Chemometric Methods for Simultaneous Determination of Candesartan Cilexetil and Hydrochlorothiazide in Binary Combinations

Simple, accurate, precise, and cost-effective chemometric techniques for the measurement of candesartan cilexetil and hydrochlorothiazide in synthetic mixtures were improved and validated. H-point standard addition, Q-absorption ratio, and correction absorbance spectrophotometric techniques were utilized for the simultaneous determination of both medicines in real pharmaceutical formulations. A new calibration approach was implemented based on chemical H-point standards. This approach was developed to resolve significantly overlapping spectra of two analytes and provide direct correction of both proportional and constant errors caused by the matrix of the sample. The first method of simultaneous determination of candesartan cilexetil and hydrochlorothiazide was carried out using the H-point standard addition method at wavelengths 239 and 283. For the ratio of the absorption at two selected wavelengths, one of which is the isoabsorptive point and the other being the maximum of one of the two components, the second method absorption ratio method was utilized. In distilled water, the isoabsorptive point of candesartan cilexetil and hydrochlorothiazide occurs at 258 nm. λmax of hydrochlorothiazide is 273 nm, which is the second wavelength used. Lastly, the absorbance correction method was implemented. This approach is based on absorbance correction equations and uses distilled water as the solvent for the examination of both medicines. In NaOH/EtOH solvent, the absorbance maxima of candesartan cilexetil and hydrochlorothiazide are 250 nm and 340 nm, respectively. For both wavelengths, candesartan cilexetil and hydrochlorothiazide exhibited linearity over a concentration range of 1–46 μg/ml and 1–44 μg/ml, respectively, for H-point standard addition. The Q-absorption ratio approach provides linearity over the concentration ranges of 1–46 μg/ml at 273 nm for candesartan cilexetil and 1–29 μg/ml for hydrochlorothiazide, 1–46 μg/ml at 258 nm for candesartan cilexetil, and 1–44 μg/ml for hydrochlorothiazide. For hydrochlorothiazide, the linearity for the correction absorbance method was obtained throughout a concentration range of 1–46 μg/ml at wavelengths 250 and 340 nm and 1–44 μg/ml at wavelength 250 nm. The results of the analysis have been statistically and empirically supported by recovery studies. All methods yielded recoveries in the range of 96 –102% for both medications. The LOD ranged from 0.46 –0.94 μg/mL for hydrochlorothiazide and from 1.26 –2.40 μg/mL for candesartan cilexetil. The approaches were then used to quantify candesartan cilexetil and hydrochlorothiazide in pharmaceutical tablets.


Introduction
Candesartan cilexetil (CAN) 2-ethoxy-1-((4-[2-(2H-1,2,3,4tetrazol-5-yl) phenyl] phenyl, methyl))-1H-1, 3-benzodiazole-7-carboxylic acid is an angiotensin receptor blocker used mainly for the treatment of high blood pressure and congestive heart failure. Candesartan has a very low maintenance dose. Hydrochlorothiazide (HCT), 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1dioxide, is one of the oldest thiazide diuretics. Recently, CAN has been difcult to be separated from HCT in most tablets [1,2]. Candesartan is an efective, irreversible antagonist because it has the highest known receptor afnity of all the ARBs, and high doses of angiotensin II (Ang II) don't push it of the receptor. Study after study has shown the positive efects of candesartan cilexetil in the treatment of high blood pressure and heart failure (HF) [3]. For more than 50 years, thiazide-type diuretic hydrochlorothiazide (HCT) has been available for clinical usage [4]. HCT is also used to lower blood pressure while walking, which is mostly caused by a drop in blood pressure at night [5]. In clinical trials lasting anywhere from 8 weeks to 3 years, the fxeddose combination medication candesartan and hydrochlorothiazide has emerged as a key option in the treatment of hypertension due to its great efcacy in lowering blood pressure and preventing damage to target organs [6].
Various mathematical techniques have been developed for the use of chemometric strategies, such as partial least squares, so it is possible to study drug ̶ excipient interactions in a single sample without resorting to costly and timeconsuming chemical separation [7], and multiple linear regression [8,9], Te H-point standard addition method (HPSAM) is also used to assess binary mixtures in chemometric techniques [10]. It was afterward changed to multicomponent analysis [11][12][13]. Te Q-absorption ratio method [14,15], and the correction absorbance method have also been used for binary mixture analysis [16,17].
In this work, HPSAM, the Q-absorbance ratio technique, and the correction absorbance method were used for the simultaneous assessment of CAN and HCT. Tese processes were accurate, selective, sensitive, and reasonably priced. Tese new techniques eliminate strongly overlapped spectra. Te outcomes were contrasted with those attained using the HPLC technique. A simple graphical description of the suggested method is depicted in Figure 1.

