socIAL AND HeAL tH IMPoR tANce AND ANALYtIcAL ...bsphs.org/wp-content/uploads/2014/04/Pankova.pdf · 68 PHARMAcIA, vol. 61, No. 1/2014 sava ognjanov, stefka Pankova socIAL AND HeAL

68 PHARMACIA, vol. 61, No. 1/2014 Sava Ognjanov, Stefka Pankova

socIAL AND HeALtH IMPoRtANce AND ANALYtIcAL cHARActeRIstIcs oF LosARtAN

sava ognjanov1, stefka Pankova2

1„Polfarma LtD“; 2Faculty of Pharmacy, Medical University – Sofia

summary.The aim of current study is to summarize and comprise the different developed methods for determination of angiotensin receptor blocker Losartan Роtassium alone and in combination with calcuim antagonists, ACE – inhibitors and diuretics (Hydrochlorothiazide) in dosage pharmaceutical preparations (tablets, capsules) and in biological materials (plasma, urine). The reported techniques for the determination of Losartan Potassium include: 1) high performance liquid chromatography (HPLC) with UV – detection (tablets, capsules, plasma, urine); 2) HPLC with fluorescence detection (plasma, urine); 3) HPLC with mas detection (tablets, plasma); 4) supercritical fluid chromatography (tablets); 5) high performance thin layer chromatography (HPTLC) (tablets); 6) capillary zone electrophoresis (tablets); 7) capillary electrochroma-tography; 8) micellar electrokinetic capillary chromatography (tablets); 9) UV – derivative spectrophotom-etry (tablets); 10) spectrofluorimetry (urine). From the results is obvious, that for determination of Losartan Potassium, HPLC is the most widely used method and spectrofluorimetry is specific for analysis in urine.

Key Words: Losartan Роtassium, analytical methods, analysis, HPLC, HPTLC, UV – spectropho-tometry.

Hypertension is one of the most important social disease, from which are suffering more than 1 billion people all over the world. In the regulation of blood pressure the rennin – angiotensin system plays very important role. The cascade begins with the cleavage of angiotensinogen by renin to the decapeptide angio-tensin I. Angiotensin converting enzyme produces the powerful vasoconstrictor angiotensin II by clipping a dipeptide unit [1].

Sartans are angiotensin II receptor antagonists which reduce the pressor effects of vasoconstrictor angiotensin II. The therapy with sartans is with higher social importance because offers a good quality of life for hypertensive patients due to the absence of side ef-fects [2]. In comparison with monotherapy the combi-nation therapy is more effective in reducing the blood pressure due to synergistic effects [3]. Because of their variety of pharmacological activities sartans are widely applied in clinical practice alone or in combi-nations with other antihypertensive drugs such as beta blockers, calcium antagonists, diuretics, angiotensin – converting enzyme inhibitors for treatment of hy-pertension (mild, severe and associated with diabetes mellitus) [4, 5]. For therapy of hypertension sartans are very often applied in fixed combinations with thi-azide diuretic Hydrochlorothiazide (HCTZ): Cande-

sartan [6]; Eprosartan [7]; Irbesartan [8, 9]; Losartan Рotassium [6]; Olmesartan, Valsartan, Telmisartan [9]. Losartan is non – peptide antihypertensive agent with a gradual and long – lasting effect and exerts its effective blood pressure lowering action by specific blocking of angiotensin II receptors. Losartan is pre-scribed alone or combined with diuretic Hydrochlo-rothiazide (HCTZ) for therapy of essential hyperten-sion [10, 11], stroke prevention [10, 12, 13], moderate heart failure [11, 14], atrial fibrillation [12, 15, 16], hypertensive patients with type 2 diabetes mellitus [17]. Losartan improves endothelial function in non insulin dependent diabetes [18] and possesses antial-buminuric effect in hypertensive patients with type 2 diabetes mellitus [19]. Oxidative stress is involved in the initiating of pathogenesis of hypertension [20], stroke [21], vascular disease [22] and atherothrombo-sis [23]. Important antioxidants are alfa lipoic acid for diabetic neuropathy and flavonoids diosmin and hes-peridin for chronic venous insufficiency, coumarins [24], Galantamine [25], peptide esters of Galantamine [26]. In this connection it is reported one new role of Losartan as antioxidant in renal transplant recipients with renin – angiotensin system polymorphisms [27].Losartan is applied in combination with inhibitors of angiotensin – converting enzyme (Enalapril, Lisino-

