SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION OF TELMISARTAN AND HYDROCHLOROTHIAZIDE IN PHARAMACEUTICAL PRODUCT BY LEAST-SQUARE METHOD USING FULL SPECTRA
Tran Thuc Binh1, Nguyen Thi Quynh Trang1,2, , Vo Thi Kim Truc1, Ngo Van Tu3
1Hue University of Sciences
2Sai Gon University
3Hue University of Education
ABSTRACT
A procedure for simultaneous determination of Telmisartan (TEL) and Hydrochlorothiazide (HYD) in pharmacy was established based on the full spectrum spectrophotometric method combined with classical least squares written by C++ language. The procedure proved reliable with good repeatability and recovery when real analyzing drug samples: RSDTEL(%) = 0.94; RevTEL% = 101.07; RSDHYD(%) = 0.57; RevHYD(%) = 103.74. The received results were suitable with HPLC method.
1. INTRODUCTION
(TEL) is chemically designated as 4'-[(1,4’-dimethyl-2'-propyl [2,6'-bi-1H-benzimidazol]-1'-yl) methyl] [1,1'- biphenyl]-2-carboxylic acid. It is an angiotensin II type I blocker and is used as an antihypertensive along with Hydrochlorothiazide. It is a thiazide diuretic which reduces the reabsorption of electrolytes from the renal tubules, thereby increasing the excretion of sodium and chloride ions and consequently of water. Chemically Hydrochlorothiazide (HYD) is 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7sulfonamide 1, 1-dioxide. The combination of Hydrochlorothiazide and Telmisartan is useful in treatment of mild to moderate hypertension [8].
The widespread use of these drugs in combination requires development of analytical methods for their simultaneous determination. Several analytical procedures have been proposed for determination of Telmisartan and Hydrochlorothiazide separately and in combination with other drugs. Until now, almost in our country have not any literatures concerning to simultaneous determination of TEL and HYD in pharmaceutical products. In foreign countries, many literatures have been publiced for their simultaneous determination mainly by HPLC [7], spectrophotometric method using Vierordt equation system [6], first derivative spectrum method [5],….
To our knowledge, simple and economical analytical method for simultaneous determination of Telmisartan and Hydrochlorothiazide has not been reported so far. In this paper, we present a simple, accurate, rapid and economic method for simultaneous estimation of Telmisartan and Hydrochlorothiazide in tablet formulation. We apply classical least square method (CLS) [1], [4] using full spectra with selfmade software named SIMULAN written by C++
language. The developed method were validated and found to be accurate, precise and reproducible.
2. EXPERIMENT
2.1. Instruments and chemicals 2.1.1. Instruments
A double beam UV/Vis spectrophotometer, V630 Jasco, Japan was employed with a pair of 1 cm quartz cells for all analytical work.
Double water distiller Fistreem Cyclon; glassware: volumetric flask, beaker, pipet, buret,...
2.1.2. Chemicals
Standard chemical TEL, 98,97%, HYD 98,10 %, Central pharmaceutical test Centre; Sodium hydroxide of analytical grade and double distilled water were used throughout the analysis.
Prepare standard solutions
Prepare standard TEL solution
- Prepare 1000 µg/ml TEL stock solution: Weigh exactly 0.10104 g TEL, put in 100 mL volumetric flask, dissolve by 0.1 M NaOH solution, shake carefully and add 0.1 M NaOH solution to the mark.
- From TEL stock solution, prepare 50 µg/mL TEL intermediate solution and 10 µg/mL and 8 µg/mL TEL working solutions.
Prepare standard HYD solution
- Prepare 1000 µg/ml HYD stock solution: Weigh exactly 0.1019 g HYD, put in 100 mL volumetric flask, dissolve by 0.1 M NaOH solution, shake carefully and add 0.1 M NaOH solution to the mark.
- From HYD stock solution, prepare 50 µg/mL HYD intermediate solution and 10 µg/mL and 8 µg/mL HYD working solutions.
2.2.Analytical object and method 2.2.1. Analytical object
Micardis(R) Plus tablets with content written on label: TEL 40 mg and HYD 12.5 mg, lot number: 505392, productive day: 26/06/2015, expiry day: 26/06/2018, serial number: VN-16587-13, producted at Boehringer Ingelheim Pharma GmbH & Co. KG Binger Str. 173 55216 Ingelheim am Rhein, Germany.
2.2.2. Analytical method
Classical least square method (CLS) using full spectra with selfmade software named SIMULAN written by C++
language were used to determine simultaneously TEL and HYD in mixture solutions.
