65 Abstarct Atenolol was used with povidone iodine to prove the efficiency, reliability and repeatability of the long distance chasing photometer (NAG-ADF-300-2) using continuous flow injection analysis. The method is based on reaction between atenolol and povidone iodine in an aqueous medium. Optimum parameters was studied to increase the sensitivity development of method. Calibration graph was linear in the range of 2- 19 mmol/L for cell A and 5-19 mmol/L for cell B. Limit of detection 146.4848 ng/55 µL and 2.6600 µg/200 µL respectively to cell A and cell B. Correlation coefficient (r) 0.9957 for cell A and 0.9974 for cell. Relative standard deviation (RSD %) was lower than 1%, (n=8) for the determination of atenolol at concentration (5, 9 and 17) mmol/L for cell A and (5, 13 and 17) mmol/L for cell B respectively. The results were compared with classical method UV-spectrophotometric at λ max=270 nm by using method standard addition via t-test, at 95% confidence level. The comparison of data explain that long distance chasing photometer (NAG-ADF-300-2) is the choice with excellent extended detection and wide application. Keyword: Atenolol, Attenuation of light, Continuous flow injection analysis, Turbidity, Fluorescence. 1. Introduction Atenolol is a beta (ꞵ1) selective (cardio selective) adrenoceptor blocking agent. The main uses of atenolol are in the treatment of hypertension and coronary heart disease. Atenolol is also used to lower the risk of death after a heart attack. The chemical name of atenolol is 4-[2- hydroxy-3-[(1-methylethyl) amino] propoxy] benzene acetamide or called Tenormin, white powder, molecular formula C14 H22 N2O3, molecular Weight 266.336 g/mol, structure of atenolol is shown in Figure 1. Atenolol are mostly hydrophobic compounds therefore their limited aqueous solubility is the most challenging problem in atenolol development that causes their poor bioavailability. Many published works could be found regarding the improvement of the low bioavailability of poorly soluble atenolol including solubilization techniques. Among the elham19690@gmail.com Department of Chemistry, College of Education for Pure Sciences (Ibn-Al- Haitham), University of Baghdad, Baghdad, Iraq. Elham N. Mezaal Doi: 10.30526/33.1.2383 Nagam S. Turkey Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq. Ibn Al Haitham Journal for Pure and Applied Science Journal homepage: http://jih.uobaghdad.edu.iq/index.php/j/index Assessment of Long Distance Chasing Photometer (NAG-ADF-300-2) by Estimating the Drug Atenolol with Povidone Iodine Via CFIA Article history: Received 13 June 2019, Accepted 28 Augest 2019, Publish January 2020. file:///C:/Users/Mustafa/Desktop/2020/elham19690@gmail.com 66 broad variety of methods proposed for enhancing atenolol solubility, the addition of pharmaceutic cosolvents is the most widely used technique for atenolol in aqueous media [1-4]. The solubility enhancement of poorly soluble atenolol can be achieved by the changes of temperature [5]. Solubility enhancement by using alkaline medium [6], stability of atenolol in acidic environment depending on diversified polarity [7]. Previous studies applied spectrofluorometry to estimation of atenolol in aqueous solution and samples human urine because of its sensitivity, selectivity and low cost instrumentation [8]. Photoluminescence of metal nanoparticles that offer emission of light without requiring conjugation with luminous dyes is the basis for the new method [9]. Determination of atenolol by using high performance liquid chromatographic (HPLC) [10-14], Uv-vis spectrophotometry [3, 15-19]. Chromatographic densitometry [7]. Reflectance spectroscopy [20]. Spectrofluorometry method [2].Potentiometric titration [21]. And GC-Mas [4]. Figure 1. Structure of atenolol. Flow Injection Analysis (FIA) as “technique based on the sequential injection of a discrete liquid sample into a moving, no segmented continuous carrier stream, several methods depend on continuous flow injection analysis [22-33]. Infection can slow down the wound healing process. However, the infection can be effectively prevented using an antiseptics. Povidone iodine is the most effective antiseptic to prevent infection .It has been widely used on 10% concentration. However, either in vivo or in vitro studies indicated that the povidone iodine, in 10% concentration, is toxic to fibroblast, povidone iodine brown powder, molecular formula (C6H9NO) n. x I [34, 35]. The aim of this work was to assessment of a new long distance chasing photometer (NAG- ADF-300-2) by determination of atenolol with povidone iodine. The method is based on the quenching of the povidone iodine by using drug atenolol via a new long distance chasing photometer for 300 mm length with 2 mm path length to chase and to accumulate output resulted from attenuated incident light 0-180 0 via two flow cells of 110 mm and 60 mm length (NAG- ADF-300-2 analyzer) [36]. 