Simultaneous determination of the antihypertensives hydrochlorothiazide,losartan potassium,irbesartan and valsartan in bulk powders and pharmaceutical preparations by high performance liquid chromatography

Abstract

A simple, rapid and accurate, routine-LC method is described for simultaneous determination of four antihypertensive drugs, hydrochlorothiazide, losartan potassium, valsartan and irbesartan in bulk powders and pharmaceutical preparations. The chromatographic separation of the four pharmaceuticals was achieved on a reversed phase C18 “Intersil^® ODS-3” column (5 μm, 4.6×250 mm) using a binary mobile phase of 45% ACN and 55% 50 mM KH₂PO₄ (pH 4.5) at 1 mL/min flow rate. The detection-wavelength was 210 nm. The total separation time was less than 12 min. The method was validated for system efficiency, linearity, accuracy, precision, limits of detection and quantitation, specificity, stability and robustness. The limits of detection were 0.02, 0.12, 0.14 and 0.01 μg/mL for hydrochlorothiazide, losartan potassium, valsartan and irbesartan, respectively. Linearity ranges were 5–50 μg/mL for hydrochlorothiazide, 10–100 μg/mL for losartan potassium, irbesartan and valsartan. The recovery values of this method were between 97 and 103% and the reproducibility was within 1.67%. Statistical analysis proved that the method enabled reproducible and selective quantification of all four analytes as the bulk drug and in pharmaceutical preparations.

Keywords

HPLC simultaneous determination antihypertesives pharmaceutical preparations

1 Introduction

Losartan potassium, valsartan and irbesartan are the first drugs of new class of orally active, non-peptide angiotensin II (type AT1) receptor antagonists for the treatment of hypertension. Because of their enhanced specificity, selectivity and tolerability they are considered to be superior to previous angiotensin converting enzyme (ACE) inhibitors [1, 2]. Studies have shown an increase in the efficacy when a low dose of hydrochlorothiazide (HCTZ) is administered with these non-peptide angiotensin II (type AT1) receptor antagonists due to stimulation of the renin–angiotensin system [1, 3]. In this combination therapy, the effects of both drugs appear to be additive and synergistic for patients whose elevated blood pressure is not well controlled with either substance alone [1, 3].

HCTZ, 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide 1,1-dioxide (Scheme 1), is a thiazide diuretic. It increases the rate of urine excretion by the kidney. Thiazide diuretics are extremely useful in the treatment of oedema associated with mild to moderate congestive heart failure and control of hypertension [4].

United states pharmacopoeia (USP) describes a high performance liquid chromatography (HPLC) method for analysis of HCTZ [5], while British pharmacopoeia describes a potentiometric titration using 0.1 M tetra butyl ammonium hydroxide in 2-propanol, for its assay in bulk and dosage forms [6]. Gradient HPLC method is reported for related compounds [6]. In addition to previously mentioned methods, there were other methods including spectrophotometric methods applied to the simultaneous estimation of HCTZ in combination with other drugs in tablet dosage form [7 –19]. A novel chemiluminescence method for determination of HCTZ was developed based on its reaction with Ce (IV) in acid medium in the presence of rhodamine 6G as sensitizer [20]. Electrochemical study of HCTZ and its determination in urine and tablets was developed [21]. Concerning with chromatographic methods, a stability-indicating LC method for the simultaneous determination of ramipril and HCTZ in dosage forms was reported [22] and an HPLC method was reported for simultaneous determination of HCTZ, xipamide and triamterene [23].

Losartan potassium (LOS), (2-n-butyl-4-chloro-5-hydroxymethyl- 1-((2’-(1H-tetrazol-5-yl)(biphenyl-4-yl)methyl) imidazole, potassium salt (Scheme 1), is an angiotensin II receptor antagonist indicated for the treatment of hypertension. Oxidation of the 5-hydroxymethyl group on the imidazole ring results in the active metabolite of LOS. It may be used alone or in combination with other antihypertensive agents, including diuretics. It is also indicated to reduce the risk of stroke in patients with hypertension and left ventricular hypertrophy [4].