The basic theory of CLS method is as follow:
Beer’s Law can be rewritten for solutions containing components: A = K.C, where, K is the matrix of absorptivity constants having m rows, n columns; A is a vector of spectral absorbances having m rows and 1 column; and C is the vector of constituent concentrations, having n rows, 1 column. Using matrix algebra, it is trivial for a computer to solve these equations and produce the K matrix in the above equation (the matrix of absorptivity coefficients).
Just by the nature of matrix algebra, the solution gives the best fit least squares line(s) to the data. Once the equation is solved for the K matrix, it can be used to predict concentrations of unknown samples. For those familiar with linear algebra, to solve for the K matrix requires computing the matrix equation: K= AC-1 where C-1 is the inverse of the constituent concentration matrix. Unfortunately, computing the inverse of a matrix requires that the matrix be square (having the same number of rows and columns). This does not mean that the above equation cannot be solved. An alternative to computing the true inverse of the C matrix is to compute its "pseudo-inverse", as follows: K = AC’(CC’)-1. Where C’ is the matrix transpose (pivot the matrix so that the rows become the columns) of the constituent concentrations matrix. Depend on matrix of absorbance A0, matrix of absorptivity constants K, the concentrations of components in sample are computed as follows:
C0 = A0K-1 = A0K’(KK’)-1 In which, K’: matrix transpose of K matrix; K-1: inverse matrix of K matrix.
Procedure for measurment and computing:
Step 1: Prepare standard solutions of TEL and HYD and their mixture solutions.
Step 2: Measure absorbance spectra in suitable wavelength area, save data files in the form *.txt or *.dat.
Step 3: Run SIMULAN software to compute concentration of TEL and HYD in mixture solutions and their relative error, RE%.
3. RUSULTS AND DISSCUSION
3.1. Investigate absorbance spectra of TEL and HYD and examine absorbance addition property of mixture solution on the full spectrum
Preparing standard solutions TEL 10 g/mL, HYD 10 g/mL, mixture solution containing TEL 10 g/mL and HYD 10
g/mL with 0.1M NaOH medium. Sanning spectra of solutions in the wavelength area from 240 t0 340 nm. The spectra of these solutions were showed in 1.
(nm)
240 260 280 300 320 340
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
( 4) ( 3)
( 2) ( 1)
A
( 1) HYD 10 ( 2) TEL 10
( 3) Theory HYD10 - TEL10 ( 4) Practice HYD10 - TEL10
Figure 3.1 shows that absorbance spectra of standard solutions of TEL (10 g/mL) and HYD (10 g/mL) in 0.1 M NaOH medium overlap severly in the wavelength area from 240 to 335 nm and have addition property.
3.2. Reliability Assessment of the method on laboratory samples
From standard solutions of TEL, HYD 50 g/mL, prepare solutions in 50 mL volumetic flasks: 8 g/mL TEL, HYD standard solutions and mixture solutions in which the concetrations of TEL and HYD were given in table 2. Every mixture solution was prepared 3 times. Scanning spectra of the standard solutions and the mixture solutions in the wavelengths area from 240 to 335 nm. The spectra of standard solutions of TEL, HYD and some mixture solutions were showed in figure 2.
Concentrations of TEL and HYD in the mixture solutions were computed by selfmade software named Simulan.
Calculate relative experiment errors REexp, relative standard deviation RSDexp and RSDH. If RSDexp 1/2RSDH then the repeatybility of the result was accepted. The results of accuracy and repeatbility of the method were presented in table 1 and table 2.
