Determination of Losartan Potassium, Quinapril

Home , Quinapril

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 70 No. 6 pp. 967ñ976, 2013 ISSN 0001-6837 Polish Pharmaceutical Society

DETERMINATION OF LOSARTAN POTASSIUM, QUINAPRIL HYDROCHLORIDE AND HYDROCHLOROTHIAZIDE IN PHARMACEUTICAL PREPARATIONS USING DERIVATIVE SPECTROPHOTOMETRY AND CHROMATOGRAPHIC-DENSITOMETRIC METHOD

MARIUSZ STOLARCZYK, ANNA MAåLANKA, ANNA APOLA and JAN KRZEK

Department of Inorganic and Analytical Chemistry, Collegium Medium, Jagiellonian University, 9 Medyczna St., 30-688 KrakÛw, Poland

Abstract: Two methods, spectrophotometric and chromatographic-densitometric ones, were developed for determination of losartan potassium, quinapril hydrochloride and hydrochlorothiazide in pharmaceutical preparations. Spectrophotometric method involved derivative spectrophotometry and zero order spectrophotometry. The measurements were carried out at λ = 224.0 nm for quinapril, λ = 261.0 nm for hydrochlorothiazide and λ = 270.0 nm for losartan when the derivative spectrophotometry was applied and λ = 317.0 nm when zero order spectrophotometry was applied for the determination of hydrochlorothiazide. In chromatographic-densitometric studies high performance thin layer chromatography (HPTLC) plates were used as stationary phase and a mixture of solvents n-butanol : acetic acid : water (15 : 5 : 1, v/v/v) as mobile phase. Under the established conditions good resolution of examined constituents was obtained. Retardation factor for quinapril hydrochloride

was Rf ~ 0.70, for losartan potassium Rf ~ 0.85 and for hydrochlorothiazide Rf ~ 0.78. The developed methods are characterized by high sensitivity and accuracy. For quantitative analysis, densitometric measurements were carried out at λ = 218.0 nm for quinapril, λ = 275.0 nm for hydrochlorothiazide and λ = 232.0 nm for losartan.

Keywords: quinapril, losartan, hydrochlorothiazide, derivative spectrophotometry, HPTLC

Losartan (Losartanum) belongs to sartans, i.e., consequence to increase their antihypertensive angiotensin receptor blockers recommended in activity. hypertension therapy. Numerous analytical methods were used in the Quinapril belongs to the group of angiotensin- studies on losartan, quinapril and hydrochlorothi- converting enzyme inhibitors. It is used in a form of azide analysis in pharmaceutical preparations and prodrug. Its active form is quinaprilat which is body fluids (1). formed as a result of biotransformation in the liver. Hydrochlorothiazide and photodegradation It is recommended in therapy of hypertension and products were determined spectrophotometrically heart failure. In hypertension treatment it is used using first derivative, and high performance liquid both in monotherapy and in combined therapy. chromatography (HPLC) method often combined Hydrochlorothiazide is qualified to the group with mass spectrometry (2-5). Thin layer chro- of saluretics, i.e., thiazide diuretics. In terms of matography (TLC) method was used in an analysis chemical structure it is sulfonamide derivative. This of hydrochlorothiazide and other substances from drug exhibits diuretic and blood pressure lowering diuretics group (6). Good results in hydrochlorothi- activity. azide determinations in pharmaceutical preparations Main indication for this drug administration is were obtained using voltammetry method (7), hypertension (primary and renovascular). In hyper- HPTLC (8), reversed phase high performance liquid tension therapy, this drug is used both in monother- chromatography (RP-HPLC) (9) or capillary elec- apy and in therapy combined with other antihyper- trophoresis (10) methods tensive drugs of various targets. An application of Liquid chromatography method with fluorimet- antihypertensive drugs of separate activity mecha- ric (11) or mass detection (12, 13) were used in losar- nisms allows to obtain synergistic effect and in a tan potassium determination in biological material.

* Corresponding author: e-mail: [email protected]

967 968 MARIUSZ STOLARCZYK et al.