United states pharmacopoeia (USP) describes a high performance liquid chromatography (HPLC) method [5], while British pharmacopoeia (BP) describes a potentiometric titration method using 0.1 M perchloric acid for its assay in bulk and its dosage forms [6]. It was also determined in the presence of other drugs by several techniques including spectrophotometric methods [24 –26]. Capillary electrochromatography has been used to determine LOS and HCTZ [27]. Novel HPLC assays were developed for determination of LOS and its main active metabolite [28] while stability indicating methods were developed for simultaneous determination of LOS and its degradation products [29, 30]. Other chromatographic methods were used for determination of LOS either alone or in the presence of other analytes [31 –35].

Irbesartan (IRB), 2-butyl-3-({4-[2- (2H-1, 2, 3, 4-tetrazol-5-yl) phenyl] phenyl) methyl)-1, 3-diazaspiro [4, 4] non-1-en-4-one (Scheme 1), is an angiotensin II receptor antagonist with actions similar to those of LOS. It is used in the management of hypertension and treatment of renal disease in hypertensive diabetic patients [4]. United States Pharmacopoeia (USP) describes a high performance liquid chromatography (HPLC) for its determination [5], while British Pharmacopoeia describes a potentiometric titration method using 0.1 M perchloric acid for its assay in bulk powders [6]. A first-derivative spectrophotometric method was used for the determination of IRB alone and in the presence of HCTZ [36] and a sensitive high-performance liquid chromatographic technique for their assay was described [37]. Liquid chromatography with fluorescence detection for determination of IRB and LOS was described [38]. Identification and characterization of degradation products of IRB using gradient HPLC method and mass spectrum were reported [39]. Spectrofluorimetric and spectrophotometric methods were reported for determination of IRB [40]. A stability-indicating liquid chromatography method is developed and validated for the quantitative simultaneous estimation of IRB and HCTZ in combined pharmaceutical dosage form [41]. Other chromatographic methods used for determination of IRB in the presence of celiprolol and bisoprolol [42], olmesartan medoxomil and HCTZ [43] or with HCTZ only [44].

Valsartan (VAL), (2S)-3-methyl-2-[pentanoyl[[2’-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]amino]butanoic acid (Scheme 1), is an angiotensin II receptor antagonist with actions similar to those of LOS. It is used in the management of hypertension to reduce cardiovascular mortality in patients with left ventricular dysfunction after myocardial infarction and in the management of heart failure [4].

United states pharmacopoeia (USP) describes a high performance liquid chromatography (HPLC) method for its determination [5], while British pharmacopoeia describes potentiometric titration method with 0.1 M tetrabutylammonium hydroxide in 2-propanol [6].

For the simultaneous estimation of VAL and other drugs in tablet dosage form, spectrophotometric methods were applied [16 , 45–47] and also HPLC technique [16, 48]. A high- performance liquid chromatographic procedure for the quantitation of VAL and its enantiomer has been developed [49].

Electrochemical behaviour of VAL was established and then used for its determination in capsules [50]. Ultra-performance liquid chromatography (UPLC) was investigated for the simultaneous analysis of chlorthalidone, VAL, its metabolite and fluvastatin in human plasma using an RP C18 in gradient mode [51].

There are no previous reports describing the chromatographic determination of the four medicaments simultaneously. Most of the previously published papers about chromatographic determination of antihypertensives reported about the determination of a combination of only two pharmaceuticals of these four drugs [16 , 46] or even described the determination of angiotensin converting enzyme (ACE) receptor antagonists alone [38 , 52]. There is only one report about separation and determination of the drugs under investigation simultaneously using capillary electrophoresis [53]. In which two different methods were applied to enable separation of all analytes [53]. In a previous study we reported about the qualitative chromatographic separation of the antihypertensives [54]. This study describes the development and validation of a RP-HPLC simultaneous determination of HCTZ, LOS, VAL and IRB and can be considered as an application of our previous study [54]. The proposed method enabled fast separation of the analytes if it is compared with the previously published capillary electrophoresis method [53].

