Nghiên cứu phát triển phương pháp chemometric để xác định đồng thời các chất có phổ hấp thụ phân tử xen phủ nhau và áp dụng trontom tat LA trang (tieng anh) (ban chinh thuc)

HUE UNIVERSITY HUE UNIVERSITY OF SCIENCES NGUYEN THI QUYNH TRANG RESEARCH ON THE DEVELOPMENT OF A CHEMOMETRIC METHOD FOR THE SIMULTANEOUS DETERMINATION OF MOLECULAR ABSORPTION SPECTRUM OVERLAPPING AND APPLICATION IN DRUG ANALYSIS THE ABSTRACT OF DOCTORAL DISSERTATION HUE - 2018 HUE UNIVERSITY HUE UNIVERSITY OF SCIENCES NGUYEN THI QUYNH TRANG RESEARCH ON THE DEVELOPMENT OF A CHEMOMETRIC METHOD FOR THE SIMULTANEOUS DETERMINATION OF MOLECULAR ABSORPTION SPECTRUM OVERLAPPING AND APPLICATION IN DRUG ANALYSIS MAJOR: ANALYTICAL CHEMISTRY CODE: 62 44 01 18 THE ABSTRACT OF DOCTORAL DISSERTATION SCIENTIFIC SUPERVISORS: Assoc.Prof.Dr TRAN THUC BINH Assoc.Prof.Dr NGO VAN TU HUE - 2018 The abstract of doctoral dissertation Nguyen Thi Quynh Trang INTRODUCTION The term chemometric was first introduced in 1972 by Svante Wold (Swede) and Bruce R Kowalski (American) The establishment of the Chemometric Association in 1974 provided the first definition of chemometrics, the application of mathematical, statistical, graphical methods….for experimental planning, optimize the chemical information extracted from the data set and provide the most useful information from the original data set Chemometric is widely used in fields such as environmental chemistry, organic chemistry, biochemistry, theoretical chemistry, statistics in chemistry and has especially established an important position in analytical chemistry Analytical chemistry is an effective tool in the fields of science and technology, such as chemistry, biology, agronomy, medicine, food especially in the pharmaceutical industry Chemometric methods have been used by researchers at domestic and foreign for many years to concurrently analyze a mixture of substances in a variety of subjects, including pharmaceuticals Studies have shown that the most commonly used chemometric methods are the partial least squares (PLS) method, the Polimerase chain reaction method (PCR), classic least squares method (CLS), artificial neural network method (ANN), derivative method, kalman filter method Each method has its own advantages and disadvantages The CLS method can use the entire spectral data to set up the m equations in n unknowns (m> n) The transformation matrix based on the principle of least squares will produce the results of the error satisfying the requirements However, if there is a lot of noise (or spectral error) in the spectrophotometer and/or when the constituents interact with each other, it produces an optical effect that changes the absorbance of each constituent, this method can not eliminate the noise leading to the analysis results have big errors The ANN method has the disadvantage that the training time is long and it requires a lot of different algorithms, so that when building an analytical model, it requires testing different models to find the optimal network The abstract of doctoral dissertation Nguyen Thi Quynh Trang structure Derivative spectrophotometric method does not apply when the sample contains many constituents with absorbing optical spectrum overlapping or similar, since it is difficult to select an appropriate wavelength to determine a particular constituent, or their derivative spectrophotometric still have the same maximum absorption Kalman filter method can eliminate most of the noise and therefore minimize errors, but the disadvantage of this method is that the initial values for the filter must be selected that means must choose the appropriate initial value of the content of analytes in their mixtures and the associated error (expressed by the variance) If the initial values (concentration and variance) not match, the end result is a large error In the world there have been some studies applying Kalman filter method to chemometric to simultaneously determine mixtures of or substances in the pharmaceutical However, these studies neither offer a suitable initial value nor cover initial values and are therefore difficult to apply to analytical laboratories In