INTRODUCTION:

The development of chemical analysis is always determined by conducting observational analysis. Therefore, the development of software based on knowledge of mathematical theory with a computerized system can be utilized as an integral part of performing chemical analysis. Thus, quantitative chemical analysis methods were also developed without requiring manual calculations to determine compounds¹. As new analytical techniques and technologies are developed through the revolution of computer technology, chemometrics has evolved as a result of the enormous changes in data obtained in chemistry^1-5.

HPLC^6-11 and UV spectrophotometry^12-18 has been used in various journals to analyze antihypertensive drugs containing hydrochlorothiazide, amlodipine, and valsartan. Either alone or in combination with other hypertension drugs^17-18.

But so far, the journals haven't published a determination simultaneous of Amlodipine, Valsartan, and Hydrochlorothiazide mixture with the UV spectrophotometry chemometric method. Different researchers have developed spectrophotometers and other spectroscopy techniques, including FTIR.The multicomponent analysis by spectrophotometry used chemometric techniques which are applied in the manufacture of quantitative calibration curves for adjacent spectral analysis considered very important in controlling the quality of drug component levels in a mixture of active ingredients in drug preparations^9,10-

Chemometrics is a subsidiary of mathematics which uses software that has been developed and widely used in various fields of pharmacy^9,10,11. This method is a combination of chemical data processing with mathematical and statistical calculations by using various mathematical models and software in order to provide good Analytical results.

Currently, chemometrics is the most developed subsidiary within various fields of pharmacy. Chemical data are the basis of chemometrics, and mathematical models are necessary to get information from chemical data, Hence, the cross-validation is required to make sure the result of the information is accurate. Experiment and observation are integral parts of this method, as they must be performed in such a manner. In the aspect of quantitative analytical chemistry, chemometric techniques have applied in spectroscopic methods such as UV-Visible spectrophotometry, Near infra red spectroscopy,and fluorescence spectroscopy, especially testing of mixtures of finished drug components in pharmaceutical preparations^9,16,17 One of the multivariate methods that is often used is Partial Least Squares because this method supports the calculation of the concentration of each drug component in the mixture. Validation with parameters RSMEC, PRESS, and R2^19-22. Combinations of hypertension drugs lower blood pressure more effectively than one hypertension drug alone. Combination therapy in one tablet not only reduces blood pressure but also offsets the side effects of one drug with another. Amlodipine, valsartan, and hydrochlorothiazide are a three-component drug combination used to treat high blood pressure^6,18.

USP 43 - NF 38 (2020) has provided assays for a mixture of Amlodipine (AML), Valsartan (VAL), and Hydrochlorothiazide (HCT) tablets by High-Pressure Liquid Chromatography (HPLC) is used acetonitrile and water as the mobile phase (50:50)⁵. Sharma, et al.. (2012) determined the levels of a mixture of AML, VAL, and HCT using a HPLC⁶. However, this method also has a fairly high cost because it uses a lot of solvents, so it is also necessary to use a practical method. With the development of software on a computer that is applied to a spectrophotometer^19-22. It can analyze multicomponent compounds with overlapping UV spectrophotometric^18,19. In most cases, UV spectrophotometry is combined with chemometric methods via the partial least squares calibration method ^19-22.

The researchers aimed to study the ultraviolet spectrophotometry method with chemometrics assisted by partial least squares calculation to determine the concentrations of a mixture of AML, VAL, and HCT in tablet preparations.

Experiment:

Equipment and Materials:

UV-1800 Shimadzu spectrophotometer with computer and software UV. Probe version 2.42 and Minitab version 18.2.4.4, Raw material of AML, VAL, and HCT. Tablets of Exforge HCT^®, Methanol p.a (E.Merck).

Preparation of working solution:

Weighed each 50mg of AML, Val, and HCT carefully and transfer into a 50ml volumetric flask, fill with methanol to the marked line, and obtain a solution with a concentration of 1000ug/ml. Five ml of each solution was transferred into a 50ml graduate flask, diluted with methanol to the marked line, and then homogenized to obtain solutions with a concentration of 100ug/ml^19,21.

Measurement the absorbance matrix of raw material:

Pipette 100ug/ml of AML and 100ug/ml of VAL, and 60 ug/ml ml of HCT. Transfer each into a 10ml graduate flask, and add methanol. Each absorbance was measured at a wavelength between 200 and 400nm. Additionally, each solution was combined with 100ug/ml of AML solution, 100ug/ml ml of VAL solution, and 60ug/ml of HCT solution. Three solutions were mixed together in a 10ml volumetric flask, filled with methanol up to the marked line, and shaken until homogeneous. Also, the absorbance was measured between 200 - 400nm¹⁶.

