartis Pharma] were purchased from local markets.

Procedure for calibration graph

Accurately measured aliquots of standard solution of VRT (100 µg/mL)

corresponding to 0.5, 1.0, 1.5, 2.0, …………..5.0 mL were transferred into a series

of 10 mL calibrated flasks using a micro burette. To this solution was added 3.5 mL

0.05 % p-CA, then shaken well and the contents were diluted to the mark with

methanol and mixed well. The absorbance of the bright pink colored complex was

measured at 530 nm after 5 min against the reagent blank prepared similarly, but

without drug content.

5A.2.3 Recovery of VRT from pharmaceutical samples

In order to determine the contents of VRT in commercial dosage forms (label

claim: 40 mg tablet), the contents of ten tablets were weighed accurately and ground

into a fine powder. An amount of powder containing 10 mg of VRT was accurately

weighed and transferred into a 100 mL calibrated flask and 30 mL methanol was

added. The content was shaken for about 30 min; the volume was diluted to the mark

with methanol and mixed well and filtered using a Whatman no.41 filter paper. The

filtrate containing VRT at a concentration of 100 µg/mL was subjected to analysis by

the procedure described above.

5A.3. Results and Discussion

Chemistry of the method

The method developed involves charge-transfer complex formation between

the basic nitrogenous VRT as n-donor and p-chloranilic acid (p-CA) in polar solvents.

The formed charge transfer (C-T) complex was subsequently dissociated into radical

anions, which are colored species. The absorbance of the colored complex was

measured at 530 nm (Fig 5A.1), and it was observed as shown in the following

equation:

152

Thus, p-CA was used as a reagent in the proposed method for the estimation of

VRT. The possible reaction pathway for VRT - p-CA complex is shown in

Scheme 1.

Fig. 5A.1 Absorption spectra for VRT

0

0.1

0.2

0.3

0.4

0.5

400 420 440 460 480 500 520 540 560 580 600

Ab

sorb

ance

Wavelength, nm

Blank

20 µg/mL VRT

40 µg/mL VRT

153

Scheme 1: Proposed reaction scheme.

The influence of various factors on the formation of charge-transfer complex

viz., reagent concentration, reaction time and stability of the colored complex were

studied and maintained throughout the experiment to determine the quantity of VRT in

bulk and in dosage forms. Maximum absorbance values were obtained with 3.5 mL of

0.05 % p-CA, which remained unaffected by further addition of p-CA. Hence; 3.5 mL

of the reagent was used for the determination in the developed method. It was

observed that the reaction got stabilized within 5 min in the developed method. The

color of the C-T complex was remained stable at room temperature (27 ± 3 ºC) for a

period of 2 h.

154

5A.4 Method validation

The developed method was validated in terms of linearity and sensitivity, limit

of detection (LOD) and limit of quantitation (LOQ), precision, accuracy, selectivity

and recovery following the ICH guidelines [31].

5A.4.1 Linearity, sensitivity, limits of detection and quantitation

A calibration graph was constructed using the absorbance values against the

concentration of VRT in the ranges 5-50 µg/mL at 530 nm (Fig.5A.2). Under the

optimum experimental conditions, a linear relationship existed between the

absorbance and concentration of the drug. The regression analysis of the calibration

curve using the method of least squares was made to calculate the slope (b), intercept

(a) and correlation co-efficient (r) values are presented in Table 5A.1. The optical

characteristics such as absorption maxima, Beer’s law limit, molar absorptivity and

Sandell’s sensitivity values are also given in Table 5A.1.

Fig 5A.2 Calibration curve of VRT

The limit of detection (LOD) and limit of quantification (LOQ) were calculated

as per ICH guidelines [30] using the following equations:

LOD and LOQ

0

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40 50

Ab

sorb

ance

conc. in μg/mL

155

where σ is the standard deviation (n=5) of reagent blank determination and

s is the slope of the calibration curve.

Table 5A.1. Analytical and regression parameters of the proposed method

*y=a+bx, where x is the concentration of VRT in µg/mL and y is the absorbance at

530 nm, Sa is the standard deviation of the intercept, Sb is the standard deviation of the

slope.