PHARMACIA, vol. 61, No. 1/2014 69 Social and health importance and analytical...

pril, Ramipril, Trandolapril) [28], beta – blockers (Atenolol), calcuim antagonist Amlodipine besilate, diuretic Hydrochlorothiazide [29, 30] in dosage phar-maceutical preparations – tablets and capsules and in biological materials – plasma and urine.

Several methods have been reported for analysis of Losartan Potassium (Los) and its degradation prod-ucts in drugs and biological materials [31]: 1) spectro-photometry (SFM); 2) spectrofluorimetry; 3) high performance thin layer chromatography (HPTLC); 4) capillary zone electrophoresis (CZE); 5) multisyringe chromatography (MSC); 6) supercritical fluid chro-matography (SFC); 7) HPLC [32]. Literature review reveals that UV spectrophotometry [33, 34, 35, 36, 37, 38, 39], HPTLC [40] and HPLC [41, 42] methods are very often applied for the estimation of Losartan in pharmaceutical dosage forms. Different spectro-photometric methods for the determination of Losar-tan Роtassium in tablets or in capsules are developed. UV method at λ = 208 nm in tablets is one of the most simple [43]. A first – derivative UV quantitative ana-lytical method is used for the determination of Losar-tan [44] by measurement of the absorption at λ = 232.5 nm in Cozaar tablets 25 mg [45] or at λ = 234 nm in tablets [46] and in capsules [47]. Second – de-rivative UV spectrophotometry is described for analy-sis of Losartan in tablets [48, 49], by measuring the distance between two extremum values at λ = 219.6 nm and λ = 228.8 nm (in Cozaar tablets 50 mg) [48]. Two – wavelength ratio UV spectrophotometry (206.6 nm/270.6 nm and 236 nm/270 nm) [50] and dual – wavelength UV spectrophotometry: (A261.4 nm – A206.6 nm) are also applied [50]. Spectrophotometry in visible area for analysis of Losartan in bulk and in synthetic mixture for solid dosage forms is proposed. Method is based on measurement of the absorbtion of derivative an orange – red colour complexes of Losar-tan in acidic medium (pH = 1.2) after reaction with Calmagite at λ = 491 nm or Orange – II at λ = 486 nm [51]. In other similar methods are used derivative re-action between Losartan and 2.3 – dichloro – 5.6 – dicyano – 1.4 – benzoquinone and measuring the complex at λ = 460 nm [52] or with bromothymol blue and phosphate buffer [34]. For Losartan Potassi-um and Atenolol is described extractive spectrophoto-metric method based on the formation of coloured complexes in the presence of buffer of pH = 7 with different reagents: the purple complex bethween Lo-sartan Potassium and ferroin solution and the yellow complex from Atenolol with methyl orange solution. After quantitatively extraction with chloroform the absorbances of complexes in the organic layers are