Measurement the validation parameter:

Method validation involves demonstrating that the method consistently produces the expected results. A validation parameter are the accuracy test using the standard addition method,precision, qualitative limit, and quantitative limit^23-24. Validation was performed using parameters:

CF- CA SD

%Accuracy =-----------x 100 ; %Precision = ---------

CA* X

3x SD 10x SD

LOD = --------------- ; LOQ = -------------

Slope Slope

Note:

CF =Number of analytes measured

CA = Amount of analyte in the sample

CA*= Default number added

SD = Standard deviation

X = The data have been average

Absorbance matrix for Partial Least Squares Value:

As part of the chemometric methods, the absorption spectra of 10.0ug/ml AML, 10.0ug/ml VAL, and 6.0 ug/ml HCT were calculated with UV probe 2.42 software for absorbance values at 220-310nm, and then with Minitab 18.2.4.4 software to obtain the absorbance matrix of partial least squares value^19-20

Calibration curve in partial least squares calculation:

Calibration curves constructed using 35 sets of AML (6.0-14.0ug/ml); VAL (6.0-14.0ug/ml), and HCT (4.0-12.0ug/ml) were used, with a wavelength for 230-300 nmAML, 220-290nm for VAL, and 230-300nm for HCT with interval of 2 nm. Then the wavelength range that gives the best linearity relationship between the partial least squares value and the concentration is selected, as indicated by the correlation coefficient value ≤ 1 and a small error value. The selection of 35 set ranges based on the absorbance obtained complies with the lambert-beer law (0.2-0.8nm). Minitab 18.2.4.4 was used to process the partial least squares data, and then partial least squares regression was performed^15,20.

Cross Validation Leave-one-out:

The partial least squares calibration model was selected using the stat option, followed by regression partial least squares. There was a concentration variable for AML in the response section and the absorbance variable in the model column. Pressing the options button further determines the additional leave-one-one process.

The actual and calculated value is obtained and then copied and pasted into the Minitab 18.2.4.4 worksheet software. The value of R2 and RMSEC are obtained. VAL and HCT were treated the same way during the validation process. The PRESS value, R2 value, and RMSECV value determine a calibration model's accuracy and precision^19-22.

Determination of AML, VAL, and HCT levels in Tablets:

Tablet samples for assays contain AML 10mg, VAL 160mg, and HCT 12.5mg. Then, carefully measure the amount of a solution equivalent to 50mg of VAl, pour it into a 50ml volumetric flask, and add methanol solvent to the marked line. Then 2.5ml of solution is pipetted into a 50ml graduate flask and diluted with methanol solvent to the marked line. One ml solution was pipetted into a 100ml graduate flask and diluted with methanol solvent to the marked line. Measured absorption at a wavelength of 200-400nm^19,20..

As the absorption spectrum of the sample obtained combines VAL, AML, and HCT, it is necessary to separate the absorption of each drug using a divisor. The selection of divisor concentration is based on the orientation results which then the spectrum is processed by the UV probe application. Partial Least Squares calculated the chemometric value at the wavelength of 230-300nm for AML, 220- 290nm for VAL, and 230-300nm for HCT with an interval of 2nm, data processing was performed on Minitab software version 16.2.4.4. Then, the levels of each substance will be obtained by using the partial least squares regression equation on the standard ^19-22

Tablet data calculation by Chemometric model:

The sample concentration with coefficients from each model for AML, VAL and HCT compounds can be calculated by the formula^23-24:

𝑋 = 𝑡1𝑝1 + 𝑡2𝑝2 + . . + 𝑡𝑠𝑝𝑠 + 𝜀

Note:

X = Sample calculated concentration

Ts = Coefficient of calibration model Ps = Absorbance of each sample measurement

𝜀 = Correction of errors that may occur in the partial least squares calibration model.

RESULTS AND DISCUSSION:

Absorbance matrix:

UV spectrophotometric measurements were performed on the absorbance matrix in methanol for 10.0 ug/ml of AML, 10.0ug/ml VAL, and 6.0ug/ml HCT at wavelengths 200-400nm, respectively. Absorbance matrix of AML, VAL, and HCT and it mixture can be seen in Fig.1.

wavelength

Fig.1: The overlapping spectrum of a. AML, b VAL c, HCT and d. mixture on UV Spectrphotometric

The overlapped spectra of a single drug and mixtures of drugs do not differ (Figure 1). The absorption spectrum of AML, VAL, and HCT in methanol (Figure 1) have wavelengths of 236nm, 255nm, and 270nm, respectively. These results differ from those for AML in acidic solution at peak 239nm, as well as alkaline solution at peak 238nm, VAL in acid solution at peak 203 and 248nm, and HCT in acid solution at peak 272 nm^25-26.

Therefore, conventional spectrophotometry cannot determine it, so it is necessary to make another determination for this mixture. Kamal et al, 2016, state that chemometrics with partial least squares calculations can be employed as long as partial least squares calibration is feasible, and the constituents of the mixture can be accurately determined. Thus, the spectrum of the drug mixture can be quantified and selectively analyzed, resulting in the absorbance of each drug⁵.