Quinapril, and other angiotensin convertase ume of 10.0 mL). Quinapril hydrochloride (19.6 mg inhibitors, was determined using voltammetry of quinapril hydrochloride was weighed and com- method (14). Spectrophotometric (15, 16) and pleted with methanol up to the volume of 10 mL). HPTLC methods (17) were used for studies in com- Hydrochlorothiazide (12.4 mg of hydrochlorothi- plex drugs containing hydrochlorothiazide. azide was weighed and completed with methanol up Pharmacologically active quinapril metabolite in to the volume of 10 mL). blood serum was determined using ultra high per- For chromatographic-densitometric analysis: formance liquid chromatography-electrospray ion- Methanol standard solutions for chromatographic- ization mass spectrometry (UHPLC-MS) (18). densitometric analysis had the following concentra- This research aimed at a development of the tions: losartan 0.44 mg/mL, quinapril 0.38 mg/mL method of hydrochlorothiazide, as well as quinapril and hydrochlorothiazide 0.52 mg/mL. and losartan co-present in complex drugs, determination with an application of derivative spectropho- Sample solutions tometry and high performance thin layer chromatog- Ten tablets of the studied preparations were raphy. powdered in a porcelain mortar. Weighed amounts The study included pharmaceutical prepara- of samples in a range of 129.8 to 240.2 mg tions in which one of the components is (Accuzide 20), 99.5 to 271.3 mg (Lorista® H) were hydrochlorothiazide and the second is one of the extracted using 10 mL of methanol, shaking for 15 substances of antihypertensive activity, i.e., losartan min and centrifuged (1500 rpm). Solutions obtained potassium or quinapril hydrochloride. Accuzide 20 directly after extraction and centrifugation were containing 20.0 mg of quinapril and 12.50 mg of used in chromatographic-densitometric analysis, hydrochlorothiazide, as well as Lorista® H prepara- while in order to perform spectrophotometric analy- tion containing 50 mg of potassium losartan and sis, the solutions obtained were diluted in a ratio of 12.50 mg of hydrochlorothiazide were used in the 1 : 100. studies. Spectrophotometric analysis EXPERIMENTAL The absorption spectra in the range from 200 to 400 nm were registered for standard solutions and Apparatus studied samples solutions in the presence of Spectrophotometer UV-VIS Cary 100 Varian methanol. Spectra of zero order were transformed in (Australia), quartz cuvettes of a layer thickness 1 the first order derivative (D1). The concentration of cm. Computer ñ Dell Optiplex 755; Intel(R) studied substances in pharmaceutical preparations Core(TM)2 Duo CPU; E4500 @ 2.20 GHz; 1.18 was calculated using straight line equation based on GHz, 1.95 GB Ram (Microsoft Office 2006, the value of derivative and an absorbance read out at Statistica 7.1 edition 2007). Applicator for samples suitable wavelengths at λ = 224.0 nm and λ = 261.0 application LINOMAT 4; Camag (Muttenz, nm for quinapril and hydrochlorothiazide (in the Switzerland). Densitometer TLC- Scanner 3 with case of active substances content determination in WinCats software version 1.3.4; Camag (Muttenz, Accuzide 20 preparation), respectively, and λ = Switzerland). Microsyringe of a volume of 100 µL 270.0 nm for losartan potassium and λ = 317.0 nm (Hamilton Comp. Reno, USA). Chromatographic for hydrochlorothiazide (determined in Lorista ®H chamber 23 ◊ 9 ◊ 11 cm Nano-Desaga (Germany). preparation).

Materials Chromatographic-densitometric analysis Reference standards: losartan potassium, Suitable standard solutions and studied sam- quinaprilum, hydrochlorothiazide. Tablets: Lorista® ples were spread in amount of 3 mL in a form of 10 H (KRKA), Accuzide® 20 (Pfizer). Reagents of ana- mm band using an applicator on HPTLC chromato- lytical grade quality: methanol, n-butanol, acetic graphic plates (10 ◊ 10 cm). Chromatograms were acid. HPTLC plates 60F254 No 1.05548.0001 developed in a chromatographic chamber saturated (Merck-Darmstadt, Germany). with mobile phase composed of n-butanol : acetic acid : water ñ 15 : 5 : 1 (v/v/v), they were developed Standard solutions to a height of 9.5 cm and dried at room temperature. For spectrophotometric method: Losartan Densitometric registration was performed after potassium (19.3 mg of losartan potassium was plates drying, with the following wavelengths: λ = weighed and completed with methanol up to the vol- 218 nm for quinapril, λ = 275 nm for hydrochlorothi- Determination of losartan potassium, quinapril hydrochloride and hydrochlorothiazide in... 969

Figure 1. The zero order absorption spectra for quinapril (A), hydrochlorothiazide (B), mixture (C)

Figure 2. The zero order absorption spectra for losartan (A), hydrochlorothiazide (B), mixture (C)

Figure 3. The first (D1) derivative spectra for quinapril (A), hydrochlorothiazide (B), mixture (C) 970 MARIUSZ STOLARCZYK et al.