Viet Nam, Mai Xuan Truong has studied the application of Kalman filter method to simultaneously determine the vitamins in pharmaceuticals, rare earth elements However, the author did not introduce how to choose the initial values and thus limited the possibility of applying the proposed method in practice As a result of these issues, it is clear that the development studies of chemometric-photometric method combined with the use of Kalman filter methods is very necessary, especially to simultaneously determine mixtures of substances difficult to analyze that containing optical absorption spectrophotometer overlapping in various sample objects, including pharmaceutical samples However, the challenge is to find a suitable solution to select the initial value for the Kalman filter to produce accurate analysis results (repeatability and accuracy) or acceptable error At the same time, the analytical process needs to be developed that based on the chemometricphotometric method combined with the Kalman filter so that can be applied conveniently in the field of pharmaceutical testing in our country For these reasons, the topic "Research on The abstract of doctoral dissertation Nguyen Thi Quynh Trang the development of chemometric method for the simultaneous determination of molecular absorption spectrum overlapping and application in drug” was carried out for the following these purposes: i) Developed a chemometric-photometric analysis process in combination with Kalman filter method to simultaneously analyze mixtures of and substances with molecular absorption spectra overlapping in pharmaceutical samples; ii) Apply the process has been built to simultanneously analyze mixtures and substances in some pharmaceuticals are on the market Vietnam Master thesis structure The thesis consists of 184 pages, with 50 tables and 14 figures, of which: - Table of contents, list of abbreviations, tables and figures: 09 pages - Introduction: 04 pages - Chapter 1: Overview 43 pages - Chapter 2: Content and Research Methods 16 pages - Chapter 3: Results and discussion 67 pages - Conclusion 02 pages - The list of published research results: 01 page - References: 15 pages, with 127 references CONTENT THESIS CHAPTER LITERATURE REVIEW - The Bughe-Lambe-Bia law and Optical properties of optical absorption + The Bughe – Lambe - Bia + Optical properties of optical absorption - Some UV-VIS spectrophotometric methods combined with chemometric simultaneously determine the components with absorption spectrum overlapping each other + Vierordt method + Derivative spectrophotometric method + Full-partial method (least squares method) + The least squares method The abstract of doctoral dissertation Nguyen Thi Quynh Trang + Principal Components Regression method + Artificial neural network method + Kalman Filter Method - Overview of multi-component pharmaceuticals and research active ingredients + Profile of the development of multi-component pharmaceuticals + Overview of telmisartan (TEL), hydrochlorothiazide (HYD) + Overview of paracetamol (PAR) and caffeine (CAF) + Overview of paracetamol (PAR) and ibuprofen (IB) + Overview of amlodipine besylate (AML), hydrochlorothiazide (HYD), valsartan (VAL) CHAPTER RESEARCH SUBJECTS AND METHODOLOGY 2.1 CONTENT To achieve the objective of the thesis is to contribute to the development of chemometric-photometric method using the Kalman filtering algorithm to apply in pharmaceutical analysis, the research contents include: Study to find the suitable solution to select the initial value (concentration value and initial variance) for the Kalman filter for using the chemometric - photometric method simultaneous determine of molecular absorption spectrum overlapping (mixture contains substances and mixture contains substances) Construct a computer program based on the Kalman filter algorithm on Microsoft-Excel 2016 software with the Visual Basic for Applications programming language, it is possible to quickly calculates of the concentration of photocatalytic absorption spectra overlapped in the study system (containing or substances simultaneously) Verification of the reliability of the analytical