2. Chemicals and Apparatus 2.1 Reagents and chemicals All analytical chemicals reagent were used for all solutions and dissolved by distilled water. A standard solution of 50 mmol/L of atenolol, was prepared by dissolving 1.3317 g in a 100 ml. A series of povidone iodine solutions were prepared from the dilution of standard solution 50 mmol/L with distilled water. 2.2 Apparatus A homemade NAG-ADF-300-2 is a long distance chasing photometer as a flow cell will have 300 mm as a distance with 2 mm as a path length to chaise and to accumulate the output resulted from attenuation of incident light 0-180 0 and diverged or fluorescence light at 0-90 0 via a flow cell. The first flow cell is of 110 mm length covered with 11 white snow LED (WSLED), followed by uncovered distance of 100 mm length then attached to another with 2 solar cell at each side of (0-180 0 and 0-90 0 ), cell (cell number 2) which is covered by 6 WSLED and a single photo cell (solar) of 60 mm length at each side. Using peristaltic pump (Ismatec, Switzerland) 67 and six-port medium pressure IDEX Corporation USA injection valve with sample loop (1 mm i.e. Teflon, variable length). Potentiometric was recorder to estimate the output signals (Siemens, Germany). UV-spectrophotometric (RF-1501, shimadzu, Japan) was use for classical methods. 3. Methodology Two manifold designs were investigated to choose the optimum manifold system for the determination of atenolol by using povidone iodine system. The first is shown in Figure 2.A. The manifold reaction system is consists of one line represents the carrier stream povidone iodine (5 mmol/L) which passes through injection valve in which sample segment of atenolol (5 mmol/L) was used at 2.9 mL/min flow rate. 100 µL as a sample volume and that passes through the measuring cell (A and B) to quench of povidone iodine molecule is shown in Figure 2.C. The second manifold design of flow injection analysis is shown in Figure 2.B. The manifold reaction system is consists of two lines: first line is carrier stream (distilled water) leading to the injection valve, which allows the use of 100 µL of atenolol sample segment (5 mmol/L) and flow rate 3.2 ml/min, while the second line supply the povidone iodine molecule solution (5 mmol/L) and a flow rate of 4.6 ml/min. The two lines mixes together at a Y-junction and leading to measuring cell (A and B). The response obtained is shown in Figure 2.D. Figure 2.A, B. Schematic diagram of manifold flow injection analysis using one and two manifold design. C, D: Profile of YZ (mV) – t min (d) cm energy transducer response output using a, b total continuous Response of povidone iodine 5 mmol / L for cell A, cell B c, d total quenched of povidone Iodine for cell A, cell B by using sample volume 100 µL for one line and two line. 68 Scheme 1. Shows a proposed expected mechanism for the reaction of atenolol with povidone iodine [37-39]. Scheme 1. Proposed mechanism of reaction between atenolol and povidone iodine. 4. Results and Discussion 4.1. Study of the optimum intensity used for cell A and cell B Choosing the optimum intensity for either cells (cell no.1-cell A and cell no.2-cell B with in between distance of 100 mm. An arbitrary selected intensity was put into work (an intensity of indication approximate of selector switch for cell A was on no.3, while it was on no.2 for cell B which was based on preliminary experiment).These numbers reflect the 0-1-2-3-4 intensities which were varied a according to nature and type of reaction carried out. On the basis of obtained responses profile Figure 2.A , B. other necessary chemical and physical parameters were carried out, which describe the full detailed study supported by the recorded of