Figure 4. The first (D1) derivative spectra for losartan (A), hydrochlorothiazide (B), mixture (C)

Figure 5. Absorption spectra of hydrochlorothiazide, quinapril and losartan registered directly from the chromatogram

azide and λ = 232 nm for losartan. The wavelength Validation study at which densitometric detection was performed, Validation of the methods was performed was selected based on spectra registered directly according to ICH requirements (19) from chromatogram. Specificity: Due to lack of data concerning Content of active compounds in the prepara- placebo composition, the comparative spectrophoto- tions was calculated comparing the areas of ana- metric studies were performed for model solutions lyzed sample peaks with an area of peaks of stan- (Rm) and pharmaceutical forms being a subject of dard substances of known concentration. the study (Pf). Solutions of a comparable composi- Determination of losartan potassium, quinapril hydrochloride and hydrochlorothiazide in... 971

Figure 6. Densitogram of examined preparations solutions: quinapril (1) Rf ~ 0.70, hydrochlorothiazide (2) Rf ~ 0.78, losartan (3) Rf ~ 0.85

tion containing 75, 100 and 125% of particular com- where Sy ñ standard estimation error, a ñ gradient of pound were prepared. Measurements of derivatives a straight line. values were performed at selected wavelengths. Recovery: Determination of recovery percent- Specificity of the method for an analyte was deter- age aims to determine an accuracy of the method, mined comparing the values of absorbance and i.e., accordance between the real value and a value derivatives for standard solutions and pharmaceuti- obtained as a result of analysis. It should be in the cal preparations. range of 95-105%. Recovery for particular compo- Specificity of chromatographic-densitometric nents was provided as percents based on determined method was verified by a comparison of retardation concentration of analyte which was added to the factors (Rf) for studied substances and calculation of samples in amount from 80% to 120% with respect resolution of registered peaks (Rs ñ resolution fac- to the declared amounts. tor). Precision: Five assays were performed in order Linearity: For spectrophotometric method, lin- to determine the precision of the method. Amount of earity is maintained within the studied ranges of 3 µL of analyzed component solution was spread on concentrations. The equations of linear regression chromatographic plate. In the next stage, the plates characterizing the points of straight line cut, correla- were developed in the developing mobile phase, and tion coefficient and statistical tests were used for the then densitometric analysis was performed. The results assessment. In chromatographic-densitomet- areas of peaks were compared in order to determine ric method, linearity assessment was performed by the precision. measurements for standard solutions in concentration range of 0.0078 ñ 1.00 mg/mL for hydrochlorothiazide, 0.0313 ñ 2.00 mg/mL for quinaprilum and RESULTS AND DISCUSSION 0.0306 ñ 0.49 mg/mL for losartan. Limit of detection (LOD) and limit of quantifi- Combination of pharmacologically active com- cation (LOQ): For both methods, limit of detection pounds in complex drugs is justified in terms of clin- and limit of quantification were calculated using ical practice, since it often allows to obtain values of statistical parameters for suitable calibra- improved therapeutic effects with increased undesir- tion curves according to the following formula: able activity. It is understandable that pharmacolog-

LOD = 3.3Sy/a and LOQ = 10.0Sy/a ical properties and technological aspect with respect 972 MARIUSZ STOLARCZYK et al.

to components stability and pharmaceutical availability are considered at the stage of components selection. The components occurring in the studied drug have similar physicochemical properties, and their absorption spectra registered using Shapiro-Wilk test Shapiro-Wilk zero order spectrophotometric method inter- fere with each other, which prevents its application in direct analysis of quinapril, losartan s test í and hydrochlorothiazide. This problem is not observed in the case of an application of derivative spectrophotometry method, which was developed for the needs of direct analysis of components being a subject of the research. Equally good results ) Mandel 2 may be obtained using chromatographic-den- 0.677 0.677 0.9599 (0.819) 0.9599 0.677 0.855 0.855 0.8932 (0.335) (0.281) (0.371) 0.8932 0.855 0.8826 0.551 0.8994 0.780 0.807 0.288 0.808 0.288 0.9582 (0.803) 0.8434 (0.107) p(cx 0.0007(0.150)* 0.0007 0.8699 sitometric method in an analysis of selected

significance p preparations. The spectra of zero order in the range of 200 ñ 400 nm were registered in the first stage of the research for suitable standard 2 2 2

