Int J Pharm Pharm Sci, Vol 8, Issue 8, 243-250Original Article

ANALYSIS OF THREE CARDIOVASCULAR DRUGS IN THEIR TERNARY MIXTURE USING GREEN ANALYTICAL METHODOLOGY OF SMART SPECTROPHOTOMETRIC METHODS AND RP-HPLC METHOD

**MARWA K. EL JAMAL*, AZZA A. GAZY**

Department of Pharmaceutical Technology, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon
Email: marwa.jamal@bau.edu.lb

Received: 25 Apr 2016 Revised and Accepted: 20 June 2016

ABSTRACT

Objective: Development and validation of two new, simple and specific analytical techniques for the simultaneous determination of a ternary mixture of co-administered cardiovascular drugs ticagrelor (TICA), irbesartan (IRB) and hydrochlorothiazide (HCT).

Methods: Chemometric assisted UV spectrophotometric methods (considered as green analytical chemistry) and RP-HPLC method were developed and validated. The different applied chemometric methods are based on Fourier function convolution (FF) and Double divisor ratio spectra Fourier function (FFR). As for the HPLC method, it was developed on a C₁₈ Sunfire^© waters column with a mobile phase composed of Acetonitrile: sodium dihydrogen phosphate (adjusted to pH 3.6 using orthophosphoric acid) in the ratio of 60:40 v/v at a flow rate of 1 ml/min. Quantification was based on measuring peak areas at 230 nm.

Results: By applying the chemometric assisted methods, good percentage recoveries were obtained in quantifying each of the drugs in their triple mixture. The RP-HPLC method, peaks were well resolved with retention times 4.6, 8.0 and 10.0 min, for HCT, TICA and IRB, respectively. The established method was also applied in samples spiked with plasma; the peaks were well separated from each other and from plasma protein peaks, proving the ability of the HPLC method to be applied in biological samples. The methods (RP-HPLC and chemometric assisted methods) were validated in terms of linearity, LOD, LOQ, precision and accuracy. The results obtained from the analysis of the co-administered mixture by the proposed chemometric assisted methods were statistically compared to those obtained by the applied RP-HPLC method.

Conclusion: The applied RP-HPLC methods were proved to be more sensitive when compared to the applied chemometric methods, thus could be used for determination of drug concentration in human plasma. However, the applied chemometric methods, considered as green analytical chemistry, is a simple, time-saving method that requires minimal use of a hazardous solvent.

Keywords: Fourier Function, Double divisor Fourier function ratio spectra, Spectrophotometry, RP-HPLC, Spiked plasma

INTRODUCTION

Combination therapy (or polytherapy) is a broad term for the use of multiple medications, in order to fight a particular condition; It is a therapeutic intervention in which more than one pill (each containing separate drug) are administered to the patient.

Cardiovascular disease (CVD) is the leading cause of death and disability worldwide. A dose combination therapy for the treatment of blood pressure, diuretic and antiplatelet treatments has been proposed as one strategy to reduce the global burden of CVD by remarkable percentage. So, developing a method for the estimation of a mixture of cardiovascular co-administered drugs would be of great importance [1].

Green analytical chemistry is concerned with the development of analytical procedures that minimize consumption of hazardous reagents and solvents, and maximize safety for operators and the environment. Therefore, chemometrics addresses the constraints that happen due to reducing the solvents used and the duration of analysis by doing most of the works on the front desk using microcomputers with appropriate software on the primary data generated in the lab work. After some recent years, chemometrics has become one of the mathematical and statistical techniques for the resolution of overlapping spectra of multi-component mixtures, which is difficult to separate using the traditional spectroscopic method as well as it has a wide application in different disciplines [1].

Irbesartan (IRB), chemically described as 2-butyl-3-[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]-4-yl]-methyl1-3-diazaspiro-[4,4]-non-1-en-4-one, (fig. 1), is an angiotensin II blocker, which acts mainly by selective blockade of AT1 receptors and reduces the effects of angiotensin II. IRB may be used alone or in combination with other antihypertensive or diuretic agents [2].

Hydrochlorothiazide (HCT), chemically described as 6-chloro-3, 4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide, (fig. 2), is a thiazide diuretic. It increases sodium and chloride excretion in the distal convoluted tubule. Because of their synergistic antihypertensive action, irbesartan and hydro-chlorothiazide are available in the market as a combined dosage form [2].

Ticagrelor (TICA), chemically described as (1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino}-5-(propylthio)-3H-[1,2,3]-tria zolo [4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxycyclo pentane-1,2-diol2, (fig. 3), is an orally active antiplatelet agent, act as an inhibitor for platelet activation and aggregation mediated by the P2Y12 ADP-receptor1. Ticagrelor is indicated to reduce the rate of cardiovascular thrombotic events in patients with the acute coronary syndrome. Ticagrelor and its major metabolite reversibly interact with the platelet P2Y12 ADP-receptor to prevent signal transduction and platelet activation, which inhibits platelet aggregation and thrombus formation in atherosclerotic disease [2].

