Simultaneous Determination of Hydrochlorothiazide and Losartan Potassium in Pharmaceutical Product by UV-Vis Spectrophotometric Method with Kalman Filter Algorithm

In this paper, hydrochlorothiazide (HCT) and losartan potassium (LSP) in tablets are simultaneously determined with UV-Vis spectrophotometry and chemometrics without separation. The spectra of standard and sample solutions were recorded in the wavelength from 220 to 300 nm at 1.0 nm intervals. The concentrations of HCT and LSP in the sample solutions were computed with the Kalman filter algorithm written on the Microsoft Excel 2016 and Visual Basic for Applications (VBA) platform. The method validation was determined via the accuracy and repeatability of measurements when analyzing HCT and LSP in the Splozarsin Plus tablet and comparing the mean values of their contents in the sample with those analyzed with HPLC. The proposed method is simple with a low cost compared with the standard HPLC method.


Introduction
Molecular absorption spectrometry incorporating chemometrics is often used to determine components in a mixture with overlapping spectra simultaneously. e general principle of this combined method is that the full-spectral data are loaded into software programmed with algorithms and statistical methods, from which each component in the mixture is quantified. Due to its simplicity, the method is considered environmentally friendly and economical. Besides, full-spectral data for calculations combined with algorithms help to increase the accuracy of the determination. Standard chemometrics methods include classical least squares (CLS), partial least squares (PLS), Kalman filter, principal component regression (PCR), artificial neural networks (ANNs), and derivative spectrophotometry. ese methods have been studied and applied to various matrices [1][2][3][4][5][6][7].
In our previous study [4], we succeeded in employing the Kalman filter algorithm with full-spectral measurement to build a program (written on the Microsoft Excel 2016 and Visual Basic for Applications (VBA) platform) for determining the concentration of substances in a mixture. Inheriting these results, we continue modifying and applying this combined method (from now on, Kalman-Excel method) to determine Hydrochlorothiazide (HCT) and Losartan Potassium (LSP) in Splozarsin Plus simultaneously. Hydrochlorothiazide (C 7 H 8 ClN 3 O 4 S 2 , M � 297.7 g/mol) is a diuretic medicine (or water pills) used to treat hydronephrosis, renal failure, or nephropathy. In addition, HCT is also used effectively to prevent and treat hypertension, liver disorders, myocardial infarction, heart failure, or stroke in patients with cardiovascular problems. Losartan Potassium (C 22 H 22 ClKN 6 O, M � 461.0 g/mol) belongs to a class of medicine called angiotensin receptor blockers that dilate blood vessels, facilitating blood to flow more easily. Losartan Potassium is used to treat high blood pressure and heart failure and help to protect the kidney damage caused by diabetes [8][9][10].
For the determination of HCT and LSP in medicine, various techniques have been developed elsewhere. Maggio et al. [8] established a procedure based on the multivariate analysis of spectral data in the 220-274 nm region, with the partial least square algorithm. e results agreed well with those determined with HPLC. Rathee et al. [9] developed Vierordt's and Q absorbance ratio methods. ey were successfully applied to the assay of LSP and HCT in tablet formulations. Rao et al. [10] built three techniques for this purpose: (1) to solve the equations simultaneously; (2) to determine the absorbance ratio at 272 nm (the maximum absorption of HCT) and 266.5 nm (the isosbestic wavelength); (3) to use the first-order derivative method. ube et al. [11] also proposed three spectrophotometric methods for the simultaneous determination of LSP and HCT in a binary mixture: (1) to measure the absorbance at 235 nm and 271 nm, corresponding to the absorbance maxima of LSP and HCT, respectively (the concentration of each drug was obtained by using the absorption values calculated for both drugs at these wavelengths); (2) to use the dual-wavelength principle in which the absorbance difference between two points on the mixture spectra was directly proportional to the concentration of the component of interest and independent of the interfering components; and (3) to describe and calculate the area under the curve between 265 and 282 nm for LSP and between 229 and 242 nm for HCT. e proposed methods were applied to marketed formulation. High-performance liquid chromatography was also applied to quantify these two compounds in pharmaceutical products [12][13][14][15]. Although HPLC delivers simultaneous determination, high cost prevents its application.
As the absorption spectra of HCT and LSP are overlapped, the Kalman filter algorithm using full spectra (Kalman-Excel method) [4] is employed in this study to quantify these two substances in pharmaceutical products simultaneously. e proposed method opens up the possibility of a fast and cheap technique for pharmaceutical analysis and testing. e stock solution of HCT (50.0 μg/mL) was prepared by dissolving 12.5 mg of HCT standard in 150 mL of distilled water and ultrasonicated for 15 min. e solution was then made up to 250 mL with distilled water. Twenty-five millilitres of the stock solution was diluted with 75 mL of distilled water to make a working solution of HCT (12.5 μg/ mL). e stock solution of LSP (50.0 μg/mL) (also acted as a working solution) was prepared by dissolving 12.5 mg LSP standard in distilled water and making up to 250 mL.