2 solutions containing active substances studied. 2 2 Spectrophotometric measurements were per- 2 formed in the presence of methanol as a reference substance, and their results are presented in Figures 1 and 2. Analyzing the course of absorption spectra of quinapril and hydrochlorothiazide mixture (Fig. 1) as well as losartan and hydrochlorothiazide (Fig. 2), a clear interference of = 0.99747 = 0.99449 = 0.99747 = 0.99439 = 0.99799 = 0.99724 = 0.99981

2 2 2 2 2 2 2 absorption maxima in the range of 200-300 nm was noted, which prevents direct analysis of the studied substances. Transformation of the obtained spectra into the first order derivative curves (Figs. 3, 4) and an application of zero- crossing technique allowed to determine the wavelengths at which interference phenomena were not observed. For quantitative analysis purposes, suit- = 0.99838 = 0.99581 = 0.99870 R = 0.99566 R = 0.99837 R = 0.99696 R = 0.99812 R R R - HPTLC

2 2 2 2 2 2 2 λ

B able wavelengths were selected: = 224 nm y = 0.0036x ñ 0.0005 y = 0.0038x - 0.0013 ñ 0.00001x R R R R R R R for quinapril and λ = 261 nm for hydrochlorothiazide in their mixture. The

s test, Shapiro-Wilk test of examined substances. analysis of losartan and hydrochlorothiazide í B A mixture was possible with an application of relationship D1 = f(c) at λ = 270 nm for determination of losartan, and relationship A = f(c) λ = 317 nm for hydrochlorothiazide determi-

ñ spectrophotometric method, nation. The results obtained allow to conclude A that the method of the first order derivative spectrophotometry (D1) or combination of derivative spectrophotometry method and zero Losartan y = 17805.8x + 177.7 y = 18141.5x + 158.9 ñ 694.1x Losartan y = 0.001x + 0.005 y = 0.001x + 0.002 ñ 0.000001x order spectrophotometry in case of concurrent Quinapril Quinapril y = 0.00097x ñ 0.00129 y = 0.001x - 0.002149 ñ 0.000001x y = 2388.8x + 123.4 y = 2637.7x + 66.6 ñ 126.1x Substances Methods Linearequation Quadratic equation Linearequation Quadratic Substances Methods determination of losartan potassium and