Several analytical methods have been reported for the determination of irbesartan in pure drug, pharmaceutical dosage forms using spectrophotometry [3], RP-HPLC [4]. Also, irbesartan has been determined in human plasma by LC [5]. In the presence of hydrochlorothiazide, irbesartan has been determined by UV-Spectroscopy [6], RP-HPLC [7, 8], HPLC by the aid of chemometry [9]. Hydrochlorothiazide and irbesartan are simultaneously determined by LC-MS in human plasma [10], or in spiked human plasma using microemulsion-LC [11]. Irbesartan, in combination with other drugs, has been determined by RP-HPLC [12], HPTLC [13], LC-MS [13].

Hydrochlorothiazide has been determined individually by spectrophotometry [14], HPLC [15]. In combination with many other drugs, hydrochlorothiazide has been determined by HPLC [8, 16, 13], spectrophotometric chemometric analysis [17, 18], HPTLC with UV-absorption densiometry [19].

Ticagrelor has been assayed by spectrophotometry [20], HPLC [21]. In plasma sample, ticagrelor has been determined by LC-MS [22], LC-MS-MS [23] and UPLC-MS [24].

To the best of our knowledge, neither of the already published papers has been related to any chemometric nor RP-HPLC method for simultaneous determination of HCT, TICA and IRB. So the present study emphasizes on the determination of HCT, TICA and IRB using different chemometric methods that will be compared to a RP-HPLC method. The developed methods will be validated according to ICH guidelines [25].

MATERIALS AND METHODS

Apparatus

The spectrophotometric measurements were carried out on a Jasco V-530 double beam UV-Vis Spectrophotometer connected to a computer loaded with Jasco UVPC software and HP Deskjet 5652 printer. The absorption spectra were measured using 1-cm quartz cells. For DDRD, FF and FFR, the absorption spectra were recorded using 1 cm quartz cells.

HPLC system (waters 1525) is composed of binary HPLC pump, connected to water 2487 dual wavelength absorbance detector. The liquid chromatographic system is equipped with waters 717 plus autosampler. Liquid separations are performed on RPC Sunfire © C18 analytical column (250 x 4.6 mm x 5μm) at ambient temperature. The mobile phase used was degassed and filtered by passing through 0.5 μm pore size membrane filter at a pumping speed of 30L/min (Glassco diaphragm Vacuuum pump). The samples were also filtered using PTFE 0.2 μm Minisart SRP 15 (Sartorius Stedim) disposable filters.

Chromatographic conditions

The mobile phase was prepared by mixing potassium dihydrogen phosphate buffer (adjusted to pH 3.6 with orthophosphoric acid) and acetonitrile in a ratio (40:60) by volume.

Materials and reagents

Ticagrelor (from supplied by Omnipharma, Astrazenica, Lebanon), irbesartan and hydrochlorothiazide (from Algorithm, Lebanon) were used as working standards. Acetonitrile (SIGMA-ALDRICH CHROMASOLV^®FOR HPLC>99.9%), KH₂PO₄ (Merck KGaA), Na₂HPO₄ (BDH), human plasma was supplied by Dar Al Ajaza, Lebanon.


Fig. 1: Irbesartan	Fig. 2: Hydrochlorothiazide	Fig. 3: Ticagrelor

Chemometric assisted methods

Standard stock solutions

Standard solutions of TICA (1000 μg/ml, IRB (100 μg/ml) and HCT (400 μg/ml), were prepared separately in methanol.

Working standard solutions

Portions of the above stock solutions were separately diluted with methanol to attain the concentration of 100 μg/ml TICA, 10 μg/ml IRB and 40 μg/ml HCT.

Calibration graphs

Accurate volumes of the working standard solutions of TICA, IRB or HCT into separate sets of 10-mL calibrated flasks were transferred and completed to volume with methanol to obtain the concentration ranges stated in table 2. Absorbance values were recorded at λ range of 200-300 nm for each drug, at 1 nm interval, against methanol as blank.

Synthetic mixtures

Six validation synthetic mixtures were prepared by mixing appropriate volumes of the working standard solutions of TICA, IRB and HCT and diluting to volume with methanol. The combination of TICA, IRB and HCT are illustrated in table 4. The absorbance values for each solution at 1-nm intervals in the wavelength range 200-300 nm, were recorded.

HPLC method

Standard stock solutions

Standard solutions of TICA (100 μg/ml), IRB (100 μg/ml) and HCT (100 μg/ml), were prepared in HPLC-grade methanol.