method Chemometric-photometric method using the Kalman filter algorithm (calculated by software program has been built): The abstract of doctoral dissertation Nguyen Thi Quynh Trang Comparison of analytical methods with the chemometric- Other photometry (least squares using full spectrum and diffusion method) when analyzing laboratory standard samples (containing or analyzes) Develop a chemometric-photometric analysis using the Kalman filter algorithm (calculated by software program has been built) Apply the analysis process has been built - analysis of multi-component pharmaceutical samples (containing or ingredients) are currently in circulation in Vietnam 2.2 METHODOLOGY 2.2.1 Kalman filter method and calculation program Based on the theoretical basis, the Kalman filter method and the calculation program are performed according to the following steps (Figure 2.1): i) Record the spectrum of the analytical solution (laboratory standard solution) and the mixture of analytes, obtaining the spectral data set (optical absorption at selected wavelength k) in the form of a file txt tail (number of wavelengths selected depending on the characteristics of the components in the study); ii) Enter the mono-particle and compound material data files into a computer software program (programmed in Microsoft-Excel 2016 software) to calculate the ε (molecular absorption) values of the monomers; iii) Run the Kalman filter: - Give the initial initial value, including the first estimate of the Cest(0) and the covariance of the error Pest(0) (study content (1) will give the initial value); - Extrapolation of concentration status: C pri( k ) = Cest ( k −1) (2.1) - Extrapolation of the covariance of the error: (2.2) Ppri( k ) = Pest ( k −1) - Calculation Kalman Loop: The abstract of doctoral dissertation Nguyen Thi Quynh Trang ( K(k ) = Ppri(k )εT (k ) ε(k ) Ppri(k)εT (k ) + R(k ) −1 ) (2.3) - Updated status estimate: ( Cest (k ) = C pri( k ) + K(k ) A( k ) − ε(k )C pri( k ) ) (2.4) - Update the covariance of the error: Pest ( k ) =  INV − ε( k ) K ( k )  Ppri ( k )   (2.5) The above calculation steps are performed from the first wavelength to the last wavelength Finally, the calculation program will produce the result: the concentration of each constituent in the system and the covariance of the error This variance is usually the smallest at the last wavelength 2.2.2 Minimum squared method using simulan software [2] Step Prepare standard solutions for each constituent and their mixtures Step 2: Record the absorption spectra of the standard solution to calculate the absorption coefficient ε of the constituents: ε= (εij )mxn Step 3: Record the optical absorption spectra of the mixed solution, enter the optical absorption matrix measured A = (Ai1)mx1 Step 4: Solve the system of m equations by the least squares method: A = ε C to find the concentration of C 2.2.3 Derivative spectrophotometric method Step Prepare standard solutions for each constituent and their mixtures Step 2: Record the optical absorption spectra and the spectrum, find the appropriate wavelength at which the derivative value of a substance to be analyzed is different from zero or maximum, and the other derivative value is equal to Step 3: After determining the measured wavelength at a certain derivative, proceed to quantify the substances by the benchmark method or add standard 2.2.4 Computer programming method The abstract of doctoral dissertation Nguyen Thi Quynh Trang - Calculations to determine the concentration of substances by Kalman filter method is quite complex, so need to program on the computer to calculate fast and convenient for users; - Select open source software is Microsoft-Excel to not infringe copyright; - Select the language and the tool Visual basic for application; 2.2.5 Data processing method Application of Microsoft-Excel 2016 software with Data Analysis tool to process experimental data: Calculation of statistical data (arithmetic mean, standard deviation, RSD); Comparison of two repetitions (or two variances), using F (Ftest); Comparing two mean values, using t-test; Compare two methods, using paired-t-test CHAPTER RESULTS