Sample Preparation.
In this study, Splozarsin Plus tablets (Co. Ltd. SHINPOONG DAEWOO, Bien Hoa industrial zone, Dong Nai, Vietnam, batch code 9003, manufactured date 5 June 2019, expired date 5 June 2021) were used for the method validation. e reference amount of LSP and HCT on the label is 50.0 mg and 12.5 mg, respectively.
Twenty tablets with an average weight of 253.4 mg were ground to a fine powder and mixed well. A particular amount of the powder (m) was dissolved in 150 mL distilled water and ultrasonicated for 30 min before filtering to make up a 250 mL solution. Ten millilitres of the solution was diluted to 100 mL with distilled water for measurements. e amount of each target compound in a tablet is expressed via mg/tablet and calculated according to the following equation: where C i (μg/mL) is the concentration of compound i in the final solution; M (mg) is the average weight of the tablets; and m (mg) is the weight of the powder.

Analytical Procedure.
To determine HCT and LSP simultaneously, we utilized the Kalman filter algorithm with full-spectral measurements. e Visual Basic for Applications, a programming language written on the basis of Microsoft Excel 2016, was used for data treatment [4]. Briefly, the standard solutions of individual compounds and their actual sample solutions were subjected to record molecular absorption spectra (scanned from 220 to 300 nm wavelength at 1.0 nm intervals).
e achieved data were saved as .csv files and extracted to the computer as Excel sheets. e developed Kalman-Excel program was applied to determine the concentrations and respective errors.

Relative Error (RE%).
e relative error (RE%) between concentrations is calculated according to the following equation [16]: where C is the measured concentration (μg/mL) and C 0 is the concentration of the standard solution (μg/mL).

Limit of Detection (LOD) and Limit of Quantification (LOQ).
Measurements are made on a minimum of seven aliquots (n ≥ 7) of a prepared sample solution that has a concentration near the expected limit of detection [16]. e developed Kalman-Excel program was applied to determine the concentrations of HCT and LSP in the seven aliquots when using their spectrum data and spectrum data of the standard solutions of HCTand LSP. e LOD and LOQ were then calculated by the following formula: where SD is the standard deviation of seven replicates.

Repeatability (RSD%).
e repeatability of the analytical method is assessed via relative standard deviation [16][17][18]: where SD is the standard deviation and C m is the mean concentration of n replicates. e RSD value is acceptable if lower than 1/2RSD Horwitz [19], in which RSD Horwitz is calculated according to the following equation: where C is expressed as a fraction (e.g., C � 5 μg/ mL � 5 × 10 −6 ).

Trueness.
e trueness of the analytical method was verified via the method's recovery and the mean values of the developed method and a standard method.
(i) Recovery. e mean recovery is determined by assessing spiked samples at three different levels (three replicates per level) of each target compound, namely, 0.500, 1.000, and 1.500 μg/mL for HCT and 2.000, 4.000, and 6.000 μg/mL for LSP. e recovery rate is calculated by the following equation: where C 2 (μg/mL) is the measured concentration of the spiked sample; C 1 (μg/mL) is the measured concentration of the original sample (sample without spiking); and C s (μg/ mL) is the spiked concentration.
In this study, Splozarsin Plus tablets were used as the original sample. ree-fifths of the average tablet weight (3/ 5 × M) were taken for each sample. e standard solutions of HCT (5.000 μg/mL) and LSP (20.000 μg/mL) were also prepared at the same time for spectral measurements. e developed Kalman-Excel program was applied to quantify the concentration of HCT and LSP in the samples simultaneously. e acceptable recovery rate is from 85% to 110% [18].
(ii) Comparing Two Sample Means Performed By Kalman-Excel Method and Standard HPLC Method. According to Harvey [20], the trueness of an analytical method could be evaluated by analyzing n 1 and n 2 replicates with the developed method and the standard method. e mean values achieved from the two analytical methods are compared with the t-test. e Splozarsin Plus tablets were sent to the Drug, Cosmetic and Food Quality control Center of ua ien Hue Province for analysis with the HPLC method. Hydrochlorothiazide and Losartan Potassium were quantified following the Vietnam Pharmacopoeia V guideline [20]. e Splozarsin Plus solutions were prepared (described in Section 2.2) and analyzed with the developed Kalman-Excel method (described in Section 2.3) with three replicates. e t-test was used for evaluation: when the variances for the two analyses are not significantly different if the experimental t value is lower than the t value at risk of 0.05 and (n 1 + n 2−2 ) degrees of freedom, the mean values achieved from the two methods are not significantly different.