Working standard solutions

Portions of the above stock solutions were separately diluted with methanol to attain the concentration range of 10 μg/ml TICA, 1 μg/ml IRB and 10 μg/ml HCT.

Calibration graph

Pure drugs

Into series of 10-mL measured flasks, volumes from working solutions of TICA, IRB and HCT were transferred and diluted with methanol to give the final concentrations stated in table 3. The above solutions were filtered using 0.2 μm disposable filters. 20μL portions of the working solutions of TICA, IRB and HCT were injected in triplicates and chromatographed under the chromatographic conditions mentioned above. The peak area values of each drug were plotted against the corresponding concentrations to obtain the calibration graph for each drug. The concentrations of TICA, IRB and HCT from the corresponding calibration graphs were computed.

Spiked plasma

To calculated volumes of the working solutions of TICA, IRB and HCT, 100 μL of human plasma and 100 μL of acetonitrile were added (to deproteinize biological sample), to produce a final concentration in the range stated in table 3. After vortex mixing and centrifugation (6000 rpm/min), the supernatant solutions were filtered using 0.2 μm disposable filters transferred to separate Eppendorf tubes and 20 uL of each were injected into the HPLC column. The peak areas for each drug were measured and the corresponding concentrations in the mixtures were derived referring to calibration graph.

Synthetic mixtures

Pure drugs

Accurate volumes of working standard solutions of TICA, IRB and HCT were transferred into a four separate 10-mL calibrated flasks and diluted to the mark with methanol to give synthetic mixtures containing IRB, HCT and TICA in the ratios stated in table 5. The solutions were filtered using 0.2 μm disposable filters. Volumes of 20 μL portions of the mixture solutions were injected in triplicates and chromatographed under the chromatographic conditions mentioned above. The peak areas for each drug were measured and the corresponding concentrations in the mixtures were derived referring to calibration graph.

Spiked plasma

Accurate volumes (from the supernatant layer obtained from centrifugation) of each drug were pipetted into Eppendorf tubes to give synthetic mixtures containing TICA, IRB and HCT in the ratios stated in table 5. Volumes of 20 μL portions of the mixture solutions were injected in triplicates and chromatographed under the chromatographic conditions mentioned above. The peak areas for each drug were measured and the corresponding concentrations in the mixtures were derived referring to calibration graph.

RESULTS

Chemometric assisted methods

Spectral characteristics

Fig. 4 represents the absorption spectra of TICA, IRB and HCT and their mixtures, recorded over the range of 200-300 nm against methanol.

Fig. 4: Absorption curves of 1 μg/ml IRB in methanol, 8 μg/ml of HCT, 25 μg/ml TICA in methanol and their mixture

Two chemometric methods, FF (Trigonometric Fourier function) and hybrid double divisor ratio spectra (FFR) were applied and validated.

TICA was determined in presence of IRB and HCT by applying FF and FFR. The following equations were applied for FF (eq. 1) and FFR (eq. 2), respectively:

tˊ_j = [(1.707) A₀₊(0.707) A₁+(-0.707) A₂+(-1.707) A₃+(-1.707) A₄+(-0.707) A₅+(0.707) A₆₊(1.707) A₇]/4……. (1)

Where A₀–A₇ stand for eight absorbance values at 4-nm intervals for 20 μg/ml TICA, 2 μg/ml IRB+6 μg/ml HCT (that is the double divisor) and their mixtures within the range (240-268 nm). The number in brackets are the selected combined Fourier functions and Tˊ= [cos x_i+cos (x_i+45 °)] (fig. 5).

tˊ_j = [(-0.866) FFR₀+(0) FFR₁+(0.866) FFR₂+(0.866) FFR₃+(0) FFR₄-(0.866)FFR₅]/3…. (2)

Where R₀–R₅ stand for six ratio values at 4-nm intervals for 20 μg/ml TICA, 2 μg/ml IRB+6 μg/ml HCT (that is the double divisor) and their mixtures within the range (244-264 nm). The number in brackets are the selected combined Fourier functions and Tˊ= [sin x_i-sin (x_i+60 °)] (fig. 6).

IRB was also determined in the presence of TICA and HCT by applying FF and FFR. The following equations were applied for FF and FFR, (eq. 3, 4) respectively:

tˊ_j = [(-0.866) A₀₊(1.732) A₁-(00866) A₂-(0.866) A₃+(1.732) A₄-(0.866) A₅]/4…………. (3).