AND DISCUSSION 3.1 CHOOSE THE INITIAL VALUE 3.1.1 Select a random initial value In this way, selecting a random initial value can select any P C value for the concentration est (0) and variance est (0) [27], [112] For a mixture containing or substances (a mixture of laboratory standard reagents), the initial values for each substance were randomly selected at a concentration of Cest (0) = 0,3 µg/mL and variance Pest (0) = Table 3.1 Results of determination of TEL and HYD concentration in Kalman method with with random selection of initial value (*) Mixture Co (µg/mL) TEL C (µg/mL) RE (%) Co HYD (µg/mL) H1 H2 H3 H4 H5 H6 H7 H8 H9 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 0,30 0,30 0,30 0,30 0,30 0,30 0,30 0,30 0,30 -70 -85 -90 -93 -94 -95 -96 -96 -97 9,00 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 The abstract of doctoral dissertation C (µg/mL) RE(%) (*) Nguyen Thi Quynh Trang 0,30 0,30 0,30 0,30 0,30 0,30 0,30 0,30 0,30 -97 -96 -96 -95 -94 -93 -90 -85 -70 Co: Concentration in standard mixed solution; C: Determined concentration Table 3.1 shows that with different concentration ratios, between the concentration of the standard solution and the concentration determined to be relatively high RE% error (in the range of 69.7% - 96.7%) The concentration values determined in all mixtures are equal to the initial concentration (0.3 µg / mL) Table 3.2 Results of determination of AML, HYD and VAL concentrations in Kalman method with random selection of initial value (*) AML HYD VAL (*) Mixture Co (µg/mL) C (µg/mL) RE (%) Co (µg/mL) C (µg/mL) RE (%) Co (µg/mL) C (µg/mL) RE (%) H1 0,250 0,300 20 0,325 0,307 -6 4,00 0,301 -93 H2 0,50 0,300 -40 0,65 0,304 -53 8,00 0,300 -97 H3 1,00 0,300 -70 1,30 0,302 -77 16,00 0,300 -98 H4 5,00 0,304 -94 5,00 0,299 -94 5,00 0,299 -94 Co: Concentration in standard mixed solution; C: Determined concentration Table 3.2 shows that with different concentration ratios, the concentration of the standard solution and the concentration determined were very high (-5.5% - 98.1%) The lowest RE value (-5.5%) corresponds to the standard concentration of 0.325 (close to the initial concentration x = 0.3) The higher the initial concentration, the greater the RE % value Thus, with the results of the tests in Table 3.1 and Table 3.2, it can be seen that the initial method of selecting the concentration value and the random variance are incomplete, the calculated results still have a relatively large error 3.1.2 Select the assumed initial value In this study, we investigated a different set of assumed initial value in comparison with previous studies (for aquation of or substances) The abstract of doctoral dissertation HYD (*) RE (%) Co (µg/mL) C (µg/mL) RE (%) -70,0 -84,9 -89,8 9,00 8,00 7,00 0,30 0,31 0,31 -96,6 -96,2 -95,6 Nguyen Thi Quynh Trang -92,3 6,00 0,31 -94,8 -93,5 5,00 0,31 -93,7 -94,2 -94,6 -94,7 -94,6 4,00 3,00 2,00 1,00 0,31 0,31 0,31 0,30 -92,2 -89,7 -84,7 -69,7 Co: Concentration in standard mixed solution; C: Determined concentration The results in Table 3.3 and 3.4 show that: - According to option 1, the Kalman method gives reliable results on the concentration of substances in the mixture with the error of RE 0.30) However, based on p (statistically significant) values, it can be observed that the Kalman method is closer to the results of the HPLC method (p = 0.55 - 0.96) than With the BPTT method (p = 0.38 - 0.66) or in other words, the Kalman method achieves better accuracy than the BPTT method (when compared to the HPLC method) 19 Table 3.40 The results confirm the accuracy of the method when analyzing the actual sample of Exforge (*) AML Sample Method Ct Cx (µg/mL) (µg/mL) HYD Rev (%) Ct (µg/mL) Cx (µg/mL) 0,965 1,168 1,200 94,0 0,30 1,451 Sample 1,415 90,0 0,60 1,710 B1 0,967 1,171 BPTT 1,202 94,0 0,30 1,457 1,418 90,2 0,60 1,719 0,980 1,186 Kalman 1,214 93,6 0,30 1,470 Sample 1,450 94,0 0,60 1,759 B2 0,981 1,189 BPTT 1,217 94,4 0,30 1,474 1,454 94,6 0,60 1,762 0,937 1,134 Kalman 1,171 93,6 0,30 1,416 Sample 1,397 92,0 0,60 1,698 B3 0,939 1,137 BPTT 1,173 93,6 0,30 1,422 1,400 92,2 0,60 1,697 RevTB (%)-Kalman 92,9 94,0 RevTB (%)-BPTT 93,2 