5A.4.2 Intra and inter day precision and accuracy

The accuracy of an analytical method expresses the closeness between the

proposed method and reference method. Further, the accuracy and precision (intra-

day) of the proposed method were evaluated by replicate analysis (n=5) of calibration

standards at three different concentration levels in the same day. Precision and

accuracy of inter-day were measured by performing the calibration standards at cited

three concentrations on five consecutive days. Both precision and accuracy were based

on the calculated percent relative standard deviation (RSD, %) and percent relative

error (RE, %) values, respectively for the developed method were found to be

Parameter Method

λmax nm 530

Beer’s law range (µg/mL ) 5.0 – 50

Molar absorptivity (ε), (L/mol/cm) 0.22 x 10 4

Sandell sensitivity (µg/cm2) 0.0940

Intercept (a) -0.0027

Slope (b) 0.0111

Correlation coefficient (r) 0.999

Sa 0.0116

Sb 0.0003

LOQ (µg/mL ) 1.273

LOD (µg/mL ) 0.3818

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satisfactory. The analytical results obtained from this investigation are summarized in

Table 5A.2.

Table 5A.2. Evaluation of intra-day and inter-day accuracy and precision results

Intra-day a

VRT

taken,

µg/mL

VRT found c,

µg/mL

Precision d

(RSD %)

Accuracy e

(RE %)

Method

10 9.93±0.10 1.02 0.66

20 19.84±0.06 0.30 0.17

40 40.07±0.33 0.83 0.18

Method

Inter-day b

10 9.97±0.25 2.50 0.21

20 19.84±0.13 0.63 0.81

40 39.79±0.29 0.74 0.52

Mean value of five determinations, b. Mean value of five determinations, c.

Mean value of three determinations, d. Relative standard deviation (%), e. Bias%:

(Found-taken/taken)×100.

5A.5 Application to analysis of pharmaceutical samples

The validity of the proposed method was ascertained by the statistical

comparison of the results obtained by a reference method [29] with the proposed

method by applying Student’s t-test for accuracy and F-test for precision in some

commercial formulations. The results were compared with those of the reported

method. Statistical analysis of the results using the Student’s t- and F-tests revealed no

significant difference between the reported method at the 95% confidence level with

respect to accuracy and precision (Table5A 3).

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Table 5A.3. Results of determination of VRT in tablets and statistical

comparison with the reference method

Tablet brand name Nominal amount

mg/tablet

Found*(% of nominal amount±SD)

Reference method

[29]

Proposed method

Valzaar a 40 100.88±0.48 99.62±0.704.

t=1.68,F=2.15

Diovan b 80 100.80±0.46 100.38±0.854.

t=0.51,F=3.45

Marketed by: a. [Torrent], b. [Novartis Pharma]

*Mean value of five determinations

Tabulated t and F-values at 95 % confidence level are 2.77 and 6.39, respectively.

Recovery study

To test the applicability of the proposed method, recovery experiments were

carried out by standard addition method. In this study, pre-analyzed tablet powder was

spiked with pure drug at three different concentrations and the total was found by the

proposed method. Each determination was repeated three times. The recovery of the

pure drug added was quantitative and revealed that co-formulated substances did not

interfere in the determination. The results of recovery study are compiled in Table

5A.4.

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Table 5A.4. Results of recovery experiments via the standard addition technique

Tablet brand

name

VRT in

tablet

solution

mg/tablet

VRT

tablet

solution

added in

µg/mL

Pure VRT

added

(µg/mL)

Total found

( µg/mL)

Pure VRT

recovered*

% ± SD

Valzaar a 40 10 10 20.02 100.22±0.77

10 20 30.05 99.16±0.88

10 30 40.05 99.49±0.46

Diovan b 80 10 10 20.08 99.13±0.77

10 20 30.10 100.98±0.85

10 30 40.03 101.03±0.95

a. * Mean value of three determinations

b. Marketed by: a. [Torrent], b. [Novartis Pharma]

159

Section-5B

Rapid Determination of Valsartan Using LC–MS/MS in Human Plasma and

Application to Pharmacokinetics

5B.1 Introduction

In this section, the author has described LC-MS/MS method for the analysis of

VRT in human plasma.

5B.2.Experimental

5B.2.1 Materials and Reagents

Valsartan, obtained from Clearsynth Labs (P) LTD Andheri-West, Mumbai.

purity 99.46% (by HPLC). Telmisartan (internal standard) was purchased from Vivan

Life Sciences (P) Ltd, (Thane-West, Mumbai). Sodium acetate (Sigma, St. Louis,

MO, USA). HPLC-grade acetonitrile (JT Baker (Phillipsburg, NJ, USA), and all the

other chemicals and solvents were of the highest analytical grade were used.