Where A₀–A₅ stand for six absorbance values at 4-nm intervals for 3 μg/ml IRB, 8 μg/mlTICA+20 μg/ml HCT and their mixtures within the range (204-224 nm). The number in brackets are the selected combined Fourier functions and Tˊ= [sin x_i-sin (2x_i+60 °)] (fig. 7).

tˊ_j = [(-0.866) FFR₀₊(0) FFR₁+(0.866) FFR₂+(0.866) FFR₃+(0) FFR₄-(0.866) FFR₅]/3 ….…(4)

Where R₀–R₅ stand for six ratio values at 4-nm intervals for 3 μg/ml IRB, 8 μg/ml TICA+20 μg/ml HCT (that is the double divisor) and their mixtures within the range (240-260 nm). The number in brackets are the selected combined Fourier functions and Tˊ= [sin x_i-sin (x_i+60 °)] (fig. 8).

Fig. 5: Fourier functions convoluted curves for 25 μg/ml TICA in methanol, 2 μg/ml IRB+6 μg/ml HCT in methanol and their mixture

Fig. 6: Fourier functions convolution of ratio for 25 μg/ml TICA in methanol, 2 μg/ml IRB+6 μg/ml HCT in methanol and their mixture

Fig. 7: Fourier functions convoluted curves for 3 μg/ml IRB in methanol, 8 μg/ml HCT+20 μg/ml TICA in methanol and their mixture

HCT was finally determined in presence of TICA and IRB by applying FF and FFR. The following equations were applied for FF and FFR, (eq. 5, 6) respectively:

tˊ_j = [(1.5) A₀₊(0) A₁+(-1.5) A₂+(-1.5) A₃ (0) A₄+(1.5) A₅]/3…………. (5)

Where A₀–A₅ stand for six Absorbance values at 4-nm intervals for 10 μg/ml HCT, 2 μg/ml IRB+20 μg/ml TICA (that is the double divisor) and their mixtures within the range (212-232 nm). The number in brackets are the selected combined Fourier functions and Tˊ= [sin x_i-sin (x_i+60 °)] (fig. 9).

tˊ_j = [(-0.866) FFR₀₊(0) FFR₁+(0.866) FFR₂+(0.866) FFR₃+(0) FFR₄-(0.866) FFR₅]/3 … (6)

Fig. 8: Fourier functions convoluted curves of ratio for 3 μg/ml IRB in methanol, 8 μg/ml HCT+20 μg/mlTICA in methanol and their mixture

Where R₀–R₅ stand for six ratio values at 4-nm intervals for 10 μg/ml HCT, 2 μg/ml IRB+20 μg/ml TICA (that is the double divisor) and their mixtures within the range (240-260 nm). The number in brackets are the selected combined Fourier functions and Tˊ= [sin x_i-sin (x_i+60 °)] (fig. 10).

Fig. 9: Fourier functions convoluted curves for 10 μg/ml HCT, 2 μg/ml IRB+20 μg/ml TICA in methanol and their mixture

Fig. 10: Fourier functions convoluted curves of ratio for 10 μg/ml HCT, 2 μg/ml IRB+20 μg/ml TIC in methanol and their mixture

DISCUSSION

Chemometric assisted methods

As fig. 4 shows, there is clear spectral overlapping, preventing their resolution by direct traditional spectrophotometric measurements. Thus, the applied a chemometric assisted methods showed their ability to quantify each of the drugs in their ternary mixture.

The FF method depends on the convolution of absorption spectra of TICA, IRB and HCT using combined trigonometric Fourier function (FF). Following the general rules for the use of Fourier function in the processing of the absorption spectral data [26, 27], the associated different parameters were optimized. FFR method that is considered as a hybrid double divisor ratio spectra method depends on the convolution of the double divisor ratio spectra (to cancel the interfering effect of other drugs in the mixture, and obtain better resolution) using trigonometric Fourier functions, wherein this method, Fourier functions are applied to double divisor ratio spectra.

The optimum parameters were selected for each drug in order to give precise and accurate results for the analyte in the presence of the other interfering drugs. These include the selection of function order, number of points and wavelength intervals corresponding to spectral characteristics. Both methods gave good % recoveries. However, the application of the hybrid method (FFR) proved its excellence in resolving this triple mixture (table 4).

HPLC method

Optimization of chromatographic conditions

To establish and validate an accurate method for the analysis of these drugs in pharmaceutical formulations and plasma, C₁₈ column was used. The effect of mobile phase composition, flow rate, and pH were studied. The best resolution with reasonable retention time was obtained with a mobile phase composed of acetonitrile: phosphate buffer in the ratio of (60:40).

Upon studying the effect of pH of the aqueous mobile phase, strongly acidic pH values gave peaks of short retention time that interfered with plasma protein peak. However, alkaline pH gave very long retention time. The best separation in terms of system suitability and the resolution was achieved by using the value of pH of 3.6. The optimum wavelength for detection was 230 nm at which much better detector response for HCT, TICA and IRB was obtained.