measured at λ = 571 nm and λ = 426.0 nm for Losar-tan Potassium and λ = 426 nm for Atenolol [53]. For the simultaneously determination of Losartan in com-bination with HCTZ or Amlodipine (Aml) or Enal-april are described different spectrophotometric meth-ods. First derivative UV spectrophotometry is reported for analysis of Losartan and HCTZ in tab-lets [54] by measurement of the absorbtion at λmaxLos = 271.6 nm (zero crossing wavelength for HCTZ) and λmaxHCTZ = 335.0 nm (zero crossing point for Losartan [55] or at λmaxLos = 222 nm and λmaxHCTZ = 332 nm [56]. First derivative UV meth-od is olso used for Losartan and Amlodipine in tab-lets [57, 58] and for Amlodipine 5 mg/Losartan 12.5 mg/HCTZ 50 mg in Trilopace® tablets by measure-ment of the absorbtion at λmaxAml = 236.5 nm, λmaxLos = 254 nm and λmaxHCTZ = 271 nm [59]. First derivative spectrophotometry of the ratio spec-trum is described for quantification of components in Hyzaar® filmtablets: Losartan 50.0 mg/HCTZ 12.5 mg at λ = 238.36 nm [60] and for combination from Losartan Potassium and Atorvastatin in tablets [61]. Compensation technique by calculation the ra-tios of the maxima and minimum of the first deriva-tive spectra is applied for Losartan 50.0 mg and HCTZ 12.5 mg in Hyzaar® filmtablets: 218 nm/236 nm (Los), 230 nm/261nm (HCTZ) [60]. Absorbance ratio (Q – analysis method) for analysis of Losartan and Amlodipine in tablets is based on measurement of absorbtion at an iso – absorptive point λ = 242.5 nm and λmaxAml = 237.5 nm [58]. The same method is appropriate fot Losartan and HCTZ in tablets by measurement of absorbances at isosbestic wave-length (λ = 266.5 nm) and wavelength of maximum absorption of HCTZ (λ = 272 nm) [56]. Simultane-ous UV equation method in tablets employs forma-tion and solving of simultaneous equation by meas-urement the absorbance at: 1) λ = 208 nm (Amlodipine and Losartan) and at absorbance maxi-mum of Amlodipine λ = 237.5 nm [58]. 2) λLosartan = 250 nm and λEnalapril = 222 and [62] in tablets Envas (25 mg Losartan/5 mg Enalapril); 3) λLos = 251.6 nm, λAtenolol = 224.2 nm, λHCTZ = 271.6 nm [63]; 4) λLosartan = 218 nm, λHCTZ = 272 nm and λ(isosbestic point) = 266.5 nm [56]; 3) λLos = 236 nm and λHCTZ = 270 nm [64]; 4) λLos = 206.6 nm and λHCTZ = 270.6 nm [50]. Dual wavelength method uses the difference of absorbance values at λ = 206.6 nm and λ = 261.4 nm for the estimation of Losartan and absorbance val-ues at 270.6 nm for the estimation of HCTZ [50]. AUC method for Losartan, Amlodipine and HCTZ is based of measurement of absorbtions at: 266 nm –


276 nm (Los); 231.5 nm – 241.5 nm (Aml); 249 nm – 259 nm (HCTZ) for Aml 5 mg, Los 12.5 mg and HCTZ 50 mg in Trilopace ® tablets [59]. Chemomet-ric algorithm methods are: 1) UV – PLS (partial least squares) algorith with variable selection of the UV zero order spectra between λ = 220 nm and λ = 300 nm: for 12.5 mg HCTZ/50 mg Losartan in tablets [65]; 2) classical least squares (CLS), 3) multiple lin-ear regression (MLR), 4) principal component regres-sion (PCR), PLS is applied by measurement of ab-sorption data in the range of 230.5 nm – 350.4 nm of zero order spectra of Losartan, HCTZ and Amlodi-pine in combination in tablets [66]. Spectrofluorime-try is used for the simultaneous analysis of Losartan and Valsartan for 30 min. in urine, λexcitation = 247 nm, λ emission = 259 nm [67]. In literature are included the thin layer chromatography densitometric methods by using Silicagel G60F254 at λ = 254 nm for Losartan on mobile phase: acetonitrile : methanol : 0.1 % acetic acid = 3.5 : 2.6 : 3.9 v/v at λ = 235 nm [68], for Losar-tan and its degradation products in Cozaar® tablets [69] and for Losartan and HCTZ in combined dosage forms [55] and at λ = 226 nm for the simultaneous estimation of Losartan Potassium and Atenolol in tab-lets using mobile phase: methanol : ethyl acetate : toluene : triethylamine = 4 : 3.9 : 2 : 0.1 v/v [70]. Lo-sartan HCTZ and Atenolol in tablets are separated and analysed on stationary phase aluminium HPTLC plates, coated with of Silicagel G60F254 at λ = 274 nm and mobile phases: acetonitrile : methanol : 0.1 % acetic acid = 35 : 25 : 40 v/v; chloroform : methanol : acetone : formic acid = 7.5 : 1.5 : 0.5 : 0.03 v/v; tolu-ene : methanol : triethylamine = 6.5 : 4 : 0.5 v/v [71]. HPTLC method is described for simultaneously de-termination of Losartan, HCTZ and Amlodipine in pharmaceutical formulations on Silicagel G60F254 plates with mobile phase chloroform : methanol : ace-tone : formic acid 7.5 : 1.3 : 0.5 : 0.03 v/v, detection at λ = 254 nm [72]. For simultaneous quality and quan-tity analysis of Losartan, Telmisartan and Valsartan is developed TLC method on plates precoated with Sili-cagel G60F254, mobile phase: 7.5 : 1.5 : 5 : 5 : 0.01 : 0.03 = CHCl3 : CH3OH : acetone : toluene : CH3COOH, detection at λ = 254 nm [73, 74, 75]. Capillary zone electrophoresis is applied for: 1) Losartan in Cozaar® tablets [76]; 2) Losartan/HCTZ in tablets [77] and in capsules [78]; 3) Losartan/Chlorthalidone in capsules [59]; 4) Losartan/Irbesartan/Telmisartan in urine with internal standard Candesartan [79]; 5) Losartan/Can-desartan/Eprosartan/Irbesartan/Telmisartan/Valsartan in tablets [80]; 6) Losartan/Candesartan/Eprosartan/Irbesartan/ Telmisartan/Valsartan /HCTZ in tablets