2 Experimental

2.1 Chemicals and reagents

All chemicals and reagents are at least analytical grade. Water (pharmaceutical grade), KH₂PO₄, KOH and H₃PO₄ were purchased from (Merck, Germany). MeOH and ACN were HPLC-grade(J. T. Baker, Holland). All active drugs were at least pharmaceutical grade and obtained from Egyptian International Pharmaceutical Industries Co. (EIPICo., Egypt). Pharmaceutical formulations: Losazide^® (50 mg LOS and 12.5 mg HCTZ per tablet), Vasotec^® (160 mg VAL per tablet) and Xtension^® (150 mg IRB per tablet) all were obtained from Egyptian market.

2.2 Instrumentation

Agilent HPLC series 1200 (Agilent technologies, Germany) consisting of solvent pump (model G1311A), autosampler (model G1329A), column compartment (model G1316A) and UV detector (model G1314A) was used for separation.

pH values were measured using pH meter Metrohm 713^® (Switzerland).

2.3 Column

Reversed phase C18 “Intersil^® ODS-3” from (GL Sciences Inc., Japan) 5 μm, 4.6×250 mm was used as a stationary phase.

2.4 Chromatography

The experiments were performed with isocratic elution. The binary mobile phase consisted of 45% ACN and 55% of 50 mM KH₂PO₄ (pH 4.5) set at flow rate 1 mL/min and column temperature at 30°C. Volume of 10 μL of samples was injected per run and eluates were detected using UV -Detector at λ= 210 nm.

2.5 Preparation of stock and standard working solutions

A 100 mg of LOS, VAL or IRB and a 50 mg of HCTZ were mixed and dissolved in 100 mL MeOH. The standard working solutions were prepared by diluting aliquots of the stock solution into (MeOH: H₂O, 1:1) to reach concentration of 5–50 μg mL^-1 for HCTZ and, 10–100 μg mL^-1 for LOS, IRB or VAL.

The calibration graph was constructed by plotting the average absorbance obtained at wavelength 210 nm of triplicate 10 μL injections of each standard solution at prescribed conditions versus its corresponding injected concentrations.

2.6 Sample preparation

Ten tablets of each (Losazide^®, Vasotec^® and X-tension^®) were accurately weighed and ground to fine powder. A portion of each tablet powder, equals to the average weight of one tablet, was weighed and mixed together then dissolved in 100 mL methanol then the solution was left in the ultrasonic bath for 10 minutes. After that the solution was filtered and the first 10 mL was rejected and 10 mL of the following filtrate was quantitatively taken and serial dilutions were performed.

3 Results and discussion

3.1 System suitability

When a sample containing the four analytes was injected into chromatograph under the mentioned isocratic conditions, the t_R values of the symmetrical peaks of HCTZ, LOS, VAL and IRB were 3.52±0.02, 6.49±0.05, 8.60±0.12 and 10.40±0.08 min, respectively and k’-values were 0.44, 1.7, 2.47 and 3.34, respectively (Table 1).

The reproducibility tests for these analytes were less than 1% of the retention times. Chromatographic parameters such as number of theoretical plates (N), resolution (R_s), selectivity (α), and retention factor (k’) were determined (Table 1). The results indicate that the described method showed adequate column efficiency, good selectivity and repeatability and could be applied for simultaneous determination of the four analytes.

Table 1 shows the column system performance on the examined drugs and their order of elution from the column.

3.2 Linearity and calibration

Assay of each analyte was linear to a concentration range described in Table 2.

The slope, intercept and regression coefficient for each compound were estimated (Table 2). The limit of detection defined as the injected quantity giving an S/N of 3.3 (in terms of peak height), were found to be 0.02, 0.12, 0.14 and 0.01 μg mL^-1 for HCTZ, LOS, VAL and IRB, respectively. Limits of quantitation defined as the injected quantity giving an S/N of 10 (in terms of peak height) were found to be 0.06, 0.37, 0.44 and 0.04 μg mL^-1for HCTZ, LOS, VAL and IRB, respectively.