Ammonium acetate was obtained from (Fluka Switzerland)

20 mM ammonium acetate (pH-3.8): Dissolved 1.568 g of ammonium acetate in

1000 mL of water and the pH was adjusted to 3.8±0.1 using formic acid. It was

sonicated before use.

Mobile phase [20 mM ammonium acetate (pH3.8) and acetonitrile (10:90, v/v)]: It

was prepared by mixing 100 mL 20 mM ammonium acetate (pH -3.8) and 900 mL of

acetonitrile in a 1000 mL reagent bottle, mixed well and filter through 0.2 µm filter

paper. It was degassed by sonication before use.

Sodium acetate (2mM): It was prepared by dissolving 15 mg of sodium acetate in a

100 mL volumetric flask using water. It was sonicated and was used as an extraction

buffer.

Methanol: water: 50:50, v/v (diluent): Appropriate volume of methanol was diluted

in a milli-Q water and mixed well. It was used as a diluent.

160

Standard solutions of valsartan and telmisartan

Accurately weighed 100 mg of pure valsartan was dissolved in and diluted to

mark in a 100 mL volumetric flask with methanol. The standard solutions of valsartan

were serially diluted with methanol: water (1:1v/v) to get a concentration of 0.04,

0.09, 9.3, 23.18, 57.93, 144.8, 362.0 and 452.5 µg/mL for valsartan. An internal

standard telmisartan was prepared similarly, and it was diluted to get 2 ng/mL using

methanol:water: 1:1v/v. These solutions were stored in a freezer until analysis.

5B.2.2 Operating conditions

Column : Thermo hypersil keystone Beta basic-8, (2.1

mm. 50 mm, 3µ).

Mobile phase : acetonitrile and 20 mM ammonium acetate

containing 0.1% formic acid (pH = 3.8) (90:10,

v/v),

Column oven temperature : 40 °C

Flow rate : 0.3 mL/min (without splitter mode)

Auto sampler tray temperature : 10 °C

Injection volume : 5 µL

Mode of ionization : positive ion mode

Total run time : 2.0 min.

MS/MS operating condition

Source temperature : 500 °C

Nebulizer : 9.0 psi

Curtain gas : 8.0 psi

IS voltage : 5000 V

CAD gas : 4 psi

DP (declustering potential) : 50eV

FP (focusing potential) : 200eV

EP (Exit potential) : 10 eV

CE (collision energy : 50 eV

CXP (collision exit potential) : 3 eV

161

5B.2.3 Procedure for the preparation of calibration curve and assay samples

The calibration curve for valsartan was prepared in human plasma at eight

concentration levels of 0.93, 1.9, 185.37, 463.41, 1158.53, 2896.34, 7240.86, and

9051.08 ng/mL. Quality control samples were also prepared in human plasma at the

following concentrations 2.449, 3711.0 and 7136.54 ng/mL (low, middle and high

level concentrations) respectively. All calibration samples were prepared by the

addition of 100 µL of stock solutions to 5.0 mL of pooled plasma taken from

volunteers and mixed well. Similarly, QC- samples were prepared by adding 500 µL

of stock solutions to 25.0 mL of pooled plasma, and then treated by following the

sample preparation procedure as indicated in Section 5B.2.4.

5B.2.4. Extraction procedure (sample pretreatment)

A volume of 200 µL plasma sample was transferred to polypropylene vials to

which internal standard (50 µL, 2 ng/mL) and 50 µL of sodium acetate (2mM) were

added,then vortex to mixing and extract the sample with 2.5 mL of ethyl acetate and

vortex-mixed for10 min. The samples were centrifuged at 4000 rpm for 5 min. The

organic phase was transferred to glass vials and the solvent was evaporated to dryness

at 40 °C under a stream of nitrogen. The residue was redissolved in 500 µL of mobile

phase of which 400 µL was transferred into 1000 µL glass vials and placed in the

autosampler for analysis. The injection volume was 5 µL.