[81]; 7) Losartan/sartans [82]. Other reported methods are: I) capillary electrochromatography [76]; II) Mi-cellar Electrokinetic Capillary Chromatography (MEKC): 1) Losartan in Cozaar® tablets [76], by sys-tem equipped with a variable – wavelength UV detec-tor, fused silica capillaries Phoenix, coated with a UV – transparent polymer; 2) Losartan and several angio-tensin – II – receptor antagonists [83]; III) Multi – sy-ringe chromatography (MSC): for Losartan/HCTZ in superficial water, groundwater and wastewater outlet samples, flow rate = 0.8 ml/min., UV at λ = 226 nm [84]; IV) Supercritical fluid chromatography (SFC) for Losartan in Cozaar® tablets with UV detector [76]. HPLC is the most common analytical technique for the determination of different drugs: 1) vitamins (B6 and C): stationary phase: column RP – C18 ODS (4.6 mm x 250.0 mm, 5 μm), column temperature: 40 oC, mobile phase: acetonitrile : distilled water = 60 : 40 v/v; flow rate:1.0 ml/min., UV – detection at λ = 254 nm [85]; 2) aminoacids on stationaty phase: column Spherisorb ODS RP – C18, column temperature 25oC, mobile phase: methanol : distilled water = 1 : 1 v/v; flow rate: 1.0 ml/min., analytic alwavelength λ = 210 nm [86]; 3) Lisinopril on column Spherisorb RP C18 ODS – 2 (4.6 mm x 250.0 mm, 5 μm), column tem-perature 40oC, mobile phase: 0.125 % solution of so-dium 1 – hexanesulfonate in phosphate solution (pH = 2) : acetonitrile = 800 : 200, isocratic flow rate: 1.5 ml/min., detection: λ = 215 nm [87, 88]; 4) combina-tion of: Atenolol (1): retention time: tR=5.4 min., So-talol (2): tR=5.7 min., Metoprolol (3): tR=7.6 min., Bisoprolol (4): tR=8.3 min., Propranolol (5): tR=8.7 min., Diltizem (6): tR=9.2 min., Flecainide (7): tR=9.3 min., Amlodipine (8): tR=9.5 min., Carvedilol (9): tR=9.6 min., Verapamil (10): tR=9.9 min., Losartan (11): tR=10.2 min., Irbesartan (12): tR=10.7 min., Tel-misartan (13): tR=10.8 min., Valsartan (14): tR=11.1 min., internal standard Diazepam (15): tR=11.5 min: (Fig 1.) [89].