3.3 Accuracy

The accuracy of the method was determined by recovery experiments using the standard addition technique. Each solution was injected in triplicate and percentage recovery was calculated Accuracy results indicated high accuracy (Table 3).

3.4 Precision

Intra-day precisions were assessed by injecting standard solution of each analyte five times during a day at four different concentrations (from low to high concentration). The resultant coefficients of variation (CV) were lower than 2.0% for all (Table 4).

Inter-day precision experiments were done after treatment of the standard solution in the same method of tablets extraction, and then analyzed every day over 5 days (Table 4). All CV values were lower than 2.0%.

3.5 Specificity of the method

Figure 1 reveals that there is no interference from the excipients. Other drugs were tested for interference with the method and it was found that some drugs could interfere such as atenolol and tadalafil, but still many others do not interfere such as apomorphine hydrochloride, sildenafil citrate, tramadol hydrochloride, clopidogrel sulfate and propranolol hydrochloride.

3.6 Application of the method

The application of the method for assay of drugs in tablets was determined by the proposed method and compared statistically to reported methods [5, 6] by use of Student’s t-test and the variance ratio F-test. The results in (Table 5) show that the calculated t and F values were smaller than the tabulated values, indicating that there were no significant differences between the results obtained from this method and from published methods.

3.7 Stability of the solution

The stability of both standard and tablets’ test sample solutions was determined by monitoring the peak areas of the mixture over a period of time and they were found to be stable for 5 days at room temperature and for 14 days at refrigerator.

3.8 Robustness of the method

The robustness of the present method was evaluated in the terms of temperature, wavelength of detection and content of ACN in the mobile phase. Also the effects of slight changes, in flow rate and pH of the mobile phase on t_R values and peak area were studied (Table 6).

The slight variations in these factors had no significant effect on resolution and peak shape. The temperature was changed by increments of 2°C from 28 to 32°C and the effect of these changes on peak area was studied. To study the effect of changes in contents of organic modifier ACN on peak area, 44, 45 and 46% ACN was used. Also analyses were done at three different wavelengths, 208, 210 and 212 nm, to examine the robustness of the method. Three different pH values were applied to see if the results will change through the change in pH value (4.4, 4.5 and 4.6).

The results in (Table 6) indicate that the small changes in the examined factors led to slight changes in the peak area and/or t_R values. The changes in ACN and detection wavelength have the least effects, while the changes in pH value have the highest effect on the analysis. This means the method is more sensitive to changes in pH value than to changes in the other factors.

4 Conclusions

The proposed method has a satisfactory separation of the analytes, extended linear range and a rapid analysis time. The four components were separated in less than 12 min. A high recovery of each analyte was achieved using C18 column for determination. Recoveries indicate good agreement with analytes amounts as stated at the label, ranged from 97 to 103%. The proposed LC method ensures a precise, accurate, sensitive and selective determination of losartan potassium, valsartan, irbesartan and hydrochlorothiazide in bulk powders and pharmaceutical preparations. No interferences from the excipients were noticed.

Footnotes

Acknowledgments

The authors thank the above-mentioned companies for the friendly supply of analytes and column.

References

Schoenberg

J.A.

, Losartan with hydrochlorothiazide in the treatment of hypertension, J Hypertens Suppl 13 (1995), S43–S47.

Goldberg

and Sweet

, Efficacy and safety of losartan, Can J Cardiol 11(Suppl F) (1995), 27F–32F.

Oparil

, Aurup

, Snavely

and Goldberg

, Efficacy and safety of losartan/hydrochlorothiazide in patients with severe hypertension, Am J Cardiol 87 (2001), 721–726.

Martindale

S.S.

, The complete drug reference, Thirty sixth edition; 2009, 1398–1399.

The united states pharmacopoeia 32- NF 28, 2011.

The British pharmacopoeia, 2011.

Kargosh

and Sarrafi

A.H.M.

, Spectrophotometric simultaneous determination of triamterene and hydrochlorothiazide in triamterene-H tablets by multivariate calibration methods, J Pharm Biomed Anal 26 (2001), 273–279.

Dinçand

and Üstünda

Öz.