H-Point Standard Addition
Method. Tis technique plots the analytical signal versus the amount of analyte at two wavelengths. Considering a sample interference, Y as the interference and X as the analyte. For HPSAM to determine X concentration, interference absorbance must be constant [10]. Two straight lines with a common point in H (−C H , A H ) are depicted in Figure 2 [13].
Te measured quantity of X is added. Finally, absorbance at two specifed wavelengths is measured according to the following equations: where A (λ1) and A (λ2) represent the absorbances at λ 1 and λ 2, respectively, b 0 and A 0 represent the analytical signals of X at A λ1 and A λ2 (b 0 ≠ A 0 ), and b and A ' represent the analytical signals of Y at A λ1 and A λ2 (b � A ' ). M λ1 and M λ2 are the slopes of the calibration lines at λ 1 and λ 2 . Lastly, C i signifes the addition of X. As illustrated in Figure 2, the H-point is dependent on the analyte concentration.
Since C i � C H is derived from equations (1) and (2), Hence, As interference Y has identical absorbance values at λ 1 and λ 2, A ' � b and Which fts within the given equation.
Tat −C H is proportional to the amount of analyte present in the mixture can be concluded [36].
A H , the intersection point's ordinate value can be expressed as follows: As b 0 � M λ1 from equation (7), then A H � b and A H � A ' . Te relationship between absorbance at specifc wavelengths and the H-point (A H ) is solely due to interference. Since this is the same as the zero point on the calibration graph for the analyte when the sample is present, the analytical signal can be used to fgure out how much Y there is from the calibration graph.
Te following guidelines were used to choose the best wavelength combination for the determination of CAN and HCT in a binary mixture by HPSAM: (1) At certain wavelengths, analyte signals must be linear to concentration, and the interferent signal must be unafected by analyte concentration (2) Te analytical signal from a mixture combining analyte and interferent should equal the sum of their individual signals 2 Journal of Analytical Methods in Chemistry (3) For reasonable sensitivity and accuracy, the slope diference between two straight lines measured at λ 1 and λ 2 must be as large as feasible [37,38] 2.2. Q-Absorption Ratio Method. Tis approach is applicable to medications that follow Beer's law at all wavelengths and have a consistent ratio of absorbance between any two Journal of Analytical Methods in Chemistry wavelengths [39]. Tis method utilizes the ratio of absorption at two chosen wavelengths. One represents the drug's maximal absorbance, while the other represents the iso-absorptive point. Assume that X and Y are the two medications.
Te following equations were combined depending on this relationship: ax 1 � ay 1 at λ 1 and L � 1. At Equation (9) divided by equation (8), we get When A 2 /A 1 � ax 2 F X /ax 1 − ay 2 F y /ay 1 + ay 2 /ay 1 because(a Equation (12), which is approximate rather than exact, yields the percentage rather than the concentration of X and Y in the mixture.
If we rearrange equation (8) to include the absolute concentrations of X and Y, we obtain the following equation: From equations (12) and (13), we get where C x and C y are the X and Y concentrations, respectively, A 1 , A 2 are the absorbances of the mixture at λ 1 , λ 2 , ax 1 , and ay 1 are absorptivities of X and Y at 261 nm, and ax 2 and ay 2 are absorptivities of X and Y at 270 nm. Equations (15) and (16) give the absolute concentration values of drug X and Y [15,40,41].