Losartan is analysed by HPLC: 1) in substance: 1 ml/min., t = 35◦C, methanol as diluent, λ = 254 nm

8

rate: 1.0 ml/min, analytic alwavelength λ = 210 nm [86]; 3) Lisinopril on column

Spherisorb RP C18 ODS – 2 (4.6 mm x 250.0 mm, 5 μm), column temperature 40 oC,

mobile phase: 0.125 % solution of sodium 1 – hexanesulfonate in phosphate solution

(pH = 2) : acetonitrile = 800 : 200, isocratic flow rate: 1.5 ml/min, detection: λ = 215

nm [87, 88]; 4) combination of: Atenolol (1): retention time: tR=5.4 min., Sotalol (2):

tR=5.7 min., Metoprolol (3): tR=7.6 min., Bisoprolol (4): tR=8.3 min., Propranolol (5):

tR=8.7 min., Diltizem (6): tR=9.2 min., Flecainide (7): tR=9.3 min., Amlodipine (8):

tR=9.5 min., Carvedilol (9): tR=9.6 min., Verapamil (10): tR=9.9 min., Losartan (11):

tR=10.2 min., Irbesartan (12): tR=10.7 min., Telmisartan (13): tR=10.8 min., Valsartan

(14): tR=11.1 min., internal standard Diazepam (15): tR=11.5 min: (Fig 1.) [89].

Fig 1. HPLc for determination of beta – blockers and sartans [70].

Losartan is analysed by HPLC: 1) in substance: 1 ml/min, t = 35◦C, methanol as

diluent, λ = 254 nm [90]; 2) in tablets [4, 28, 91, 92]; 3) in capsules [3]; 4) in biological

materials by isocratic RP HPLC with UV – detection at λ = 254 nm on Ultremex CN

(4.6 mm x 100 mm), 4.5 M sodium dihydrogen phosphate : acetonitrile :

tetrahydrofurane : methanol: 85 % phosphoric acid = 69.9 : 21 : 4 : 5 : 0.1 v/v; 7.5 mM

sodium dihydrogen phosphate buffer : acetonitrile : tetrahydrofurane : 85 % phosphoric

acid = 68.9 : 26 : 5 : 0.1 v/v, 0.6 ml/min [93]; with UV – detection in plasma on

Phenomenex C18 (2.0 mm x 250 mm), 0.01 M ammonium dihydrogen phosphate :

acetonitrile : methanol = 6 : 3 : 1, 0.3 ml/min [94] and mas detection in whole blood

Fig. 1. HPLC for determination of beta – blockers and sartans [70].