YZ (mV) – t min (d) cm response. Table 1. Shows the intensity (I) of the response for either cells from I=1 to I=4.The optimum intensity of the measuring cell A, I=3 and I=2 for cell B which was adopted in subsequent studies. Table 1.Effect of intensity on Response profile, total continuous response of povidone iodine, quenched Of povidone iodine, remained of povidone iodine for cell A and cell B by using 100 µL sample Volume concentration of the atenolol (5 mmol/L]) with povidone iodine (5 mmol/L), speed of Recorder = 60 cm/hr. Intensity (I) Total response of povidone iodine Ῡ Z i (mV) RSD% Confidence interval at (95%) Ῡ Z i (mV)±t0.05/2,n-1 σn- 1/√n Quenched of povidone iodine mV Remained of povidone iodine m V Cell A 1 368 0.1060 368±0.9689 272 96 2 544 0.1195 544±1.6148 384 160 3 816 0.1201 816±2.4346 592 224 4 200 0.6050 200±3.0060 180 20 Cell B 1 336 0.1339 336±1.1179 216 120 2 440 0.1318 440±1.4409 296 144 3 96 0.5938 96±1.4160 80 16 4 80 1.2250 80±2.4346 60 20 69 I=3 (cell A) and I=2 (cell B) 776 (cell A) and 408 (cell B) 0.1559 (cell A) and 0.3554 (cell B) 776±3.0060(cell A) and 408±3.6022(cell B) 576 (cell A) and 280 (cell B) 200 (cell A) and 128 (cell B) t0.05/2,2=4.303, Ῡ Z i (mV): (S/N) energy transducer response of cell A and cell B in mV for n=3 One line system reduction of povidone iodine is low and the response intensity is high, so that the same concentration of atenolol could not suppress the povidone iodine response significantly compared with the atenolol which echoes the povidone in the two line system. Atenolol in a two line system was able to quell the povidone almost entirely because the povidone were less intense due to the dilution and dispersion of the two line system. 4.2. Optimization of variables for one line manifold system All chemicals parameters mainly reagent concentration and kind of carrier stream for the atenolol with povidone iodine system as well as physical parameters volume of sample, flow rate were studied using one lines manifold system Figure 2.A. 4.2.1. Chemical variable effect Using the optimum concentration of povidone iodine for the determination of atenolol using one line manifold system. 4.2.1.1. Povidone iodine concentration A series of the povidone iodine solutions 1-10 mmol/L at 2.9 ml/min flow rate were prepared. Atenolol 5 mmol/L used with 100 µL volume of sample. Each measurement was sequential for three times. The increase of the concentration povidone iodine and quenching.increases of povidone iodine response is shown in Figure 3 A, B, C. (8 mmol/L) was chosen as optimal quenching intensity with low of reagent concentration. Figure 3.A. Effect of concentration variable of povidone iodine on S/N energy transducer response. B, C: Attenuation of incident light expressed as an average peak heights versus povidone iodine concentration, quenching of povidone iodine by D.W and atenolol for cell A and cell B. 4.2.1.2. Effect of Different Medium Atenolol (5 mmol/L) - povidone iodine (5 mmol/L) system was studied in solution in different media (sodium carbonate and sodium hydroxide) at 10 mmol/L concentration in addition to aqueous medium as a carrier stream. The results explain using the salt solutions lead to decrease sharpness of response and 70 change of the response format therefore, the water medium of the reaction was selected. The results were summarized in Table 2. Table 2. Effect of different medium on the measurement of energy transducer response for Determination of atenolol. Type of medium 10 mmol/L Povidone iodine response ῩZ i (mV) Response of blank ῩZ i(mV) Total quenched povidone iodine ῩZ i (mV) Quenched povidone iodine ῩZ i(mV) Remained povidone iodine ῩZ i(mV) ῩZ i (mV)±t0.05/2, 2 σn-1 /√n Cell A H2O 1656±4.7698 240±4.6953 336±2.7824 96 1320 Na2CO3 1616±3.8009 240±4.5214 176±3.6022 -64 1440 NaOH 1616±4.9188 240±2.7824 344±4.7450 104 1272 Cell B H2O 968±2.9066 56±2.4346 152±1.6645 96 816 Na2CO3 968±3.7761 56±2.7327 168±3.2792 112 800 NaOH 968±3.6767 56±3.5277 184±3.7761 128 784 t0.05/2,2=4.303, ῩZ i (mV) (S/N): energy transducer response of cell A and cell B in mV for n=3 4.2.2. Physical Variables 4.2.2.1. Flow rate Figure 2. A which shows that a one lines manifold system were used a variable flow rates. Different variable responses were obtained even different profile were described. Figure 4. A shows the different types of responses characterization by response of attenuated signal versus flow rate. While Figure 4 B, C. Shows quenched in mV, Remained of povidone in mv, Peak base width in mV and addition volume in ml for cell A and cell B, flow rate of The carrier stream 4.4 ml/min utilizations the optimum choice even it is at the edge of the curve. From the figure we notice the increase of the sharp peak and quenched povidone iodine by increasing the flow rate and decreasing the width base Δ t of the response. 