According to FDA 1994 [28], system suitability tests are an integral part of any liquid chromatographic method. System suitability uses to verify capacity factor, number of theoretical plates, asymmetry factor, selectivity and resolution (table 1) and they were found to be satisfactory and within the reported acceptance criteria listed in the reference

The optimum chromatographic conditions mentioned previously were applied for all measurements. Fig. 11, shows the separation of HCT at 4.8 min, TICA at 7.9 min and IRB at 9.8 min, at the optimized chromatographic conditions. Fig. 12, shows the separation of HCT at 4.4 min, TICA at 8.0 min and IRB at 9.9 min, in spiked plasma at the optimized chromatographic conditions.

Fig. 11: HPLC chromatogram of a 20 µl injection of a standard mixture of 0.3 μg/ml TICA, 0.05 μg/ml IRB, and 0.5 μg/ml HCT using the optimized chromatographic conditions

Fig. 12: HPLC chromatogram of a 20 µl injection of a mixture of 0.3μg/ml TICA, 0.05 μg/ml IRB, and 0.5 μg/ml HCT in spiked plasma using the optimized chromatographic conditions

Method validation and statistical analysis [25, 29]

The proposed methods were validated according to ICH guidelines [25]. The validation parameters included: linearity, limits of detection and quantification, accuracy, precision and specificity. The applied analytical methods are able to separate and quantify the studied drugs in their triple mixture.

Linearity and concentration ranges

Under the described experimental conditions, the graphs obtained by plotting the signals of the proposed methods versus concentration for TICA, IRB and HCT gave linear relationships over the concentration ranges stated in table 2 and 3. Linearity data and statistical parameters for the proposed methods were calculated, including linear regression equation parameters (intercepts, slopes, correlation coefficients, the standard deviation of intercept and standard deviation of the slope (table 2 and 3). Regression analysis confirmed good linearity as shown from correlation coefficient value (r>0.999). The high F-value proved that the linear correlation between calculated signals (chemometric methods) or peak areas (HPLC method) and concentrations in significance to a high level of confidence. The high values of the correlation coefficient (r) with negligible intercepts indicate the good linearity of the calibration graphs. The standard deviation of residuals (S_y/x), of intercept (S_a) and of slope (S_b) are presented for each drug. (S_y/x) is a measure of the extent of deviation of the found (measured) y-values from the calculated ones. Also, the small degree of scattering of the experimental data point around the line of regressions was confirmed by the small values of the variances around the slopes (S_b²).

Table 1: HPLC system suitability parameters for the determination of HCT, TICA and IRB using the proposed method

Analyte	Retention time (t_r)	Capacity Factor(kˊ)	N^o of theoretical plates (N)	Asymmetry factor (A_F)	Selectivity (α)	Resolution (R_s)
HCT	4.8	0.66	536	1.66	2.61 1.24	3.44 2.20
TICA	7.9	1.72	1198	1.16
IRB	9.8	2.38	1433	1.00

Limit of detection (LOD) and limit of qualification (LOQ)

Limits of detection (LOD) and quantification (LOQ) were calculated according to the ICH guidelines [25]. LOD was defined as 10 S_a/b, where S_a is the standard deviation of the intercept and b is the slope of the calibration curve. The sensitivity of the proposed methods can be confirmed by the low LOD and LOQ values obtained in table 2. The variance test for the regression lines revealed that, for equal degrees of freedom, the increase in the variance ratio (F-values) means an increase in the mean squares due to regression and a decrease in the mean squares due to residuals, (i. e the less is the scatter of experimental points around the regression line).

Consequently, regression lines with high F-values (low significance F) are much better than those with lower ones. Good regression lines show high values for both r and F statistical parameters [30].

Table 2: Assay parameters for the determination of TICA, IRB and HCT using the applied chemometric assisted methods

	TICA	IRB	HCT
Conc. Range(μg/ml)	10-30	1-3	4-10
Parameters	FF	FF	FF
λ or λ _range(nm)	240-268	204-224	212-232
Δλ_(nm)	4	4	4
r	0.9997	0.9993	0.9993
S _y/x	7.00 x 10^-7	2.75 x 10^-6	2.77 x 10^-[34]
F	65 x 10³	2299	1.02 x 10[33]
Significance F	1.31 x 10^-7	2.00 x 10^-5	6.75 x 10^-[50]
a (intercept)	-0.03 x 10^-2	0.28 x 10^-3	0.2 x 10^-[16]
b (slope)	-0.78 x 10^-1	0.29 x 10^-1	0.48 x 10^-1
S_a	0.68 x 10^-3	0.13 x 10^-2	1.01 x 10^-[17]
S_b	0.31 x 10^-3	0.61 x 10^-3	1.52 x 10^-[18]
LOD (μg/ml)	-0.24 x 10^-1	-0.24 x 10^-1	6.22 x 10^-[16]
LOQ (μg/ml)	-0.82 x 10^-1	0.44	2.07 x 10^-[15]
a/S_a	-0.46	0.22	1.98
(S_b)²	9.33 x 10^-8	3.66 x 10^-7	2.31 x 10^-[36]
S_b %	9.33 x 10^-6	3.66 x 10^-5	1.52 x 10^-[16]