[90]; 2) in tablets [4, 28, 91, 92]; 3) in capsules [3]; 4) in biological materials by isocratic RP HPLC with UV – detection at λ = 254 nm on Ultremex CN (4.6 mm x 100 mm), 4.5 M sodium dihydrogen phosphate : acetonitrile : tetrahydrofurane : methanol: 85 % phosphoric acid = 69.9 : 21 : 4 : 5 : 0.1 v/v; 7.5 mM sodium dihydrogen phosphate buffer : acetonitrile : tetrahydrofurane : 85 % phosphoric acid = 68.9 : 26 : 5 : 0.1 v/v, 0.6 ml/min. [93]; with UV – detection in plasma on Phenomenex C18 (2.0 mm x 250 mm), 0.01 M ammonium dihydrogen phosphate : acetoni-trile : methanol = 6 : 3 : 1, 0.3 ml/min. [94] and mas detection in whole blood [89]. Recently, there have been reports in the literature of the employment of HPLC for bioequivalence studies of tablets contain-ing losartan potassium [3]. The HPLC methods de-scribed employ different conditions and columns. For the determination of Losartan in tablets are used the following HPLC methods: A) isocratic RP HPLC on: 1) Novapack ODS (4.0 mm x 150 mm, 5 μm), phosphate buffer (pH = 6.2) : acetonitrile = 60 : 40 v/v., 1 ml/min.:, λ = 254 nm [44]; 2) Shim-pack C18 (4.6 mm x, 250 mm, 10 μm), acetonitrile : phosphate buffer (pH = 3.8) = 40 : 60 v/v, 1.1 ml/min., λ = 235 nm [50]; 3) Shimadzu CLC – C8 (4.6 mm x, 150 mm, 5 μm) or LiChrospher 100 RP – C8 (4.6 mm x, 150 mm, 5 µm); triethylamine solution (0.5%) (pH = 2.4) : acetonitrile 60 : 40 v/v; 20 µl), 1.0 ml/min.; 20 °C; λ = 225 nm [92]; 4) Spherisorh C18 (4.6 mm x 250.0 mm, 5 μm),1.5 ml/min., λ = 254 nm [95]; c) 1.0 ml/min., t = 20oC, λ = 225 nm in Cozaar® tablets [96]; 2) gradient RP HPLC [28], t = 40°C, 1.5 ml/min. in Co-zaar® tablets [97]. For analysis of Losartan in cap-sules are described isocratic RP HPLC methods [92] with column temperature: t = 35o C; 1.0 ml/min. at l = 254 nm [3] on LiChroSpher RP-C18 (4.6 mm x 100 mm, 5 μm), 58 mM potassium dihydrogen phospate (pH = 6.2) : acetonitrile = 65 : 35 v/v [31]. For Losartan and its degradation products is used HPLC with mas detection [97]. In tablets Losartan and HCTZ are analysed by gradient RP HPLC on Mi-crobondapak C18 (3.9 mm x 300 mm, 10 μm), 25°C, methanol : 0.05 potassium hydrogen phosphate, 1.0 ml/min.; at λ = 270 nm [98] or isocratic RP HPLC, 1.0 ml/min. and UV – detection [99, 100, 101] at λ = 215 nm [102]: 1) on Erbasil C18 column (4.6 mm x 250 mm, 5 μm) [65] or Erbasil C18 column (4.0 mm x 125 mm, 5 μm, 0.1 M phosphate buffer (pH = 4.0) : acetonitrile = 65: 35 v/v, λ = 230 nm [103]; 2) on RP-YMC ODS A-132 C18 (6.0 mm x 150 mm, 5 μm), column temperature 35°C, 0.01 M sodium dihy-drogen phosphate : methanol : acetonitrile = 8 : 2 : 1

v/v, λ = 265 nm [60]; 3) on C18 column; (4.6 mm x 150 mm, 5 μm), 0.01 M potassium dihydrogen phos-phate : acetonitrile = 65 : 35 v/v, 1.0 ml/min. at λ = 265 nm [104]; 4) Bondapak C18 (3.9 mm.x 300 mm, 10 µm), 10 mmol/l potassium dihydrogen phosphate (pH = 3.1) : acetonitrile = 65 : 35 v/v, 1.5 ml/min., λ = 226 nm [105]. Losartan HCTZ and their degradation products are analysed by: HPLC with UV – detection [106]. For Losartan, HCTZ and Ramipril is applied isocratic HPLC methods: with UV – detection at λ = 215 nm, internal standard Аmlodipine besylate [107] and with UV – detection at λ = 210 nm on RP C18 (4.6 mm x 250 mm, 5 μm), buffer: acetonitrile = 60 : 40 v/v, 1.0 ml/min. [108]. Losartan Potassium, Hy-drochlorothiazide and Amlodipine Besilate in tablets are determined by isocratic HPLC on Kromasil C18 column (4.6 mm x 250 mm, 5 μm), 0.02 M phosphate buffer (pH = 3.7) : acetonitrile = 57 : 43 v/v, λ = 232 nm [59]. For analysis of Losartan and Enalapril in tablets is reported gradient RP HPLC: on Kromasil C18 (4.6 mm x 250 mm, 5 μm),: methanol : water : acetonitrile = 45 : 35 : 20 v/v, 1 ml/min. detection at λ = 224 nm [62]. For quantification of Losartan and Ramipril in tablets is developed isocratic HPLC method on Hypersil ODS C18 (4.6 mm x 250 mm, 5 μm), acetonitrile : methanol : tetrabutyl ammonium hydrogen sulfate = 10 mM) = 30 : 30 : 40 v/v, 1.0 ml/min., UV – detection at λ = 210 nm [109]. For Losar-tan and Atenolol in tablets is described isocratic RP – HPLC method: 1) 1.2 ml/min., UV – detection at λ = 235 nm, tRLos = 3.767 [110=88]; 2) in Losar beta® tablets: Supelcosil ODS (4.6 mm.x 250 mm, 5 µm), acetonitrile and 25 mM potassium dihydrogen phos-phate = 45 : 55 v/v, pH = 3.0, 1.2 ml/min., UV – de-tection at λ = 227 nm, internal standard: Chlorzoxa-zone [111]. Losartan and Metolazone are determined on Thermo Hypersil BDS C18 (250 mm × 4.6 mm, 5.0 μm) with isocratic conditions, acetonitrile:water (60:40), 0.8 ml/min., UV – detection at 237 nm [112]. Losartan and Аmlodipine in tablets are analysed by HPLC with UV – detection [113].