71 Figure 4. Effect of the variation of flow rate on: A: S/N energy transducer response versus t min (d) cm B, C: Attenuation of incident light expressed as an average peak heights in mV (ῩZ i (mV)) for cell A, cell B by using atenolol (5 mmol/L) – povidone iodine (8 mmol/L) system, 100 μl volume of sample, I=3for cell A and I=2 for cell B. 4.2.2.2. Sample Volume Using atenolol (5 mmol/L) – povidone iodine (8 mmol/L) system and variable length of Teflon tube ranging (2.6-25.5) cm of diameter (D) 1 mm, that is equivalent to (20-200) µL of sample volume. It was noticed that an increase volume of sample cause the increase of response for cell B and decrease of response for cell A expressed as a quenching of povidone iodine. A compromise was made in this study to choose 55 µL for cell A and 200 µL for cell B as a suitable most convenient of sample size level. All results were tabulate in Table 3. Table 3. Effect of the variation volume of sample on the total quenched, quenched, remained of povidone Iodine by using (5 mmol/L) concentration for atenolol and (8 mmol/L) concentration of povidone Iodine, speed of recorder 60 cm/hr. and flow rate of carrier stream 4.4 ml/min. Loop length (cm) r=0.5 (mm) Sample volume (µL) Total quenched povidone iodine ῩZ i (mV) RSD% Confidence interval at (95%) Quenched povidone iodine ῩZ i (mV) Remained povidone iodine ῩZ i(mV) Δ t (sec) t (sec) V add (ml) at flow cell Concentration mmol/L at flow cell D f at flow cell Cell A 2.60 20 88 0.2614 88±0.5714 0 1512 5 12 0.3867 0.2586 19.34 3.10 24 216 0.5602 216±3.0060 88 1368 7 15 0.5373 0.2233 22.39 4.10 32 280 0.4000 280±2.7824 136 1296 9 18 0.6920 0.2312 21.63 7.00 55 416 0.2668 416±2.7575 208 1264 10 22 0.7883 0.3489 14.33 9.00 71 416 0.2452 416±2.5340 176 1296 12 25 0.9510 0.3733 13.39 12 74 100 368 0.2853 368±2.6085 152 1232 14 28 1.1267 0.4438 11.27 20.40 160 520 0.2923 520±3.7761 176 1104 20 35 1.6267 0.4918 10.17 25.50 200 560 0.3250 560±4.5214 200 1/040 30 40 2.4000 0.4167 12.00 Cell B 2.60 20 48 1.1667 48±1.3912 24 928 7 17 0.5333 0.1875 26.67 3.10 24 104 0.7885 104±2.0371 72 880 8 20 0.6107 0.1965 25.45 4.10 32 120 0.3750 120±1.1179 88 872 10 23 0.7653 0.2091 23.91 7.00 55 184 0.1141 184±0.5217 136 792 11 26 0.8617 0.3191 15.67 9.00 71 184 0.1033 184±0.4720 120 784 13 28 1.0243 0.3466 14.43 12.74 100 160 0.0750 160±0.2981 112 808 15 30 1.2000 0.4167 12.00 20.40 160 240 0.5042 240±3.0060 160 712 28 40 2.2133 0.3615 13.83 25.50 200 392 0.2602 392±2.5340 296 584 35 45 2.7667 0.3614 13.84 t: Arrival time from injection valve arrivals to measuring cell (sec) ,Δ t: Base width of peak(sec), t0.05/2,2 =4.303,Df : Dilution factor at flow cell 4.2.2.3. Effect of Reaction Loop Length Variable coil lengths 0, 20, 30, 40 and 50 cm were studied. These length comprises a volume (0-392.5 µl) which connected after valve directly in flow system. While keeping all other changeable constant (i.e.; atenolol (5 mmol/L), - povidone iodine (8 mmol/L) system, 4.4 ml/min flow rate for carrier stream (distilled water) and sample volume 55, 200 μl for cell A and cell B respectively. Figure 5. Shows the manifold design system for determination of atenolol in the presence of reaction coil. A volume of a cylinder =πr 2 L (where L= length of the used tube for e.g. 72 is ϕ=1, the r=0.5 mm for 100 mm length. The volume will be equal to 3.14 (0.05 cm) 2 × 20 cm=0.157 cm 3 =157 µL. Figure 6. A, B, C and D shows that the increase of coil volume will lead to a highly dispersed which cause a weaker signal or even undetected signal. Therefore a compromise of using a convenient reaction coil length was is not using of reaction coil. Figure 5. Manifold design system for evaluation of atenolol in the presence of reaction coil 73 Figure 6. Effect of reaction coil on: A, B: S/N- energy transducer response versus t min (d) cm for cell A and cell B respectively C, D: Total quenched, quenched and remained of povidone in mv for cell A, cell B by using atenolol (5 mmol/L) - povidone iodine (8 mmol/L) system, intensity I=3 for cell A and I=2 for cell B, flow rate 4.4 ml/min , sample volume 55,200 µL for cell A and cell B respectively. 