, Spectophotometric quantitative resolution of hydrochlorothiazide and spironolactone in tablets by chemometric analysis methods, Il Farmaco 58 (2003), 1151–1161.

Dinç

and Baleanu

, Spectrophotometric quantitative determination of cilazapril and hydrochlorothiazide in tablets by chemometric methods, J Pharm Biomed Anal 30 (2002), 715–723.

10.

Ferraro

M.C.

, Castellano

P.M.

and Kaufman

T.S.

, Chemometric determination of amiloride hydrochloride, atenolol, hydrochlorothiazide and timolol maleate in synthetic mixtures and pharmaceutical formulations, J Pharm Biomed Anal 34 (2004), 305–314.

11.

Erk

, Determination of active ingredients in the pharmaceutical formulations containing hydrochlorothiazide and its binary mixtures with benazepril hydrochloride, triamterene and cilazapril by ratio spectra derivative spectrophotometry and vierordt’s method, J Pharm Biomed Anal 20 (1999), 155–167.

12.

Joshi

S.J.

, Karbhari

P.A.

, Bhoir

S.I.

, Bindu

K.S.

and Das

Ch.

, RP-HPLC method for simultaneous estimation of bisoprolol fumarate and hydrochlorothiazide in tablet formulation, J Pharm Biomed Anal 52 (2010), 362–371.

13.

El Walily

A.M.

, Belal

S.F.

, Heaba

E.A.

and Kersh

A.El.

, Simultaneous determination of enalapril maleate and hydrochlorothiazide by first-derivative ultraviolet spectrophotometry and high-performance liquid chromatography, J Pharm Biomed Anal 13 (1995), 851–856.

14.

Parissi-Poulou

, Reizopoulou

, Koupparis

and Macheras

, Second derivative UV spectrophotometric determination of hydrochlorothiazide and hydrochlorothiazide-amiloride combination in tablets, Int J Pharmaceutics 51 (1989), 169–174.

15.

Al-Momani

I.F.

, Determination of hydrochlorothiazide and enalapril maleate in tablet formulations by reversed-phase HPLC, Turkish J Chem 25 (2001), 49–54.

16.

Atana

, Altinaya

, Günden Göera

and Özkan

S.A.

, Entürk

, Simultaneous determination of valsartan and hydrochlorothiazide in tablets by first-derivative ultraviolet spectrophotometry and LC, J Pharm Biomed Anal 25 (2001), 1009–1013.

17.

El-Gindy

, Ashour

, Abdel-Fattah

and Shaban

, Spectrophotometric and HPTLC-densitometric determination of lisinopril and hydrochlorothiazide in binary mixtures, J Pharm Biomed Anal 25 (2001), 923–931.

18.

El-Gindy

, Ashour

, Abdel-Fattah

and Shaban

, Spectrophotometric determination of benazepril hydrochloride and hydrochlorothiazide in binary mixture using second derivative of the ratio spectra and chemometric methods, J Pharm Biomed Anal 25 (2001), 299–307.

19.

Erk

, Simultaneous determination of fosinopril and hydrochlorothiazide in pharmaceutical formulations by spectrophotometric methods, J Pharm Biomed Anal 27 (2002), 901–912.

20.

Murillo Pulgarín

J.A.

, Alañón Molina

and Pérez-Olivares Nieto

, Determination of hydrochlorothiazide in pharmaceutical preparations by time resolved chemiluminescence, Anal Chim Acta 518 (2004), 37–43.

21.

Abdel Razak

, Electrochemical study of hydrochlorothiazide and its determination in urine and tablets, J Pharm Biomed Anal 34 (2004), 433–440.

22.

Belal

, Al-Zaagi

I.A.

, Gadkariem

E.A.

and Abounassif

M.A.

, A stability-indicating LC method for the simultaneous determination of ramipril and hydrochlorothiazide in dosage forms, J Pharm Biomed Anal 24 (2001), 335–342.

23.

Abd El-Hay

S.S.

, Hashem

and Gouda

A.A.

, High performance liquid chromatography for simultaneous determination of xipamide, triamterene and hydrochlorothiazide in bulk drug samples and dosage forms, ACTA Pharmaceutica 66 (2016), 109–118.