Hydrochlorothiazide hydrochlorothiazide, y = 14940.2x + 59.7 y = 17278.6x - 48.1 ñ 4851.4x allows to perform an Hydrochlorothiazide (A) y = 0.0102x ñ 0.0053 y = 0.0107x - 0.0086 ñ 0.000015x Hydrochlorothiazide (D1) analysis of active substances in the studied Table 1. Linear and quadratic equation, Mandel * - for quadratic equation; Determination of losartan potassium, quinapril hydrochloride and hydrochlorothiazide in... 973 ] 2 g/spot] g/spot] 0.85 µ µ ~ = 99.44 F = 1.6861 = 1.6876 = 0.9813 = ±2.0954 = ±2.0936 = ±1.2185 = 101.86 = 103.44 ñ x x x x ñ ñ x x 0.95 0.95 0.95 ] 5313.66 [mm 2 g/spot] 0.605 [ g/spot] 0.605 g/spot] 0.200 [ g/spot] 0.200 µ µ ~ 0.78 R = ±1.783 t F = 1.4360= 2.1478 S = 1.2207 S S = ±1.5156 t = ±2.6668 t = 101.38 = 102.74 = 102.50 x x x ñ ñ ñ x x x 0.95 0.95 0.95 ] 2874.48 [mm 2 g/spot] 0.893 [ g/spot] 0.893 g/spot] 0.295 [ g/spot] 0.295 µ µ ~ 0.70 R F = 1.2542= 0.8961 S = 1.4342 S S = 100.74 = ±1.5573 t = ±1.1127 t = ±1.7808 t = 101.04 = 100.68 x x x ñ ñ ñ x x x 0.95 0.95 0.95 g/mL] 1.912 [ g/mL] 1.912 g/mL] 0.631 [ µ µ = 2.622 S = 2.933 S = 2.177 S = 99.66 = ±2.9811 t = ±3.2120 t = ±2.4700 t = 100.48 = 100.26 x x x ñ x = 317.0nm R ñ ñ x x 95% 95% 95% λ g/mL] 6.08 [ g/mL] 6.08 g/mL] 2.01 [ g/mL] 2.01 µ µ = 2.576= 3.986 S = 1.683 S S = 99.06 x = 97.26 = ±2.9321 t = ±4.5300 t = ±1.9112 t = 100.90 x x x ñ ñ x = 270.0nm x ñ confidence interval for 95% probability. ñ x 95% 95% 95% λ 0.95 g/mL] 4.48 [ g/mL] 4.48 g/mL] 1.48 [ SPECTROPHOTOMETRY HPTLC µ µ = 0.5750 S = 0.4679 S = 2.0369 S = ±0.6538 t = ±0.5321 t = ±2.3164 t = 100.27= 104.93 x = 102.43 = 261.0nm x x x ñ ñ ñ x x x 95% 95% 95% λ g/mL] 2.06 [ g/mL] 2.06 g/mL] 0.68 [ g/mL] 0.68 µ µ = 98.28 = 97.18 = 1.2236= 0.7245 S = 2.1732 S S = ±1.3914 t = ±0.8238 t = ±2.4713 t 0.2873 0.3211 0.4223 0.4723 51.404 48.1515 21.8399 = 103.31 ±0.6011 ±0.6011 ±0.7422 ±0.8214 ±63.827 ±59.788 ±27.118 ñ ñ x x x x x = 224.0 nm Quinapril Hydrochlorothiazide Losartan Hydrochlorothiazide Quinapril Hydrochlorothiazide Losartan ñ x RSD=2.24 RSD = 1.99 RSD = 1.73 RSD = 2.17 RSD = 1.42% RSD = 1.19% RSD = 0.95% RSD = 0.74 RSD = 0.45% RSD = 3.95 RSD = 2.57 RSD = 0.89% RSD = 2.09% RSD = 1.66% 95% 95% 95% λ RSD = 1.18% RSD = 0.57 RSD = 2.60 RSD = 2.63 RSD = 1.24% RSD = 1.42% RSD = 1.70% 20.06 [mg/tab] 12.43 [mg/tab] 49.85 [mg/tab] 12.68 [mg/tab] 2609.82 [mm ñ standard deviation, RSD% - relative t x x ñ x 95% S t [%] t [%] t [%] t 80% S RSD 0.30% 0.56% 0.30% 1.44% 1.97% 1.68% 0.41% LOQ 5.91 [ LOD 1.95 [ 100% S 120% S Precision Recovery Recovery Recovery - mean value, S ñ Table 2.Validation of the developed methods with statistical evaluation. x 974 MARIUSZ STOLARCZYK et al. = 0.18 = 4.44 rel rel = 0.4931 = 1.3005 = ±0.7074 = ±1.6147 x x S S %E %E 0.95 0.95 t t RSD = 0.99% RSD = 2.72% óóóóó óóóóó- óóóóó = 0.90 = 0.30 rel rel = 0.3660 = 0.2613 = ±0.6751 = ±0.3244 x x %E %E 0.95 0.95 19.82 20.06 óóóóó 49.91 óóóóó 47.78 HPTLC SPECTROPHOTOMETRY = 1.12 = 0.08 = 0.40 = 1.52 rel rel rel rel = 0.2103= 0.2772 = 0.3192= 0.1704 S S = ±0.6751= ±0.7074 = ±0.3964= ±0.2116 t t x x x x S S S S %E %E %E %E 0.95 0.95 0.95 0.95 t t t t RSD = 2.22% RSD = 1.34% RSD = 1.07% RSD = 2.56% RSD = 1.85% RSD = 1.30% ñ confidence interval for 95% probability 0.95 12.69 12.36 12.45 12.49 [mg/ tabl.] [mg/ tabl.] [mg/ tabl.] Determined content Determined content Determined content LoristaH LogistaH preparation mean n = 10) assessment mean n = 10) assessment mean n = 10) assessment Accuzide 20 Accuzide 20 Pharmaceutical (hydrochlorothiazide, Statistical (quinapril, Statistical (losartan, Statistical ñ standard deviation, RSD% - relative t x Table 3. Determination results with statistical evaluation. S Determination of losartan potassium, quinapril hydrochloride and hydrochlorothiazide in... 975 forms of drugs. Due to characteristic course of Linear determination coefficient does not derivative curves and absence of zero sites, which determine the linearity of calibration method in an would have been used for determination of compo- unequivocal manner. Therefore, Mandelís test was nents in three-components mixture, especially with used for linearity assessment. The obtained results respect to losartan potassium, the method of deriva- of linear and square adjustment prove linear adjust- tive spectrophotometry is not recommended for such ment of calibration curve in all the cases except for mixtures analysis. This is not a factor determining determination of hydrochlorothiazide using chro- its usefulness in quantitative analysis of these active matographic-densitometric method. Normality of substances, since studied substances are not residues distribution was confirmed