Correction Absorbance Method.
In this method, λ max of analyte and interference was determined by scanning the drug solution in the UV Spectrophotometer. Which requires two wavelengths, one is the λ max of the analyte and the second one is the wavelength in which the analyte has no absorbance, the signal is only related to interference; thus, the absorbance of the interference at the frst wavelength was calculated as follows [42]: A mixλ1 , and A mixλ2 , are the absorbance of the mixture at λ 1 , and λ 2 . A corrλ1 are the net absorbances at λ 1 nm, Te slope ratios of the interference calibration graph are represented by the values r 1 and r 2 , respectively [16,43].

Apparatus.
Te UV-visible spectrophotometer (UV-VIS/VIS spectrophotometer AE-S60) was connected to an identical 1.0 cm quartz cell for the UV-VIS scanning spectrum.
All of the measurements in this study were estimated with the MetaSpec Pro software suite.

Preparation of Real Sample.
Te average mass of 10 pills was measured, they were mashed, the powder was added to a 1 : 1 NaOH : ethanol solution, and it was continuously stirred for 10 minutes. Te next step is the fltering procedure. Tree times, 10 ml of 1 : 1 NaOH : ethanol was used to wash the powder of the flter paper. After that, the solution was fnished to a fnal volume of 1 L of 1 : 1 NaOH : ethanol. Te solution was stored in a 4°C refrigerator.

Preparation of Standard Solution.
Preparing a 1000 µg/ mL HCT solution by dissolving 0.025 gm HCT in 1 : 1 NaOH : ethanol, to attain the needed analyte concentration, was diluted in a 25 mL volumetric fask. A 1000 µg/mL CAN solution was made by dissolving 0.025 gm CAN in 1 : 1 NaOH : ethanol and was diluted in a 25 mL volumetric fask. Tese solutions were stored at 4°C in darkness. By serially diluting solutions with 1 : 1 NaOH : ethanol, more diluted solutions were prepared. Tese solutions were stored at 4°C in darkness. By serially diluting solutions with 1 : 1 NaOH : ethanol, more diluted solutions were prepared.
3.4. 1 : 1 NaOH : Ethanol Preparation. 0.2 N NaOH was prepared by dissolving 4 gm of NaOH in deionized water and was diluted in a 500 mL volumetric fask. Ten mixed with ethanol one by one to make the solvent mixture 1 : 1 NaOH : ethanol.

H-Point Standard Addition Method.
Following is the general approach for determining CAN and HCT in a binary combination. An aliquot of a solution containing 15 µg/mL CAN, and 15 µg/mL HCT was added to a 2 mL volumetric fask, which was then flled to the mark with deionized water.
Te solution was then allowed to stand for fve minutes at room temperature. Te absorbance of the solution at the specifed wavelengths was then measured by transferring a portion of the solution into a quartz cell. Standard additions of CAN ranging from 3 to 13 µg/mL were done on the synthetic sample, which included a variable ratio of CAN to HCT. Simultaneous determination of CAN and HCT was conducted using HPSAM at two selected wavelengths. Te wavelengths selected depend on the principle of HPSAM as mentioned above, as well as the absorbance for the analyte was diferent and constant for interference at selected wavelengths of 239 and 283 nm, as shown in Figure 3, where C H is the unknown analyte concentration of CAN, and A H is the analytical signal of interference HCT, was determined at 283 nm in the calibration curve of standard HCT with y � 0.0247x + 0.0297 regression equation and 0.9985 correlation coefcient.

Q-Absorption Ratio Method.
Te CAN and HCT in a binary mixture were determined by the following procedure. Te mixtures of standard solutions of the drugs were prepared with a 2 mL volumetric fask, in diferent concentration ratios in the range of 11-19 µg/mL by diluting the appropriate volume of a stock solution of each drug with deionized water, then the solution was transferred to a quartz cell to scan in the range of 200-400 nm. Te determination is carried out by Q-absorption ratios at two selected wavelengths. Te selection of wavelengths was carried out related to the principle of the Q-Absorption ratio method, where one of these wavelengths is the iso-absorptive point and the other one is the max of one of the drugs. After diferent wavelengths were tested, 273 nm was selected as λ max of HCT and 258 nm as the iso-absorptive point of CAN and HCT for applying the Q-absorption ratio procedure, as shown in Figure 3. HCT and CAN were determined at 273 and 258 nm with a Q-absorption ratio in the following equations: where C x and C y are the HCT, and CAN concentrations, respectively, A 1 andA 2 are the absorbances of the mixture at λ 1 and λ 2 , ax 1 , and ay 1 are absorptivities of HCT and CAN at 273 nm, and ax 2 and ay 2 are absorptivities of HCTand CAN at 258 nm.