5B.3. Bio-analytical Method validation

Quantitation was based on the relationship between VRT peak areas and I.S.

peak areas. Selectivity was evaluated by extracting plasma samples from six different

sources, recoveries of VRT at the three QC concentrations (2.5, 3711.0, 7136.5

ng/mL) and I.S. at 2 ng/mL were determined by comparing peak areas of spiked

plasma samples with the peak area in mobile phase prepared with the same nominal

concentration. For precision (as relative standard deviation, R.S.D.) and accuracy (the

accuracy as the percentage of deviation between nominal measured concentrations.)

studies, samples were prepared at three QC with 6 replicates each, and were analysed

in the same day (intra-day precision and accuracy), and analysed in 3 consecutive

days (inter-day precision and accuracy). The stability of the plasma samples was also

evaluated during method validation. The stability of VRT was evaluated in post-

162

extracted samples kept in the autosampler in 50 h, at room temperature (23 °C) for 10

h, as well as in plasma samples kept at -70 °C for 72 days and after being submitted to

3 freeze-thawing cycles (24 h each cycle). All samples described above were

compared to freshly prepared VRT samples at the same concentration level. The

method was validated according to the FDA and ICH guidelines for validation of

bioanalytical methods.

5. B.4 Results and discussion

5. B.4.1 optimization of LC-MS/MS conditions.

Precursor ions for valsartan and telmisartan and their corresponding product

ions, were determined from spectra obtained during the infusion of standard solutions

into a mass spectrometer using an electrospray ionization source, which operated in

negative and positive ionization modes with collision nitrogen gas in Q2 of a MS–MS

system. For the valsartan, telmisartan precursor and product ions were determined

through the analysis of 50 ng/mL solution prepared with methanol: water (80:20 v/v).

Valsartan, and telmisartan were mainly produced as protonated molecules at m/z

436.0 and 515.10, (Fig 5B.1a-b). Their product ions were scanned in Q3 after

collision with nitrogen in Q2 at m/z 291.10, and 497.50 for valsartan and telmisartan

respectively. 50 eV of the collision energy was optimal to produce the fragments of

m/z 497.50 and 297.10, while 50 eV of the collision energy should be needed to detect

the fragment ion of m/z 291, The MS–MS spectra of valsartan and telmisartan showed

fragmentation patterns are depicted in Fig. 5B.2a-b.

Determination of valsartan and telmisartan, a liquid–liquid extraction was used

in this analysis. Several organic solvents including ethyl acetate, methyl-t-butyl ether

and hexane/ethyl acetate mixture were previously used to extract valsartan in plasma

samples. Among organic solvents (ethyl acetate, ether, dichloromethane, chloroform

and their mixtures) were tested, ethyl acetate was found to be optimal, which can

produce a neat chromatogram for blank plasma samples and the best recovery, and the

least matrix effect was observed. There were no interfering peaks at the elution times

for either analyte valsartan 1.70 min; IS (telmisartan 0.9 min). Fig 5B.3 Represents

the typical chromatograms for blank plasma, and plasma spiked with 111.30 ng/mL

for valsartan, respectively, and 2.0 ng/mL for IS (telmisartan).

163

Fig 5B.1 (a)Full scan mass spectra of valsartan parent ion

164

Fig 5B.1 (b) Full scan mass spectra of telmisartan parent ion

165

Fig 5B.2 (a)Full scan spectra of valsartan ( Fragment ion)

166

Fig 5B.2 (b)Full scan spectra of telmisartan ( Fragment ion)

167

A reversed-phase column such as Thermo hypersil keystone Beta basic-8,

(2.1 mm. 50 mm, 3µ) with an isocratic mobile phase consisting of acetonitrile and

20 mM ammonium acetate containing 0.1% formic acid (pH = 3.8) (90:10, v/v) was

used. The mobile phase was eluted using a Perkin Elmer 018444Y model LC at 0.30

mL/min. The turbo ion spray interface was operated in the positive ion mode at

5,500 V. The operating conditions were optimized by flow injection of a analyte and

were determined as follows: nebulizing gas flow, 9.0 L/min turbo ions pray gas

flow, 8L/min, curtain gas flow, 1.44 L/min, and source temperature 500 °C. The

mass transition used for valsartan and its IS were m/z 436.00>291.10 and

515.10>497.50, respectively. Quadrupoles Q1 and Q3 were set on unit resolution.

The analytical data were processed by Analyst software (version 4.1.2).