using Shapiro- observed in three component combination in a form Wilk test (20). Points of straight line crossing do not of a drug. The chromatographic-densitometric diverge considerably from zero (Tab. 1). method with an application of HPTLC plates and Sensitivity of developed method is high, for mobile phase composed of n-butanol, acetic acid spectrophotometric method LOD was in the range and distilled water in a ratio of 15 : 5 : 1 (v/v/v) may from 0.68 to 2.01 µg/mL and LOQ from 2.06 to 6.08 be successfully used for determination of substance µg/mL. For chromatographic-densitometric method in three-component mixture in developed conditions LOD was in the range from 0.20 to 0.63 µg/spot and of analysis. Well separated peaks were obtained in LOQ from 0.61 to 1.91 µg/spot. such conditions, which was confirmed by Rf values. Percentage of recovery of the studied compo- With wavelengths selected for measurements, nents presented as the mean values for three levels spectrophotometric and chromatographic methods are of concentrations is high, and in the range of 100 ± specific for analyte represented by the studied com- 5%, both in spectrophotometric and chromatograph- pounds. Specificity in spectrophotometric method was ic-densitometric method. The results of determina- determined comparing derivatives values according to tion for individual constituents are of similar preci- the relationship (Rm ñ first derivative value of model sion, RSD is within a narrow range from 0.30 to solution; Pf ñ first derivative value of pharmaceutical 1.97% (Tab. 2). formulation). It was found that they do not differ con- The results obtained in the study are of a prac- siderably from value of 1 and are within the range tical value and may be applied in drug quality con- from 0.96 to 1.05, which proves almost identical trol. course of derivative curves for standard solutions and The results of particular components determi- pharmaceutical preparation solutions. It may be con- nation do not diverge from declared values, and are cluded on this basis that placebo used for formation of thus characterized by good precision and accuracy, suitable drug form does not influence quantitative narrow confidence interval and beneficial values of determination of an active substance. mean standard deviation (Sx), relative error (%Erel) Well separated peaks of retardation factor val- and relative standard deviation (RSD) (Tab. 3). ues of Rf ~ 0.70, Rf ~ 0.78 and Rf ~ 0.85 for They may also be an indicator for wider application quinapril, hydrochlorothiazide and losartan, respec- of derivative spectrophotometry method and tively, are observed on registered densitograms in HPTLC for complex drugs analysis. chromatographic-densitometric method. Satisfacto- ry separation of the peaks is confirmed by resolution CONCLUSIONS factors Rs = 1.36 for quinapril and hydrochlorothiazide peaks, and Rs = 1.12 for hydrochlorothiazide Spectrophotometric and chromatographic-den- and losartan peaks (Figs. 5, 6) sitometric methods have been proposed for the Linearity is maintained in a wide range of con- determination of losartan potassium, quinapril centrations, i.e., from 4.90 to 29.40 µg/mL for hydrochloride and hydrochlorothiazide present in quinapril, and from 3.10 to 18.60 µg/mL for the complex pharmaceutical formulations. Spectro- hydrochlorothiazide, as well as from 4.83 to 28.95 photometric method utilizes a ìzero-crossingî tech- µg/mL for losartan potassium and from 1.24 to 7.44 nique and the corresponding experimentally deter- µg/mL for hydrochlorothiazide, for spectrophoto- mined wavelengths. This technique enables to elim- metric method. inate interferences between determined constituents Linearity range determined by the chromato- which are clearly visible in the zero order spectra. graphic-densitometric method was from 0.0313 to The method is characterized by a wide linearity 2.00 mg/mL for quinapril, from 0.0306 to 0.49 range confirmed by statistical tests, high precision mg/mL for losartan and from 0.0078 to 1.00 mg/mL (RSD in the range 0.30 - 1.44%), low values of LOD for hydrochlorothiazide. and LOQ and specificity in relation to the matrix 976 MARIUSZ STOLARCZYK et al. components. Separation conditions developed by a 8. Santhana-Lakshmi K., Lakshmi S.: J. Anal. chromatographic-densitometric method (stationary Methods Chem. 8, 1 (2012). phase and mobile phase) allow for a satisfactory 9. Hossen M.A., Haque M.A., Dewan I., Hamidul separation of components of the mixture (confirmed Kabir A.N.M., Hossain M.K., Ashraful Islam by Rf values). 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Received: 28. 02. 2013