For simultaneous analysis of Losartan in combi-nations with other sartans are reported gradient [114] and isocratic HPLС with UV – detection: tRLosartan = 9.0 min., tRCandesartan = 10.3 min., tRIrbesartan = 11.5 min.; tROlmesartan = 12.0 min.; tRTelmisartan = 13.4 min. Valsartan = 19.9 min. [49]. For mono – and multicomponent analysis for isolation from plasma and urine is used solid phase microextraction, com-bined with HPLC. For the quantification in plasma is used RP HPLC [52, 115] with UV – detection at λ = 225 nm, 0.9 ml/min., internal standard Diclofenac


sodium [115] and λ = 254 nm, column Chromolith RP-18 (4.6 mm x 100 mm), mobile phase: 0.01 mol/l disodium hydrogenphosphate buffer : Acetonitrile = 60 : 40 v/v [116].

The following methods have been reported for analysis in plasma of Losartan and EXP 3174 [51]: isocratic HPLC [117, 118, 119] on ULTREMEX CN (5 μm), UV – detection at λ = 245 nm, LОQ = 5 ng/ml [120]; Intersil ODS-2 (4.6 mm x 250 mm, 5 λm), 50 mM ammonium acetate : acetonitrile = 74 : 26 v/v, 1.0 ml/min. at λ = 245 nm [121]; RP C18 column and mas detection [117]; or on column Capcellpak C18 (1.5 mm x 150 mm, 5 μm), 40°C; mobile phase: 0.1 % trietanolylamine : acetonitrile = 650 : 350 v/v; flow rate = 1.5 ml/min., mas detection; 117. In plas-ma Losartan and EXP 3174 are determined olso by gradient HPLC: 1) with fluorescent detector: a) on Hypersil Phenyl column (3.2 mm x 150 mm, 3 μm); mobile phase: 25 mM potassium phosphate: acetoni-trile = 60 : 40 v/v, λexcitation = 250 nm, λemission = 375 nm, linearity for Losartan: 10¸ 1000 ng/ml; LOD = 1 ng/ml [122]; b) λexcitation = 250 nm, λemmision = 370 nm, flow rate: = 0.5 ml/min., linearity: 10 – 1000 ng/ml, LOQ = 10 ng/ml [123]; c) HPLC – Fl: column Ul-tremex CN (4.6 mm x 250 mm, 5 μm), mobile phase: 0.015 M phosphoric acid : acetonitrile = 75 : 25 v/v, 35°C, flow rate: 1.25 ml/min., t = 35°C, λexcitation = 250 nm, λemission = 370 nm [124]; 2) with mas detection on column Keystone Valuepak Cyano column 3 :m (2.1 mm x 50 mm, 3 μm), column temperature 30°C, mobile phase: acetonitrile: 0.2 % formic acid = 55 : 45 v/v; flow rate: 0.2 ml/min. [118]. For simultane-ous determination of Losartan, EXP-3174 and Hy-drochlorothiazide is developed solid-phase extrac-tion liquid chromatography method with negative electrospray tandem mass spectrometry on column Nucleosil RP C18 (4.6 mm x 150 mm, 3 μm); mo-bile phase: 5 mM ammonium acetate : acetonitrile = 22 : 78 v/v, flow rate = 0.5 ml/min. [125]. Losartan and Losartanic acid are analysed by gradient HPLC with mas detection with internal standard Irbesartan [117=95] or on column Zorbax SB C18 column, (3.5 mmx 75 mm), 45°C, water : acetonitrile = 20 : 80 v/v, flow rate: 0.3 ml/min. [126]. Losartan and HCTZ are determined by HPLC with mas detection, internal standard: Valsartan [127].