Table 1. Accuracy and exactness of the method when determining TEL in the prepared mixture solutions
N0
Prepared
concentration Computed TEL concentration CTEL (g/mL)
RETEL(%) RSDTEL(%) 1/2RSDH(%) CTEL
(g/mL) CHYD
(g/mL)
First time
Second time
Thirth time
C
TEL(g/mL)
H1 1.00 9.00 0.9912 1.0176 0.9981 1.0023 0.23 1.37 8.00
H2 2.00 8.00 1.9891 1.9618 1.9814 1.9774 -1.13 0.71 7.21
H3 3.00 7.00 2.9520 2.9534 2.9486 2.9513 -1.62 0.08 6.78
H4 4.00 6.00 3.8779 3.8825 3.8856 3.8820 -2.95 0.10 6.49
H5 5.00 5.00 5.0298 5.0068 5.0763 5.0376 0.75 0.70 6.28
H6 6.00 4.00 6.0703 6.0277 6.0088 6.0356 0.59 0.52 6.11
H7 7.00 3.00 7.1774 6.9980 7.0167 7.0640 0.91 1.40 5.97
H8 8.00 2.00 7.9924 7.9610 8.0315 7.9949 -0.06 0.44 5.85
Figure 1: Absorbance spectra of standard solutions 10 µg/mL HYD (1); 10 µg/mL TEL (2), and mixture solution 10 µg/mL TEL and 10 µg/mL HYD (3) in NaOH 0.1M medium
Figure 2. Absorbance spectra of standard solutions 8 µg/mL TEL (1); 8 µg/mL HYD (2), mixture solutions: 4 µg/mL TEL and 6 µg/mL HYD (3); 5 µg/mL TEL and 5 µg/mL HYD (4);
6 µg/mL TEL and 4 µg/mL HYD (5); 7 µg/mL TEL and 3 µg/mL HYD (6) in 0.1M NaOH medium
(nm)
240 260 280 300 320 340
0.0 0.1 0.2 0.3 0.4 0.5 0.6
6 5
3 4 2 1 A
1. TEL8 2. HYD8 3. TEL4+HYD6 4. TEL5+HYD5 5. TEL6+HYD4 6. TEL7+HYD3
H9 9.00 1.00 9.0108 8.9893 9.0228 9.0076 0.08 0.19 5.75
Table 2. Accuracy and exactness of the method when determining HYD in the prepared mixture solutions
N0
Prepared concentration
Computed HYD concentration CHYD (g/mL)
REHYD(%) RSDHYD(%) 1/2RSDH(%) CTEL
(g/mL) CHYD
(g/mL) First time
Second time
Thirth time
C
HYD(g/mL)
H1 1.00 9.00 8.9328 8.9878 9.0134 8.9780 -0.24 0.46 5.75
H2 2.00 8.00 8.0322 8.0476 8.0479 8.0425 0.53 0.11 5.85
H3 3.00 7.00 7.0480 7.0530 7.0642 7.0550 0.78 0.12 5.97
H4 4.00 6.00 6.0462 6.0494 6.0453 6.0469 0.78 0.036 6.11
H5 5.00 5.00 5.0647 5.0480 5.0750 5.0625 1.25 0.27 6.28
H6 6.00 4.00 3.9522 3.9072 3.9173 3.9255 -1.86 0.60 6.49
H7 7.00 3.00 2.9993 2.9395 2.9703 2.9697 -1.01 1.01 6.78
H8 8.00 2.00 1.9873 1.9607 2.0098 1.9859 -0.71 1.24 7.21
H9 9.00 1.00 1.0273 1.0103 1.0170 1.0182 1.82 0.84 8.00
Tables 1, 2 showed that the computed concentrations of TEL and HYD in mixture solutions from H1 to H9 were approximately equal prepared concentrations. It could be conclude that the method had good accuracy. The RSDHYD
and RSDTEL values in all the mixture solutions were smaller than the respective 1/2RSDH calculated by the Horwitz function, so we could conclude that the method have good repeatbility for TEL and HYD.
3.3. Establishing procedure for analysing TEL and HYD in tablets
Twenty tablets were weighed and crushed to a fine powder. An accurately weighed powder sample equivalent to 40mg of Telmisartan was transferred to a 250 ml volumetric flask, dissolved in 150 ml 0.1M sodium hydroxide, shaken for 2 hours with untrasonic machine and the volume was made up to the mark with 0.1M sodium hydroxide. The solution was then filtered through Whatman filter paper no.41. Reject the first 30 mL. Take 2.5 mL filtered solution into 50 mL volumetic flask and the volume was made to the mark with 0.1M sodium hydroxide, shaked well, we got sample solution. The absorbance spectrum of the sample solutions were recorded in the wavelength area from 240 nm to 335 nm. The files of absorbance spectra were transferred to data files: *.dat.
Using software written by C++ language to compute concentrations of components in mixture. The analysis procedure was repeated three times with the formulation.
Substance content in a tablet was calculated with formular:
x= C
a. M .5
m
(mg/tablet)In which:
M
: average mass of a tablet (g); Ca : concentration of the substance in the measured solution (g/mL);m : the weighed mass of fine pharmaceutical powder (g).
The analytical result of Micardis(R) Plus tablet was showed in table 3.
Table 3. TEL and HYD concentrations in sample solutions and their contents in Micardis(R) Plus tablet
N0
TEL HYD
CTEL (g/mL) Content
(mg/tablet) CHYD (g/mL) Content
(mg/tablet)
T1 7.9726 39.86 2.5805 12.90
T2 7.5809 37.90 2.5882 12.94
T3 7.4490 37.25 2.5598 12.80 Result
C
= 7.6675S = 0.2723
x
= 38.34RSD = 0.94%
C
= 2.5762S = 0.0147%
x
= 12.88RSD = 0.57%
The content of every active substance (mg/tablet) in a Micardis(R) Plus tablet: TEL: 38.341.16 mg/tablet; HYD:
12.881.36 mg/tablet. The computed results were suitable with the pharmaceutical substance content written on the label.