5. B.5. Method validation

5. B.5.1. Selectivity and specificity

Selectivity testing with blank plasma from six different individuals never

resulted in interfering signals. The six blanks screened (n=6) and LLOQ spiked for

corresponding plasma lots. A chromatogram of an extracted blank plasma sample

(Fig, 5B.3) and a representative chromatogram of extracted calibration sample at the

lowest limit of quantification (LOQ) (5B.4), IS spiked to blank plasma (5B.5 ) and

extracted high QC sample (5B.6), respectively are given here.

168

Fig, 5B.3. Chromatogram of an extracted blank plasma sample

169

Fig, 5B.4 Representative chromatogram of extracted lowest limit of quantification (LOQ)

170

Fig, 5B.5 Representative chromatogram of IS spiked to blank plasma

171

Fig, 5B.6 extracted high QC sample of valsartan and IS

172

5. B.5.2. Linearity and lower limit of quantification

The linear regression of the peak area ratios versus concentration was fitted

over the concentration range of 0.9- 9051.08 ng/mL for vlsaratan in human plasma

with a correlation coefficient r =0.999

The lower limit of quantification (LLOQ) for valsartan was 0.9 ng/mL at a

signal-to-noise (S/N) ratio of 10. This sensitivity was enough to apply for

pharmacokinetic studies of valsartan after its oral administration.

5. B.5.3. Precision and accuracy

The intra- and inter-day precision and accuracy of the method developed are

listed in Table 5B.1. The coefficients of variation of the precision of the intra- and

inter-day validation were less than 0.56 and 5.88 %, respectively. The accuracy of the

method was 91.05–99.33 %. The inter-assay precision and accuracy were determined

by analyzing three calibration curves with quality control samples at four

concentration levels on 3 different days. The intra assay precision and accuracy were

determined by analyzing six replicates of the LOQ samples, and quality control

samples at three concentration levels were extracted on the same day. Detailed results

of intra-assay precision and accuracy are listed in Table 5B.1.

Table 5B.1 Intra-day and inter-day precision and accuracy for QC samples

Nominal

concentration

(ng/mL)

Within run (n=6) %

Recovery*

Between run (n=24) % Recovery*

Measured concentration

(ng/mL).

Mean ± SD (RSD)

Measured concentration

(ng/mL).

Mean± SD (RSD)

0.93

2.50

3711.00

7136.54

0.84 ± 0.01 (1.16 %)

2.41 ± 0.05 (1.92 %)

3673.40 ± 44.02 (1.20 %)

7088.92 ± 39.63 (0.56 %)

91.05

98.41

98.99

99.33

0.88 ± 0.03 (3.19 %)

2.32 ± 0.07 (3.0 %)

3565.67 ± 159.09 (4.46 %)

6825.70 ± 401.75 (5.88 %)

95.14

94.73

96.08

95.64

*Average of six determinations

173

5. B.5.4 Recovery

The extraction recoveries were greater than 66 % of spiked valsartan and its IS

(Table 5B.2). The absolute recovery of valsartan was assessed by comparing the peak

area of extracted QC samples in six replicates at each level (at low, mid and high

range) to aqueous QCs prepared in solutions at the same concentration levels.

Concentration of six replicates at each level was 2.45, 3711.0 and 7136.55 ng/mL.

The overall recovery of valsartan was 66 %. Recovery of the internal standard

telmisartan was 68.0%.

Table 5B.2 Recovery of valsartan of all three levels

QC’s name LQC (ng/mL) MQC (ng/mL) HQC (ng/mL)

Actual concentrations 2.45 3711.00 7136.55

Unextracted

(Aqueous)

Area obtained Area obtained Area obtained

5021 337882 705613

4809 328921 661512

5128 316690 667508

5021 337882 705613

4809 328921 661512

5128 316690 667508

Mean 4986 327831 678211

SD 145 9515 21394

%CV 2.91 2.90 3.15

%Nominal 100.00 100.00 100.00

174

5. B.5.5 Matrix effect

The absolute matrix effects for analyzing standards assessed analytes spiked at

two concentrations (2.44 and 7136.54 ng/mL) into plasma extracts from blank

human plasma and comparing the peaka areas of analytes of post extracted samples to

unextracted (Aqueous) which is between 0.85 - 1.15 (Table 5B.4). The matrix effect