Calibration curves construction and Validation of multivariate calibration models:

The calibration curve produced by the construction of the spectrum of a (6.0-14.0)ug/ml AML at 230-300nm; a concentration of (6.0-14.0)ug/ml VAL at 220-290nm, and a concentration of (4.0-12.0)ug/ml HCT at 230-290 nm, and 35 points for each component were measured. AML, VAL, and HCT absorbance data were cross-validated with 35 points from the selected wavelength range using Minitab 18.2.4.4 chemometric method with partial least squares.

The select cross-validation: leave one out later on the Minitab worksheet 18.2.4.4 can calculate the PRESS value, the R2, and RMSECV values for each component. The calibration curve of AML, VAL, and HCT are shown in Figure 2- Figure 4.

Fig.2:The actual response and calculated response for basic calibration curve of AML

Fig.3: The actual response and calculated response for basic calibration curve of VAL

Fig.4:The actual response and calculated response for basic calibration curve of HCT

Table 1: Partial least squares validation calculation without cross-validation

No	Parameter	Component
No	Parameter	AML	VAL	HCT
1.	Regresion Equation	Y=22.2611 X -0.0380	Y=22.236 8X- 0.0414	Y =13.236 8X-0.0238
2.	PRESS	0.0260	0.5085	0.1764
3.	R2	0.9997	0.9995	0.9999
4.	RMSECV	0.0214	0.3018	0.0018

Based on the results at Figure 2- Figure 4 and Table 1 above, it’s shown that all of the actual response and calculated response were linear, and the value of the regression equation and coefficient of determination (R2) produced is R> 0.9, and the low the RMSECV value, the better the ability of the model to predict^19-22.. It is possible to use chemometric methods based on partial least squares calculations to study AML, VAL, and HCT.

Validation of the method:

Validation parameters included accuracy, precision, linearity, detection limit, and quantitative limit, the accuracy test based on the percentage of recovery determined by the standard addition method (Harmita, 2004). The recovery test spectrum for AML, VAL, and HCT can be seen in table 2 below.

Table 2:Parameter testing result from validation method of AML, VAL, and HCT

S. No.	Parameter	AML	VAL	HCT
1.	Linierity	0.9998	0.9997	0.9995
2.	Accuracy (%)	99.48	99.52	99.60
3.	Precision (%)	0.40	1.17	0.49
4.	LOD (μg/ml)	0.35	0.39	0.47
5.	LOQ (μg/ml)	1.18	1.30	11.56

The data from the measurement results on Table 2 is the validation parameter requirements. These results suggest that the mixed analysis method of AML, VAL, and HCT with ultraviolet spectrophotometry chemometric method using partial least squares calculations meets the validation requirements^21-22. This means that a method has met the validity requirements and can be used to determine a tablets mixture of AML; VAL, and HCT.

Analysis of AML, VAL, and HCT in Tablet:

The spectra of the absorption spectrum of the VAL, AML, and HCT mixture to obtain chemometric values calculated by Partial Least Squares and data processing was carried out on Minitab software version 18.2.4.4. Average concentrations where the absorbance obtained comply with LambertBeer law (absorption range 0.2-0.8) were calculated. The levels of AML, VAL, and HCT in Tablet can be seen in the table 3 below.

Table 3: Chemometric values calculations by PLS of AML, VAL, and HCT in tablets

No	Compo- nent	Actual Value (mg)	% Calculate Value ((Mean ± SD)	Require ments (%)
1.	AML	10.0	(99.59 ± 0.28)	90 ≤ X ≥ 110
2.	VAL	160.0	(101.92 ± 0.44)	90 ≤ X ≥ 110
3.	HCT	12.5	(103.60 ± 0,34)	95 ≤ X≥ 105

According to the calculation by partial least squares on the tablet samples that have been summarized in Table 3 above; the results obtained for AML range levels are (99.59±0.28)%, VAL range levels are (101.99±0.44)%, and HCT range levels are (103.60 ± 0.34)%. In addition, these levels meet USP 43/NF 38 (2020)⁶. Thus, the chemometric spectrophotometric method with partial least squares calculations can assay mixtures containing AML, VAL, and HCT to meet the validation parameter and cross-validation requirements for partial least squares calculations.

CONCLUSION:

That the partial least squares calculations used for chemometrics in the ultraviolet spectrophotometric method to determine the levels of AML, VAL, and HCT in tablet preparations meet the requirements of the USP 43-NF 38 (2020)⁶, and cross-validation requirements for PRESS, R2, and RMSEC.

ACKNOWLEDGEMENT:

The authors and the team would like to thank the Dean of the Faculty of Pharmacy, the University of North Sumatra for allowing us to use the Research Laboratory of the Faculty of Pharmacy for this research.

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Received on 28.08.2022 Modified on 13.12.2022

Research J. Pharm. and Tech 2023; 16(9):4314-4318.

DOI: 10.52711/0974-360X.2023.00706