Accuracy and precision

The applicability of the developed methods was tested by the analysis of TICA, IRB and HCT in several synthetic mixtures of different proportions on the same day and on different days. Good accuracy expressed as relative percentage error (Er %), and high precision, expressed as percentage RSD, were obtained. The results, summarized in tables 4, 5 and 6 show that the (Er %) and RSD % values do not exceed the accepted limits, which demonstrate the accuracy an repeatability of the developed methods.

Table 3: Assay parameters for the determination of HCT, TICA and IRB using the proposed HPLC method

Parameters	HCT	IRB	TICA	HCT	IRB	TICA
	In pure drugs	In spiked plasma
Conc. Range (μg/ml)	0.1-0.9	0.01-0.05	0.1-0.5	0.1-0.9	0.01-0.05	0.1-0.5
Δλ_(nm)	230	230	230	230	230	230
r	0.9999	0.9993	0.9996	0.9999	0.9858	0.9996
S _y/x	25.51 x 10⁴	74.04 x 10⁶	48.35 x 10²	76.73 x 10⁴	78.62 x 10⁶	11.04 x 10³
F	74.53 x 10³	23.34 x 10²	40.40 x 10³	24.40 x 10³	103.69	16.29 x 10³
Significance F	1.08 x 10^-7	1.95 x 10^-5	2.71 x 10^-7	5.78 x 10^-7	2.02 x 10^-3	1.06 x 10^-6
a (intercept)	-17.20	23.33 x 10²	-29.40	255.75	-16.36 x 10³	-87.3
b (slope)	12.58 x 10⁴	24.00 x 10⁴	25.52 x 10⁴	12.49 x 10⁴	28.55 x 10⁴	24.48 x 10⁴
S_a	264.84	16.47 x 10²	42.11	459.34	92.99 x 10²	63.61
S_b	461.04	49.67 x 10²	12.69 x 10²	799.62	28.04 x 10³	19.18 x 10²
LOD (μg/ml)	0.631 x 10^-2	0.205 x 10^-1	0.495 x 10^-3	0.11 x 10^-3	9.77 x 10^-2	7.79 x 10^-4
LOQ (μg/ml)	0.21 x 10^-1	0.686 x 10^-1	0.165 x 10^-2	3.67 x 10^-2	0.33	2.59 x 10^-3
a/S_a	-0.64 x 10^-1	1.41	-0.698	0.556	-1.76	-1.37
S_b²	21.25 x 10⁴	24.67 x 10⁶	16.11 x 10⁵	63.93 x 10⁴	7.86 x 10⁸	36.78 x 10⁵
S_b %	46.10 x 10³	13.94 x 10⁴	12.69 x 10⁴	79.96 x 10³	28.04 x 10⁵	19.18 x 10⁴

Table 4: Accuracy for the simultaneous determination of TICA, HCT o IRB in laboratory-made mixtures using the proposed chemometric assisted methods

TICA: IRB: HCT μg/ml	Mean recovery±SD^a, RSD %^b, Er %^c
	FF
20:1:4	100.38±1.59
	1.58
	0.38
20:2:4	101.38±1.35 1.33 1.38
10:3:4	98.66±1.56
	1.58
	-1.34
20:3:10	97.99±0.92
	0.94
	-2.01
3:30:10	101.24±0.92
	0.90
	1.24
15:2:10	97.00±0.89
	0.91
	-3.0

^amean±SD for the three determinations, ^b% Relative standard deviation, ^c% Relative error

Table 5: Accuracy for the simultaneous determination of IRB, HCT and TICA in laboratory-made mixtures and spiked plasma using the proposed HPLC method

HCT: IRB: TICAμg/ml	Mean recovery±SD^a, RSD%^b, Er%^c
	HCT
	In laboratory prepared mixtures
0.1:0.01: 0.1	98.75±0.86
	0.87
	0.39
0.3:0.02:0.2	98.92±0.98
	0.99
	0.58
0.5:0.03:0.3	99.47±1.18
	1.18
	-0.53
0.7:0.04:0.4	99.6±1.09
	1.09
	-0.40
0.9:0.05:0.5	98.98±1.09
	1.10
	-1.02

^amean±SD for the five determinations, ^b% Relative standard deviation, ^c% Relative error

Table 6: Intra-day and inter-day precision for the simultaneous determination of TICA, IRB and HCT in laboratory-made mixtures using the proposed chemometric and HPLC methods