QC’s name LQC (ng/mL) MQC (ng/mL) HQC (ng/mL)

Actual concentrations 2.45 3711.00 7136.55

Extracted

Area obtained Area obtained Area obtained

3061 227949 453108

3382 231434 444658

3186 228501 419404

3061 227949 453108

3382 231434 444658

3186 228501 419404

Mean 3210 229295 439057

SD 145 1675 15685

%CV 4.51 0.73 3.57

%Nominal 64.37 69.94 64.74

Recovery

LQC 64.37

MQC 69.94

HQC 64.74

Mean 66.35

SD 3.12

%CV 4.70

Overall% recovery 66.35

175

was evaluated for analyte and IS. There is no suppression or enhancement to analyte

or IS.

Table 5B.4 Results of the matrix factor of analyte and IS

Analyte peak area Internal standard peak area

Sample LQC HQC LQC HQC

Aqueous

sample(un

extracted)

5151 815521 431584 432645

5025 771111 422591 433945

4998 789958 427040 432943

5091 755891 426129 434859

4989 800151 425173 441945

5056 757221 424236 434956

Mean 5051 781642 426126 435216

Analyte peak area Internal standard peak area

Sample LQC HQC LQC HQC

Post extracted

samples

4951 779632 403791 427003

4895 780520 372910 432259

5021 788819 399772 435846

5001 766991 414757 430455

4999 779192 416724 435432

4856 767668 407127 433496

176

Matrix factor

Matrix factor of

Analyte

Matrix factor of

Internal standard

Sample LQC HQC LQC HQC

Post extracted samples

0.98 0.100 0.95 0.98

0.97 0.990 0.88 0.99

0.99 0.101 0.94 1.00

0.99 0.980 0.97 0.99

0.98 0.99 0.98 1.00

0.96 0.98 0.96 1.00

Mean 0.98 0.99 0.947 0.99

SD 1.31 1.07 0.036 0.008

% CV 1.33 1.08 3.76 0.88

5. B.5.6 Stability

Stability of valsartan in human plasma was evaluated; peak area measurements

obtained from the analysis of stored samples were compared to those obtained from

the analysis of freshly prepared plasma samples. The analyte was considered to be

stable in the biological matrix when 85-115 % deviation was found.

Bench top stability: triplicate aliquots of plasma spiked with valsartan at 2.45

and 7136.54 ng/mL (low and high quality control samples) were left at room

temperature for 10 hours, and then analyzed. It was investigated to ensure that

valsartan was not degraded in plasma samples at room temperature for a time period

to cover the sample preparation. The results are tabulated in Table 5B.5

In injector or processed sample stability: due to the need of occasional

delayed injection of extracted samples, this was carried out at two different

concentrations (low and high) were extracted, loaded on to the auto sampler and kept

up to 50 hours before injection. There was no appreciable changes occurs. Ensure that

the drug was not degraded in plasma samples. The results are shown in Table 5B.6.

177

Freeze thaw stability: This stability was carried out at two concentrations

(low and high). And it was assessed by exposing them to three freeze thaw cycles,

each cycle consists of removing the QC samples from the freezer, thawing them

unassisted to room temperature, kept at room temperature up to thawing i.e 1.0 h and

re-freezing about -70 °C even thaw and freeze, no changes were found in the analysis.

The results are given in Table 5B.7.

Table 5B.5 results of bench top stability of VRT

HQC(7136.543 ng/mL) LQC(2.449 ng/mL)

Bench top stability

7090.21 2.322

7055.15 2.350

7082.33 2.289

7100.59 2.305

7058.44 2.241

7012.43 2.282

Mean 7066.53 2.30

Nominal value (ng/mL) 7136.543 2.449

% Change -0.98 -6.08

Table 5B.6 Results of in-injector stability of VRT

HQC(7136.543 ng/mL) LQC(2.449 ng/mL)

In-injector stability

7011.21 2.298

7034.99 2.287

7091.52 2.198

7052.77 2.199

7018.46 2.232

7052.48 2.205

Mean 7043.57 2.24

Nominal value (ng/mL) 7136.543 2.449

% Change -1.30 -8.53

178

Table 5B.7 results of freeze-thaw stability of VRT

HQC (7136.543 ng/mL) LQC(2.449 ng/mL)