Relative Errors and
Repeatability. Seven mixtures of HCT and LSP standards with different concentration ratios were prepared from the standard solutions of HCT (12.500 μg/mL) and LSP (50.000 μg/mL). e mixtures and the standard solutions were subjected to analysis with the Kalman-Excel method, as described in Section 2.3. e absorption spectra are shown in Figure 1.
At different HCT and LSP concentration ratios, the RE values between reference concentrations and their measured concentrations vary from −1.55 to 1.93% (Table 1). In addition, the RSD values are far lower than the respective 1/ 2RSD H values. is finding indicates the good performance of the Kalman-Excel method for the simultaneous determination of HCT and LSP in standard mixtures.

Quantification of HCT and LSP in Splozarsin Plus
Tablet and Method Repeatability. A Splozarsin Plus powder weighting 253.4 mg (after grinding and mixing) was prepared as described in Section 2.2 and analyzed as described in Section 2.3. e amount of HCT and LSP in the Splozarsin Plus tablet was determined following equation (1). e absorption spectra of the sample and standard solutions are illustrated in Figure 2.

Trueness, LOD, and LOQ of Method
(1) Recovery of the Method. e absorption spectra of standard solutions and spiked samples are demonstrated in Figure 3. e results of recovery are shown in Table 3.
In addition to that, the LOD and LOQ values achieved in this method were 0.037 and 0.12 μg/mL for HCT and 0.228 and 0.76 μg/mL for LSP (Table 4). Accordingly, it was an advance over the results from the studies of Rao et al. [10] who used three different spectrophotometry methods including simultaneous equation, ratio of absorbance, and first-order derivative method to quantify HCT and LSP in tablets, or the studies of Mohammed et al. [13] and Srinivasu et al. [14] who applied RP-HPLC (Table 4).
Besides the good performance of the developed method, another highlight was its simplicity in sample preparation, in which H 2 O was the only solvent used for sample dilution and preparation, while most of the other methods consumed various organic solvents (Table 4).
(2) Comparing Two Sample Means Performed by Kalman-Excel Method and HPLC Method. To continue evaluating the method trueness, we compare the concentration of HCT and LSP in the Splozarsin Plus tablets quantified with the Kalman-Excel method with that of the HPLC method. e t-test is used for data comparison. e data in Table 5 demonstrate that t exp is lower than the t (0.05; 4), implying that the analytical results of the two methods are statistically consistent with the significance level of 0.05. In other words, the Kalman-Excel method exhibits good trueness. In addition, the F exp values are lower than the F (0.05, 2, 2) when α � 0.05, revealing that the developed analytical method and the standard method (HPLC) produce the same exact repeatability.

Conclusions
We successfully apply full-spectral measurements with the modified Kalman filter algorithm method for the simultaneous determination of Hydrochlorothiazide and Losartan Potassium in pharmaceutical formulation tablets. e method minimizes the time needed for sample preparation as well as the amounts of solvent used and the waste generated. e analytical procedure exhibits good repeatability, good trueness, and low relative errors for the studied compounds. ere is no statistically significant difference in analytical results between the Kalman-Excel method and the standard HPLC procedure used as reference. e simplicity and good sample throughput of this method make it suitable for routine quality control testing of the pharmaceutical products containing simultaneously Hydrochlorothiazide and Losartan Potassium.

Data Availability
e data used to support the finding of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.  Journal of Analytical Methods in Chemistry 7