Correction Absorbance
Method. Te following procedure was applied for the determination of HCT and CAN with the correction absorbance method. Te series standard solution was prepared by transferring the aliquot amounts of stock solution to a 2 mL volumetric fask and completed to the mark with deionized water. Te solution was then poured into the 1 cm quartz cell and scanned in the range of 200-400 nm. HCT, and CAN were determined by the correction absorbance method when the selected pair of wavelengths returned to the principle of the method as explained above. Te frst wavelength is 250 nm λ max of CAN, and the second one is 340 nm for direct determination of HCT and applying the correction absorbance equation for the removal of the absorbance of HCT at 250 nm. Finally, CAN was determined at the calibration curve of standard CAN with y � 0.0295x + 0.0648 regression equation and 0.9926 correlation coefcient, and HCT was determined at the calibration curve of standard HCT with y � 0.0054x + 0.0067 regression equation and 0.9979 correlation coefcient.

Linear Range
Te calibration curve was drawn for selected wavelengths related to the procedures of the techniques, namely, 239 and 283 nm for the HPSAM, 273 and 258 nm for the Qabsorption ratio method, and 250 and 340 nm for the correction absorbance method. As shown in Figures 4 and 5. Table 1 shows the linear range of drugs for all methods at all wavelengths. (20) and (21) provide the computations for the limit of detection (LOD) and limit of quantifcation (LOQ) for the H-point standard addition method and correction absorbance method.

H-Point Standard Addition Method and Correction Absorbance Method. Equations
where X b represents the concentration of fve replications (n � 5) and SD b is the standard deviation of the blank [13].
where σ is the standard deviation of the blank and S is the slope of the calibration curve. Table 6 shows the LOD and LOQ for those drugs [14].
Journal of Analytical Methods in Chemistry 5

Accuracy and Precision
By using the methods for assessing various ratios of the drug combination, the suggested methods' accuracy was evaluated, by preparing the following combinations for CAN and HCT, respectively ( Tables 7-9.
Additionally, the accuracy of the suggested procedures was examined by measuring the drug concentrations in a 15 g/mL combination fve times in a row. For the H-point standard addition method, Q-absorption ratio method, and correction absorbance technique, respectively, the accuracy of each approach is shown as a percentage of the relative standard deviation in Tables 10-12.

Interferences
Te tolerance limit was described as the concentration of the added species interference (such as lactose monohydrate, magnesium stearate, stearic acid, polyethylene glycol, starch, sucrose, Na 2 CO 3 , and NaHCO 3 ) causing an error of more than ±5% on the analytical signal, and then, before the beginning of the process with the analysis of the compound under study in pharmaceutical dosage forms, it was conducted to discover its efect. Samples were prepared by mixing known quantities of the investigated drugs with diferent quantities of mutual excipients. Te result shows magnesium stearate, stearic acid, and Na 2 CO 3 were insoluble in 1 : 1 NaOH : ethanol, also, the result of the methods in the determination of drug in the presence of soluble interferences shows a good percentage recovered shows that there is no interference from these supplement additives with the methods applied. Te results obtained in Tables 13-15 reveal a great degree of accuracy for all methods.