94,2 (*) Co: Concentration in the sample (µg / mL) (AML: HYD: VAL is 1.0: 1.25: 16) Kalman 0,25 0,50 0,25 0,50 0,25 0,50 0,25 0,50 0,25 0,50 0,25 0,50 20 VAL Rev (%) 94,3 90,3 95,3 91,3 94,7 95,5 95,0 95,5 95,0 94,5 95,0 93,3 Ct (µg/mL) Cx (µg/mL) 4,0 8,0 4,0 8,0 4,0 8,0 4,0 8,0 4,0 8,0 4,0 8,0 16,997 21,112 24,876 17,086 21,251 25,067 17,249 21,363 25,497 17,340 21,505 25,697 16,506 20,603 24,567 16,589 20,736 24,754 101,8 103,0 Rev (%) 102,9 98,5 104,1 99,8 102,9 103,1 104,2 104,5 102,4 100,8 103,7 102,1 Tóm tắt Luận án Tiến sĩ Nguyễn Thị Quỳnh Trang Compared to the HPLC method: Table 3.41 Comparison of chemometric methods with HPLC method for determining the content of AML, HYD and VAL in Exforge HCT(*) analytical substance AML HYD Statistics xi (mg/tablet) TB (mg/tablet) S (mg/tablet) Fexp/ F(0,05;2;2) Sp Texp/ t(0,05; f) P xi (mg/tablet) TB (mg/tablet) S (mg/tablet) Fexp/ F(0,05;2;2) Sp texp/ t(0,05; f) P xi (mg/tablet) VAL (*) TB (mg/tablet) S (mg/tablet) Fexp/ F(0,05;2;2) Sp texp/ t(0,05; f) P Analytical methods Kalman BPTT HPLC 9,65/9,80/9,37 9,67/9,81/9,39 9,54/9,41/9,59 9,61 9,62 9,51 0,22 0,21 0,09 5,30/19 5,30/19 0,16 0,16 0,53/4,3 0,63/4,3 0,65 0,59 11,68/11,86/11,34 11,71/11,89/11,37 11,72/11,76/11,41 11,66 11,66 11,63 0,26 0,26 0,19 1,9/19 1,9/19 0,34 0,34 -0,06/4,3 0,51/4,3 0,96 0,66 169,97/172,49/ 170,86/173,40/ 166,35/168,81/ 165,06 165,89 167,82 169,17 167,66 3,78 3,82 1,24 9,32/19 9,5/9 0,10 0,10 0,71/4,30 1,11/4,30 0,55 0,38 The results of the analysis are repeated (i = 1-3); Fexp = Variance of the Kalman method (or BPTT)/ Variance of the HPLC method; F(0,05;2;2): The critical value of F is 0.05 and the degrees of freedom of the two numerator and denominator variants; Sp: pooled variance, calculated from two covariates of two methods when two covariates of the two methods are the same (ie when Ftính< F(0,05;2;2)); t (0.05; f = 4): The critical value of t is statistically significant p = 0.05 and the degree of freedom f = CONCLUSION From the results of theoretical and empirical research, the thesis has the following main conclusions: 21 Tóm tắt Luận án Tiến sĩ Nguyễn Thị Quỳnh Trang 1) Based on the survey of options for initial values for the Kalman filter algorithm, a new solution has been found for the first time - selecting the approximate initial value of the concentration (by means of the quadratic least squares) and variance (calculated by the Horwitz equation) This new solution allows for the convenient application of the chemometric-photometric method using the Kalman filter algorithm (Kalman method) to simultaneously determine two or three substances with an opaque absorption spectrophotometer in their mixture 2) Kalman method test results for three standard solutions (two solutions containing each) and a mixture of three substances (molecular absorption overlapping) showed that when the measurement of optical absorption has a significant error (or large measurement noise), especially for a mixture containing three substances, the Kalman method is less error-prone and has a better repeatability than the least squares method using the full spectrum 3) It was first established the process of analyzing concurrent photocatalytic absorption spectrometry in multi-component pharmaceutical formulations containing two or three active ingredients by the Kalman method On the other hand, a computer program that uses the Visual Basic for Applications programming language is included in the Microsoft software - Excel 2016, which is included in the analysis and thus allows for quick and convenient calculations when applied Practical testing of pharmaceuticals in our laboratories The process is not only simpler to implement, but also reduces the cost of analysis compared to the standard method of High Performance Liquid Chromatography (HPLC) (4) Correctness and repeatability of the analytical process (or methodology) was examined when analyzing drug samples containing two or three active subtances (active substances with molecular absorption overlapping): For drugs containing two active subtances, the method was well tolerated with recovery of 93% 102% and good repetition with RSD