24.

Rakesh

S.U.

, Patil

P.R.

, Dhabale

P.N.

and Burade

K.B.

, New spectrophotometric method applied to the simultaneous estimation of losartan potassium and amlodipine besylate in tablet dosage form, J Pharmacy Research 7 (2009), 1252–1255.

25.

Erk

, Analysis of binary mixtures of losartan potassium and hydrochlorothiazide by using high performance liquid chromatography, ratio derivative spectrophotometric and compensation technique, J Pharm Biomed Anal 24 (2001), 603–611.

26.

Prabhakar

A.H.

and Giridhar

, A rapid colorimetric method for the determination of losartan potassium in bulk and in synthetic mixture for solid dosage form, J Pharm Biomed Anal 27 (2002), 861–866.

27.

Quagli

M.G.

, Donati

, Carlucci

, Mazzeo

and Fanali

, Determination of losartan and hydrochlorothiazide in tablets by CE and CEC, J Pharm Biomed Anal 29 (2002), 981–987.

28.

Soldner

, Spahn-Langguth

and Mutschler

, HPLC assays to simultaneously determine the angiotensin-AT1 antagonist losartan as well as its main and active metabolite EXP 3174 in biological material of humans and rats, J Pharm Biomed Anal 16 (1998), 863–873.

29.

Hertzog

D.L.

, Finnegan McCafferty

, Fang

, Jeffrey Tyrrell

and Reedd

R.A.

, Development and validation of a stability-indicating HPLC method for the simultaneous determination of losartan potassium, hydrochlorothiazide, and their degradation products, J Pharm Biomed Anal 30 (2002), 747–760.

30.

Satyanarayana

D.R.V.

, Sait1

S.S.

, Ramakoti Reddy

and Mukkanti

, Simultaneous determination of losartan potassium, atenolol and hydrochlorothiazide in pharmaceutical preparations by stability-indicating UPLC, Chromatographia 70 (2009), 647–651.

31.

Argekar

A.P.

and Sawant

J.G.

, A gradient reversed phase high performance liquid chromatography method for simultaneous determination of hydrochlorothiazide (HCTZ) and losartan potassium (LOS) from tablets, Anal Lett 33 (2000), 869–880.

32.

Jalalizadeh

, Souri

, Farsam

and Ansari

, A high-performance liquid chromatographic assay for the determination of losartan in plasma, Iranian J Pharmacol Therapeutics 2 (2003), 18–21.

33.

Jin-Ki Kim

Y.Ch.

, Ban

, Park

and Kim

Ch.

, Determination of losartan in human plasma by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS): Application to bioequivalence study, J Liq Chromatogr Rel Technol 31 (2008), 2643–2656.

34.

Yeung

P.K.F.

, Jamieson

, Smith

G.J.

, Fice

and Timothy Pollak

, Determination of plasma concentrations of losartan in patients by HPLC using solid phase extraction and UV detection, Int J Pharm 204 (2000), 17–22.

35.

Goswami

, Kumar

, Khuroo

A.H.

, Monif

, Thudi

N.R.

, Shrivastav

V.K.

, Dubey

S.K.

, Shingla

A.K.

, Prakash

and Mehra

, Pharmacokinetic estimation of losartan, losartan carboxylic acid and hydrochlorothiazide in human plasma by LC/MS/MS validated method, Clinical Research and Regulatory Affairs 25 (2008), 235–258.

36.

Albero

, Ródenas

, García

and Sánchez-Pedreño

, Determination of irbesartan in the presence of hydrochlorothiazide by derivative spectrophotometry, J Pharm Biomed Anal 29 (2002), 299–305.

37.

Erk

, Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by liquid chromatography, J Chromatogr B 784 (2003), 195–201.

38.

Shakya

A.K.

, Al-Hiari

Y.M.

and Alhamami

O.M.O.

, Liquid chromatographic determination of irbesartan in human plasma, J Chromatogr B 848 (2007), 245–250.

39.

Shah

R.P.