Quantification of Losartan, Candesartan, Irbesar-tan and Telmisartan is by HPLC with 1.0 ml/min. at λ = 250 nm [123], or fluorescent detection: λexcitation = 250 nm, λemission = 380 nm [128]. Determination of Losartan, Candesartan, Eprosartan, Irbesartan, Tel-

misartan, Valsartan, metabolite EXP 3174 is based on HPLC with mas detection [129]. For analysis of Losartan, Candesartan, Irbesatan and Valsartan [130] is reported RP HPLC on Betasil C18 (4.6 mm x 250 mm, 5 μm), acetonitrile : 5 mM sodium acetate = 40 : 60 v/v, 1.0 ml/min., UV – detection at λ = 250 nm, and fluorescent detection at λexitation = 250 nm, λemision = 380 nm [128]. Atenolol, Bisoprolol, Carvedilol, Pro-pranolol, Metoprolol, Sotalol, Diltizem, Flecainide, Amlodipine, Verapamil, Losartan, Irbesartan, Tel-misartan, Valsartan: internal standard Diazepam are determined by gradient RP HPLC: Atlantis C18 (2.1 mm x 150 mm, 5 μm), column temperature 25°C, 10 mM ammonium acetate (pH = 3.1 with formic acid) : acetonitrile, 0.3 ml/min., mas detection [89].

Losartan in combination with sartans is deter-mined by gradient HPLC fluorescent method [88, 131] on Bondapak C18 column, (3.9 mm x 300 mm, 10 μm) 5 mM acetate buffer : acetonitrile, λexcitation = 250 nm, λemission = 375 nm [131] or with HPLC with mas detection [132, 133]. In urine Losartan and EXP 3174 are quantified by gradient HPLC with UV – de-tection [98], with fluorescent detection: λexcitation = 250 nm, λemission = 370 nm on Ultremex CN (4.6 mm x 250 mm, 5 μm), 0.015 M phosphoric acid: acetonitrile = 79 : 21 v/v, 35°C, 1.25 ml/min. [124] or with mas de-tection MS [118]. For analysis in urine of Losartan in combinations with sartans are reported the following methods: I) Losartan, Тelmisartan, Valsartan: Chro-molith (4.6 mm x 250 mm), 5 mM phosphate buffer (pH = 3.8): acetonitrile : methanol = 65 : 20 : 15 v/v, 3 ml/min., λexitation = 259 nm, λemission = 399 nm [134]; II) Losartan, Irbesartan, Valsartan, Candesar-tan cilexetil and its metabolite Candesartan M1: gra-dient HPLC with UV – detection on Bondapak C18 (3.9 mm x 300 mm, 10 μm), 5 mM acetate buffer : acetonirile [135]; ІII) Losartan, Candesartan, Irbesar-tan, Telmisartan [134]; HPLC with UV – detection at λ = 250 nm, 1.0 ml/min., and HPLC with λexcitation = 250 nm, λemission = 380 nm [123]; IV) HPLC with UV – detection at λ = 232 nm, 1 ml/min., t = 35oC [136]. For Losartan and HCTZ in superficial water, ground-water and wastewater is described HPLC at λ = 226 nm, 0.8 ml/min. [47]. For simultaneous Losartan and its metabolite in influent wastewater is reported RP HPLC/MS [137].

conclusionFirst-derivative spectrophotometric method is

found to be an excellent alternative to an HPLC method for the determination of Losartan in tablets. For analysis of Losartan and HCTZ in binary mix-


tures methods based on HPLC with UV – detection, fluorimetric or mas detection and first-derivative spectrophotometry are most applied [32].

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corresponding author:Stefka PankovaFaculty of Pharmacy, Medical University2 Dunav Str. 1000 SofiaFax: +359 2 987 98 74 e-mail: [email protected]

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