Freeze thaw stability

7030.52 2.101

7000.59 2.195

7005.33 2.256

7012.76 2.309

7008.59 2.148

7019.44 2.222

Mean 7012.87 2.21

Nominal value (ng/mL) 7136.543 2.449

% Change -1.73 -9.75

5B.6. Applications

The method has been successfully used to quantitate estimation of valsartan on

human volunteers under fasting conditions after administration of a single

combination tablet containing 80 mg of valsaratn. After dosing of valsartan totally

twenty six time points were collected from volunteer, after the blood collection was

done post dosing inclusive of the pre-dose sample. The blood samples were collected

in separate vacutainers containing K2EDTA anti-coagulant. The plasma from these

samples was separated by centrifugation at 4000 rpm and kept within the range of 2–

8 °C. The plasma samples thus obtained were stored at −20 °C till analysis. Post

analysis, the pharmacokinetic parameters were computed using SAS 9.1.3 software.

The statistical data which were evaluated as Cmax (maximum observed drug

concentration during the study) 3000 ng/mL, only 80 % bio availability was observed.

In this LC–MS/MS method, each plasma sample was chromatographed within

2.0 min. This method is highly selective and suitable for bioavailability and bio-

equivalence studies of different formulations containing valsartan. The sensitivity and

specificity of the method were sufficient for characterizing the pharmacokinetics of

valsartan.

179

Conclusion

There are several methods are available for the analysis of antihypertensive

drugs, (angiotension II receptor antagonist). The method developed is simple,

extraction free and cost-effective method. The uses of p-chloranilic acid are easily

available and cheaper than other reagents, which are used in the existing methods. The

method did not require any extraction or heating steps for the color development. The

color of the C-T complex remained stable at room temperature (27 ± 3 ºC) for 2 h.

The proposed method is simple and sensitive and in addition, the method has wider

linear dynamic range with good accuracy and precision that could be applied for the

determination of valsartan in bulk drug and dosage forms.

In section -5A the developed LC-MS/MS procedure for the valsartan using

human plasma is simple, sensitive, inexpensive, accurate and more precise than other

instrumental methods. The methods developed are compared with reported methods

and are presented in Table 5B.8. The proposed procedures offer the advantage of

simple extraction solvents used for extraction of sample, and total run time 2.0 min

per sample.

180

Table 5B.8: Comparison of proposed methods with some of the reported

spectrophotometric and LC-MS/MS methods

S.l.No Reagents Beer’s Law

limit (µg/mL)

Remarks Ref

1 bromophenol blue

bromocresol green

5 – 40

1 – 50

Costly reagents used [18]

2 LC-MS/MS-method

Sample extraction from

SPE technique

- Cost is more [24]

3 LC-MS/MS-method

500µLplasma used. Total

run time was 2.8 min.

0.5-50 ng/mL Sample consumption

more and run time also

2.8 min.

[26]

4 LC-MS/MS-method

Sample extract from

precipitation.

1.0-2000

ng/mL

Less sensitive. In

precipitation method

sample cleanup is not

good enough compared

to liquid-liquid or SPE

extractions

[27]

5 LC-MS/MS-method

TBME as extraction

solvent. Runtime 2.3 min

- Carcinogenic extraction

solvent used. run time

also more.

[28]

6 LC-MS/MS-method LLOQ-20

ng/mL.

Less sensitive [29]

7 LC-MS/MS-method. solid

phase extraction

2-2000 ng/mL Less sensitive. Cost is

more for SPE method.

[30]

8 (a) LC-MS/MS-method.

Ethyl acetate (100%) used

as extraction solvent. Run

time is 2.0 min

(b) p-chloranilic acid

(a) 0.9-

9051ng/mL.

(b) 5.0 – 50

µg/mL

(a) 0.2 mL plasma is

sufficient to get good

response. Run time is 2.0

min.

(b) Extraction free and

cheap reagent used.

Proposed

methods

181

Reference

[1]. Mehtap Saydam, Sevgi Takka, J. Pharm. Sci, 2007, 32, 185.

[2]. S.C. Benson, H. Pershdesingh, P. Desai, Scientific contributions,2004, 43, 993.

[3]. D.G.T. Parambi , M. Mathew, V. Ganesan. J. Appl. Pharm. Sci., 2011, 1, 97.