HCT: IRB: TICA μg/ml	Analytical method	Intra-day precision, Mean recovery±SD^a RSD%^b, Er%^c	Inter-day precision, Mean recovery±SD^a RSD%^b, Er%^c
		TICA	IRB
10: 3: 30	FF	100.5±0.42 0.42 0.50	99.3±0.99 0.99 -0.70
10: 3: 30	FFR	100.22±1.32 1.32 0.22	101.90±1.34 1.32 1.90
0.9: 0.05:0.5	HPLC	99.0±0.61 0.61 -1.00	99.94±1.52 1.52 -0.06

^amean±SD for the five determinations, ^b% Relative standard deviation, ^c% Relative error

Table 7: Assay results for TICA, IRB and HCT in their laboratory made mixture using the proposed methods

RatioTICA+IRB+HCTμg/ml9:1.5:0.5	Mean recovery±SD^aRSD %^bEr %^c
	HPLC
TICA
Mean Recovery±SD^a	98.76±0.45
RSD %^b	0.45
Er %^c	-1.01
**t-test	––
**F-test	––
IRB
Mean Recovery±SD^a	98.99±0.87
RSD %^b	0.87
Er %^c	-1.01
**t-test	––
**F-test	––
HCTMean Recovery±SD^aRSD %^bEr %^ct-testF-test	99.08±0.610.61-0.92––––

^amean±SD for the five determinations, ^b% Relative standard deviation, ^c% Relative error, **Theoretical values of t-and F-at P = 0.05 are 2.13 and 6.93, respectively.

Laboratory made mixture

Table 7 present a statistical comparison between the assay of TICA, IRB and HCT in the laboratory made a mixture by the proposed methods using the student’s t-test and the variance ratio F-test. Since the calculated t-and F-values for each drug did not exceed the theoretical ones, this indicated that there was no significant difference between the applied methods for determination of the two drugs in commercial tablets.

CONCLUSION

For analytical purposes, it is always important to establish methods capable of analyzing co-administered drugs in a short period of time with acceptable accuracy and precision. The chemometric assisted methods are simple, inexpensive and require easy treatment of the samples. The use of ratio spectra in the chemometric methods can magnify the prediction ability of the usual Spectrophotometric techniques. A comparative study of the use of RP-HPLC and chemometric assisted methods for resolution of ticagrelor, irbesartan, hydrochlorothiazide has been accomplished. Showing that chemometric assisted methods provide, with adequate software support, a clear example of the high resolving power of this technique. Although the RP-HPLC method is more sensitive, it needs expensive equipment and materials. The chemometric assisted methods are less expensive and do not require sophisticated instruments or prior separation steps. The proposed RP-HPLC were found to be suitable for the assay of this ternary mixture in plasma and can be used for further bioequivalence studies.

ACKNOWLEDGMENT

This wok has been supported by Beirut Arab University, Lebanon.