3.4. Reliability Assessment of the method for analysing real samples 3.4.1. Precision
From table 3, we could see that RSD% values for TEL and HYD contents in Micardis(R) Plus tablet were very small. So we could conclusion that the method have high precision for TEL and HYD contents (RSDTEL = 0.94%, RSDHYD = 0.57
%).
3.4.2. Accuracy
Accuracy of the method when analysing real samples was evaluated by recovery and comparing the analytical result of studied method with that of HPLC standard method.
- Recovery:
Spiking a standard substance amount of TEL and HYD into a weighed sample powder amount. Sample and standard added sample were treated as part 3.3 to get working solutions. The standard substance amount of TEL and HYD were added so that the standard added concentrations were 1µg/mL, 2 µg/mL, 3µg/mL. Repeat experiment 3 times with every standard addition amount. Scanning spectra of these working solutions in the wavelength area from 240 to 335 nm and computed TEL and HYD concentration in the solutions.
The formula for recovery is: Rev% = (Ct – Ca).100/a. Here, Ct: concentration of TEL or HYD computed after spiking;
Ca: concentration of TEL or HYD computed before spiking; a: standard added concentration into sample.
The results were presented in table 4. The data in the table 4 showed that Revmean of TEL and HYD were 101,07%
and 103,74% respectively. So the analysis procedure had good accuracy with the two components and it could be said that excipient in the tablet was not influence to analytical results.
Table 4. Recovery of TEL and HYD
- - Comparing analytical result of studied method with HPLC standard method
The medicine sample was sent to the Analytical Center of Pharmaceutical Product in Thua Thien Hue province to compare. At the centre, the sample was analyzed by HPLC standard method. The studied results were compared statistically with the results received from this center and were presented in table 5.
N0
Standard added concentration a
(g/mL)
TEL HYD
C0(TEL)
(g/mL) Ccal(TEL)
(g/mL) Rev (%) C0(HYD)
(g/mL) Ccal(HYD) (g/mL) Rev (%) 1
1.00
7.9969
8.9514 97.88
2.5786
3.6285 104.80
2.00 10.0142 102.08 4.6553 103.74
3.00 11.0651 103.08 5.6701 102.99
2
1.00
7.5962
8.5962 101.53
2.5894
3.6391 105.09
2.00 9.5689 99.40 4.6437 102.78
3.00 10.6005 100.65 5.6603 102.40
3
1.00
7.4637
8.4818 103.28
2.5643
3.6244 106.46
2.00 9.4797 101.54 4.6242 103.22
3.00 10.4558 100.23 5.6241 102.14
Average Recovery (%) Re v(%) = 101.07 Re v(%) = 103.74
Table 5 showed that the tcal values of TEL and HYD contents were smaller than ttable , so the two method gave the analytical results identically with P = 0.95. Another way, we could conclude that the results of the studied procedure and method were identical with that of standard HPLC method.
Table 5. Comparing the analytical result by Simulan with HPLC method when analysing TEL and HYD contents in Micardis(R) Plus tablet
N0
TEL content (mg/tablet) HYD content (mg/tablet)
Simulan HPLC Simulan HPLC
1 39.86 38.36 12.90 12.90
2 37.90 38.18 12.94 12.76
3 37.24 38.41 12.79 12.97
mean 38.33 38.32 12.87 12.88
SD 1.36 0.12 0.08 0.11
compare
tcal = 0.01; ttable(0,05;6)=2,45 tcal < ttable
tcal = 0.13; ttable(0,05;4)=2,78 tcal < ttable
4. CONCLUSIONS
With the aim of studying and applying chemometric-spectrophotometric method for simultaneous determination of Telmisartan and Hydrochlorothiazid in pharmaceutical product, we had as following results:
- Developed the chemometric-spectrophotometric method using full spectra based on CLS method with selfmade software named SIMULAN for simultaneous determination of TEL and HYD in tablets.
- Established the analytical procedure to determine TEL and HYD in tablet: suitable conditions were chosen for simultaneous determination of TEL and HYD in their binary mixture with 0.1M NaOH solvent, range of wavelengths used to compute from 240 to 335 nm with interval 0.2 nm.
- The method and analytical procedure were applied to analyze the drug named Micardis(R) Plus tablet containing TEL and HYD. The result was suitable with the contents of TEL and HYD which was written on the medicine label.
The procedure proved reliable with good repeatability and recovery when real analyzing drug samples: RSDTEL(%) = 0.94; RevTEL% = 101.07; RSDHYD(%) = 0.57; RevHYD(%) = 103.74. The results were also identical with the analytical results conducting by the HPLC standard analytical method with P=0.95.
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