[4]. S.S Chitlange, K. Bagri and D.M.Sakarkar. As. J. Res. Chem., 2008, 1, 15.

[5]. C.V.N. Prasad, Ch.S. Kumari, J.S. Ramulu, Inter. J. Res. Pharm and Chem.,

2011, 1, 102

[6]. D. Jothieswari, D. Priya, S. Brito Raj, E. Mohanambal, S. Wasim Raja, Inter. J

Novel Tren. In Pharm. Sci., 2011, 1,18

[7]. A. Zarghi , A. Shafaati, S.M. Foroutan, H. Movahed , Sci. Pharm., 2008,76,

439.

[8]. Zong-Zhu Piao, Eung-Seok Lee, H.T.T. Tran, Beom-Jin Lee. Arch. Pharm.

Res., 2008, 31, 1055.

[9]. D.U. Vinzuda, G.U. Sailor, N.R. Sheth, Intern. J. Chem Tech. 2010, 2, 1461.

[10]. B.R. Yadav, M. Moinuddin, R. Battu, G. Samanthula, J. Anal. Chem., 2012, 2,

14

[11]. M. Manoranjani, T. Bhagyakumar. Intern. J. Sci. Innov. and Discov., 2011, 1,

101.

[12]. B. S. Patel, G. Bharat, Chaudhari, K. M. Buch, A. B. Patel, Intern. J. Chem Tech

Res.. 2009, 1, 1257.

[13]. S. Edam, A. Altınay , N. G. Goger , S. A. Ozkan, Z. Senturk, J. Pharm. Biomed.

Anal., 2011, 25, 1009.

[14]. S. Tatar, S. Saglk, J. Pharm. Biomed. Anal., 2002, 30, 371

[15]. S. N. Meyyanathan, A. S. Birajdar, B. Suresh, Indian J .Pharm Educ. 2010, 2,

44.

[16]. K. R. Gupta, A. R. Wadodkar, S. G. Wadodkar. Intern. J. Chem Tech Res.,

2010, 2, 985.

182

[17]. M. M. Deshpande, M. P. Mahajan. S. D. Sawant. Intern. J Pharm. Sci. Res.,

2012, 3, 2.

[18]. S. Ramachandran, B. K. Mandal, G. Navalgund, Trop. J. Pharm. Res., 2011, 10, 809.

[19]. N. Erk . Anal. Lett., 2002, 35, 283.

[20]. Erdaldinc, D. Baleanu. Rev. Chim. (Bucureti), 2010, 61, 290

[21]. N. G. Mohamed . Anal. Chem. Insig., 2011, 6, 53.

[22]. G. A. Va, R. V. Ga, Shivakumar, D. G Ya, T. H La, Indo-Global J. Pharm. Sci.,

2011, 1, 99.

[23]. B. R. Kadam, S. B. Bari, Acta Chromat, 2007, 18, 25.

[24]. H. J. Shah, N. B. Kataria, G. Subbaiah, C. N. Patel, Chromatographia, 2009, 69,

1055.

[25]. A.V. Ramani, P. Sengupta, R. Mullangi, Biomed Chromatogr, 2009, 23, 615.

[26]. P. S. Selvan, K. V. Gowda, U. Mandal, W. D. S. Solomon, T. K. Pal. J. Pharm.

Biomed. Anal., 2009, 49, 780.

[27]. X. Hu, Y. Zheng, J. Sun, L. Shang, G. Wang, H. Zhang, Chromatographia.

2009, 69, 843.

[28]. N. Koseki, H. Kawashita, H. Hara, M. Niina, M. Tanaka, R. Kawai, Y. Nagae,

N. Masuda, J Pharm. Biomed. Anal,2007. 11, 1769

[29]. A. Patnaik, M. Shetty, S. Sahoo, D. K. Nayak, S. K. Veliyath, Intern. J. Pharm.

Sci., 2010, 2, 43.

[30]. S. B. Chattaraj, K. Sharma, A. Chakraborthy, S. C. Lahiri. Spectrochim. acta A,

2012, 95, 637.

[31]. International Conference On Harmonization of Technical Requirements for

Registration of Pharmaceuticals for Human Use, ICH Harmonised Tripartite

Guideline, Validation of Analytical Procedures: Text and Methodology Q2(R

1), Complementary Guideline on Methodology, dated 06 November 1996,

incorporated in November 2005, London.