CONFLICT OF INTERESTS

Declared none

REFERENCES

John Wiley, Sons. Ltd E. Handbook of Green Analytical Chemistry; 2012.
Charles L, Lora A, Morton G, Leonard L. Drug Information Handbook, edition. Twelfth; 2005.
Ramakrishna VS, Rambabu C. Visible Spectrophotometric methods for the determination of irbesartan in pharmaceutical formulations. Int J Pharm Pharm Sci 2012;3 Suppl 4:86.
Baskararaju VLA. Validated RP-HPLC method for the estimation of irbesartan in bulk and tablet dosage form. Int J Res Pharm Chem 2011;1:25–8.
Jain P, Bhardwaj YR, Kishore D. Irbesartan: A review on analytical method and its determination in pharmaceuticals and biological matrix; 2014. p. 1–8.
Patel KR, Patel SA, Darji V. CSRN simultaneous spectrophotometric estimation of irbesartan and hydrochlorothiazide in tablets. Int Res J Pharm 2011;2:202–7.
Raja B, Himasri P, Ramadevi B. RP-HPLC method for the simultaneous estimation of irbesartan and hydrochlorthiazide in pharmaceutical dosage form. Int Res J Pharm Appl Sci 2012;2:29–38.
Elshanawane AA, Abdelaziz LM, Kamal MM, Hafez HM. Quantitative determination of four angiotensin-ii-receptor antagonists in presence of hydrochlorothiazide by a gradient technique HPLC in their pharmaceutical preparations. J Liq Chromatogr Relat Technol 2013;37:171–86.
Zorica Vujić, Nedžad Mulavdić, Miralem Smajić JB, Stankovic P. Simultaneous analysis of irbesartan and hydrochlorothiazide: an improved HPLC method with the aid of chemometric protocols. Molecules 2012;17:3461–74.
Peeyush Jain, Bhardwaj YR, Kishore D. A liquid chromatography tandem mass spectrometry-based method for the simultaneous determination of irbesartan and hydrochlorothiazide in human plasma. J Drug Dev Res 2013;5:169–73.
Hammouda MEA, El-enin MAA, El-sherbiny DT, El-wasseef DR, El-ashry SM. Simultaneous determination of irbesartan and hydrochlorothiazide in pharmaceutical preparations and spiked human plasma using microemulsion liquid. Int J Adv Pharm Res 2013;4:1944–59.
G Kumara Swamy, JMR Kumar JVLNSR. A validated reverse phase HPLC method for the simultaneous estimation of irbesartan and amlodipine in pharmaceutical dosage form. World J Pharm Pharm Sci 2014;3:996–1007.
Virani P, Sojitra R, Raj H. Chromatographic method for irbesartan and its combination with other drug. J Crit Rev 2015;2015;2:4–8.
Hapse SA, Wagh VS, Kadaskar PT, Dokhe MD, Shirsath AS. Spectrophotometric estimation and validation of hydrochlorothiazide in tablet dosage forms by using different solvents. Pharm Chem 2012;4:10–4.
Bhagwate S, Gaikwad NJ. Stability indicating HPLC method for the determination of hydrochlorothiazide in pharmaceutical dosage form. J Appl Pharm Sci 2013;3:88–92.
Bhatia R, Katoch S, Kumar D. Review article determination of hydrochlorothiazide and drugs in its combination. HPLC 2015;7:184–90.
Sivasubramanian L, Ks L. Spectrophotometric multicomponent analysis of telmisartan, hydrochlorothiazide and ramipril in pharmaceutical formulations by chemometric. World J Pharm Pharm Sci 2015;4:536–50.
Khadiga M Kelani, Abdallah A Shalaby, Magda Y Elmaamly MKH. Spectrophotometric and chemometric methods for simultaneous determination of two anti-hypertensive drugs in their combined dosage form. Pharm Anal Acta 2015;6:2153–435.
Santhana Lakshmi K, Lakshmi S. Simultaneous analysis of losartan potassium, amlodipine besylate, and hydrochlorothiazide in bulk and in tablets by high-performance thin layer chromatography with UV-absorption densitometry. J Anal Methods Chem 2012. Doi:10.1155/2012/108281. [Article in Press]
Pandya D, Patel M, Ghediya R, Shah A, Khunt R. Research article UV-Vis spectrophotometric assay determination of oral antiplatelet ticagrelor drug in the pharmaceutical formulation. Appl Content Uniformity 2016;8:316–21.
Kalyani L, Rao AL. UV-Vis spectrophotometric assay determination of oral antiplatelet ticagrelor drug in the pharmaceutical formulation. Appl Content Uniformity 2013;3:634–42.
Sillén H, Cook MDP. Determination of ticagrelor and two metabolites in plasma samples by liquid chromatography and mass spectrometry. J Chromatogr B: Anal Technol Biomed Life Sci 2010;25:2299–306.
Sillen H, Cook MDP. Determination of unbound ticagrelor and its active metabolite (AR-C124910XX) in human plasma by equilibrium dialysis and LC-MS/MS. J Chromatogr B 2011;879:2315–22.
Kale P, Agrawal Y, Soni G, Patel P. Simultaneous quantification of ticagrelor and its metabolite deshydroxyethoxy ticagrelor in human plasma by ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry. World J Pharm Sci 2015;3:37–45.
Validation of Analytical Procedures: Text and Methodology. International Conference on HarmonisationI CH Harmonised Tripartite Guideline. Q2(R1). Geneva; 1996.
AM Wahbi, H Abdine MK. Changes in the expression of hepatic cytochrome P450 isoenzymes 2E1, 2B1/2, 4A, and 2C6 IN mice infected with different levels of schistosoma mansoni cercariae. Pharmazie 2005;278:14–33.
Mohamed A Korany, Mahmoud A Elsayed, Mona M Bedair, Hoda Mahgoub, Ezzat A Korany. Computer-assisted spectrophotometry: Multicomponent analysis with a discrete fourier transform. Talanta 1990;37:1183–8.
FDA Center for Drug Evaluation Research (CDER), Reviewer Guidance: Validation of Chromatographic Methods, Washington, USA; 1994.
Miller JN, Mileer JC. Statistics and chemometrics for analytical chemistry. 5th ed. London, Pearson Prentice Hall; 2005.
P Berry G. Statistical methods in medical research. Armitage Black Well, Oxford, UK; 2001. p. 832.

How to cite this article

Marwa K. EL Jamal*, Azza A. Gazy. Analysis of three cardiovascular drugs in their ternary mixture using green analytical methodology of smart spectrophotometric methods and RP-HPLC method. Int J Pharm Pharm Sci 2016;8(8):243-250.