Application
Tese procedures and methods have been used in pharmaceutical formulations (tabs) and synthetic lab mixtures to assess the analytical applicability of the intended methodologies. Tese methods are frequently used for simultaneous determination. All of our methods' results were contrasted with the HPLC result, which served as the benchmark. Te HPSAM was used for the simultaneous estimation of HCT and CAN in the synthetic mixture and pharmaceutical formulation. Te results are listed in Table 16. Te Q-analysis technique procedure was efectively used to determine the amounts of HCT and CAN by being repeated three times within the synthetic lab mixture and pharmaceutical formulation, as shown in. Table 17, the results of the correction absorbance technique for simultaneous determination of HCT and CAN in the pharmaceutical formulation, are shown in Table 18. Te value of the real samples was calculated for each of the tablets by the HPLC method. According to the tables, the methods presented in this work are sufciently general to be applied to fgure out the HCT and CAN of a real sample of tablets simultaneously.

Results and Discussion
Based on the results, we made the following observations. Experimental evaluation of the HPSAM, Q-absorption ratio, and correction absorption methods in this work        14.87 −0.9 250 * 1 calculated using the regression equation of Y � 0.0054x + 0.0067 and the HCT calibration curve at 340 nm. * 2 calculated using the regression equation of Y � 0.0295x + 0.0648 and the HCT calibration curve at 250 nm. led us to consider these methods efective for the simultaneous determination of HCT and CAN. Our results that were presented in this work are generally sufcient to be applied to real samples in pharmaceutical formulations. Te efectiveness of the proposed methods has been substantiated in Table 19. Te spectra of the binary mixture that was prepared in accordance with Section 3.3 are shown in Figure 1. As can be seen, the samples' analytes and interference spectra exhibit signifcant wavelength range overlap. Following the testing of numerous wavelength pairings for the use of HPSAM, Qabsorption ratio, and correction absorbance methods, HCT and CAN function in this technique as analyte and interference. Te fndings indicate that 239 and 283 nm are best for determining CAN and HCT by HPSAM, while 273 and 258 nm are best for the Q-absorption ratio, fnally, 250 and 340 nm were chosen for the correction absorbance method, because there is no interference at these wavelengths. In light of this, we proposed new methods: HPSAM, Q-absorption ratio, and absorbance correction to simultaneously determine HCT and CAN. We can come up with some hypotheses regarding the reproducibility of the procedure based on the outcomes of the fve separate measurements. Te proposed methods were validated according to the ICH recommendations [44]. Tese methods were utilized successfully to estimate the quantities of candesartan, cilexetil, and hydrochlorothiazide in commercially available tablet formulations containing candesartan cilexetil and hydrochlorothiazide. Tree tablet formulations were used as samples in this study, one of these samples is Candex which contains in its composition 12.07 mg per tablet of HCT and 15.89 mg per tablet of CAN as analyzed by the standard method using HPLC. Using the H-point standard addition method, the amount of HCT was found to be 12.08 mg and the amount     of CAN was found to be 15.79 mg, which correspond to 99.917 percent and 100.64 percent of the w/w label claim, respectively. Using the Q-absorption ratio method, the amount of HCT was found to be 12.1 mg and the amount of CAN was found to be 15.9 mg, which corresponds to 100.2 percent and 100.06, respectively. Te last method used in this study is the correction absorbance method, and the amount of HCT found in the tablet formulation was 12.56 mg for HCT and 16.21 mg for CAN, which corresponds to 104.1 and 102 percent, respectively. For all medicines, recovery and error percentages were used to calculate accuracy. Te data comparison between our methods and the standard HPLC method is shown in Tables 16-18.

Conclusion
A brand-new, straightforward, quick, and sensitive approach is suggested for the analysis of two binary mixtures with overlapping spectra. Te process starts with the creation of absorbance ratio spectra, then moves on to measuring peakto-trough amplitudes. Te suggested methods have various advantages over conventional spectrophotometric methods for the resolution of binary mixtures, including the lack of a need for complex mathematical handling of the absorption data. In an ongoing study, straightforward and efective chemometric methods like H-point standard addition, qabsorption ratio, and correction absorbance methods were devised for the simultaneous measurement of    hydrochlorothiazide and candesartan in bulk and in the pharmaceutical dosage form. It was found that the validity of these methods could be demonstrated through the accurate and precise determination of drug combinations in a variety of laboratory-prepared mixtures and pharmaceutical tablets. Consequently, the methodology proposed here is suitable for routine quality control of these set mixtures.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te author declares that they have no conficts of interest.