, Sahu

and Singh

, Identification and characterization of degradation products of irbesartan using LC–MS/TOF, MSn, on-line H/D exchange and LC–NMR, J Pharm Biomed Anal 51 (2010), 1037–1046.

40.

Abdelmonem

A.A.

, Ragab

G.H.

, Hashem

H.A.

and Bahgat

E.A.

, Spectrofluorimetric and spectrophotometric determination of irbesartan and bisoprolol hemifumarate independently in their tablets, UK J Pharm Biosci 4(2) (2016), 43–52.

41.

Rane

V.P.

, Patil

K.R.

, Sangshetti

J.N.

, Yeole

R.D.

and Shinde

D.B.

, Stability indicating LC method for simultaneous determination of irbesartan and hydrochlorothiazide in pharmaceutical preparations, J Chromatogr Sci 48 (2010), 595–600.

42.

Caudrona

, Laurenta

, Billauda

E.M.

and Prognon

, Simultaneous determination of the acid/base antihypertensive drugs celiprolol, bisoprolol and irbesartan in human plasma by liquid chromatography, J Chromatogr B 801 (2004), 339–345.

43.

Sultana

, Saeed Arayne

, Shahid Alia

and Sajid

Sh.

, Simultaneous determination of olmesartan medoxomil and irbesartan and hydrochlorothiazide in pharmaceutical formulations and human serum using high performance liquid chromatography, Chinese J Chromatogr 26 (2008), 544–549.

44.

Tutunji

L.F.

, Tutunji

M.F.

, Alzoubi

M.I.

, Khabbas

M.H.

and Aridad

A.I.

, Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma using HPLC coupled with tandem mass spectrometry: Application to bioequivalence studies, J Pharm Biomed Anal 51 (2010), 985–990.

45.

Erk

, Spectrophotometric Analysis of valsartan and hydrochlorothiazide, Anal Lett 35 (2002), 283–302.

46.

Satana

, Altinay

, Günden Göger

Nil.

, Özkan

S.A.

and Sentürk

, Simultaneous determination of valsartan and hydrochlorothiazide in tablets by first-derivative ultraviolet spectrophotometry and LC, J Pharm Biomed Anal 25 (2001), 1009–1013.

47.

Cagigal

, González

, Alonso

R.M.

and Jiménez

R.M.

, Experimental design methodologies to optimise the spectrofluorimetric determination of losartan and valsartan in human urine, Talanta 54 (2001), 1121–1133.

48.

Levia

, Wuerznerc

, Ezana

and Pruvosta

, Direct analysis of valsartan or candesartan in human plasma and urines by on-line solid phase extraction coupled to electrospray tandem mass spectrometry, J Chromatogr B 877 (2009), 919–926.

49.

Francotte

, Davatz

and Richert

, Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester, J Chromatogr B 686 (1996), 77–83.

50.

Yan

, Wanga

, Chena

and Chena

, Electrochemical behavior of valsartan and its determination in capsules, Biointerfaces 67 (2008), 205–209.

51.

Iriarte

, Gonzalez

, Ferreirós

, Itxaso Magureguia

, Maria Alonso

and Maria Jiménez

, Validation of a fast liquid chromatography–UV method for the analysis of drugs used in combined cardiovascular therapy in human plasma, J Chromatogr B 877 (2009), 3045–3053.

52.

Demiralay

, Cubuk

, Ozkan

S.A.

and Alsancak

, Combined effect of polarity and pH on the chromatographic behavior of some angiotensin II receptor antagonists and optimization of their determination in pharmaceutical dosage forms, J Pharm Biomed Anal 53 (2010), 475–482.

53.

Hillaert

and Van Den Bossche

, Simultaneous determination of hydrochlorothiazide and several angiotensin-II-receptor antagonists by capillary electrophoresis, J Pharm Biomed Anal 31 (2003), 329–339.

54.

Elhenawee

, Hashem

and Ibrahim

A.E.

, Comparison between calixarene and conventional hplc-stationary phases concerning with separation of antihypertensive drugs, J Liq Chromatogr Rel Technol 37 (2014), 1–25.