5. Study of the Optimum Intensity Used for White Snow Light Emitting Diodes (WSLEDs) in NAG-ADF-300-2 analyser for one line A study was conducted for the effect of intensity of incident light for the irradiation sources on the S/N – response of the energy transducer via the selector switch (C.F front panel diagram of NAG-ADF-300-2 Figure 2 A.The selector switch gives 0-1-2-3-4 i.e.; four choices plus the off position for both cell individually controlled. It was noticed that a selection of 3 position (i.e.; I=3) was very convenient intensity for cell no.1 (Cell A) (Larger number of the selector switch means more light intensity), while position 2 (I=2) of the selector switch was a convenient intensity .It was same intensity chosen in first of atenolol study of assessment. The higher intensity (I=3) for cell A, while it was not necessary to use high light intensity for cell B due to quenching of povidone iodine. Therefore, a low intensity of height is required for cell B. The results summed up in Table 4. Table 4. Effect of intensity on attenuation of incident light expressed as an average peak heights (mV) For total quenched, quenched and remained of povidone in mV by using atenolol (5 mmol/L) – Povidone iodine (8 mmol/L) system, speed of recorder 60 cm/hr., flow rate carrier stream 4.4 ml / min and sample volume 55,200 µL for cell A and cell B respectively . Intensity (I) Total quenched povidone iodine ῩZ i (mV) RSD% Confidence interval at (95%) Quenched of povidone iodine mV Remained of povidone iodine mV Cell A 1 160 0.6125 160±2.4346 80 528 2 208 0.4760 208±2.4594 96 776 3 416 0.2668 416±2.7575 208 1264 Cell B 1 256 0.3711 256±2.3601 200 440 2 392 0.2602 392±2.5340 296 584 3 376 0.3511 376±3.2792 224 272 4 96 1.5833 96±3.7761 56 72 The intensity of output response is varied for cell A and cell B 74 6. Estimating the Linear Dynamic Range From Scatter Plot For The Variation Of Atenolol Versus S/N Energy Transducer Response A series of atenolol solutions (0.03-40, 0.07-40 mmol/L) using the optimum chemical and physical parameters; for cell A and cell B respectively were prepared and this will represent the x-axis (Independent variable).The attenuation of incident light was measured and gave the following S/N energy transducer responses as Y here represent the dependent variable as shown in Figure 7 A, B. In which, the height of response increased for cell A and B when the analyte of concentration is increased. It can be seen from the Figure 8 A, B. Explains the variance ranges for each cells. (i.e.; scatter plot at range (0.03-40) mmol/L, dynamic range (0.03-30) mmol/L, working range (0.03-25) mmol/L and linear dynamic range (2-19) mmol/L for cell A and scatter plot at range (0.07-40) mmol/L, dynamic range (0.07-30) mmol/L, working range (0.07-25) mmol/L and linear dynamic range (5-19) mmol/L for cell B). Figure 7. Some of response profile versus time using povidone iodine (8 mmol/L), sample volume 55,200 µL respectively to cell A and cell B .I=3 for cell A, I=2 for cell B. A: for cell A, B: for cell B. 6.1. Limit of Detection In general terms, the LOD of an analyte may be characterization as that: concentration which gives an instrument signal y9 significantly different from the blank or back ground signal. This characterization gives the analyst a perfect deal of freedom to decide the exact definition of L.O.D. There is an increasing trend to define the L.O.D. as: the analyte concentration giving a signal equal to the blank signal, y B plus three standard deviation of the blank SB. LOD = y B + 3SB 1. Gradual dilution Practically based on consecutive dilution of the lowest concentration used in calibration graph, this should be regarded as the real, and trustable value of D.L. i.e. Reliable D.L. for the proposed method. 2. Theoretically (method of slope) 75 L.O.D. =3SB / slope SB = σ n-1 B standard deviation of blank n=13 3. Theoretically (equation of linear method) Ŷ = y B + 3SB YB: average response for the blank solution, this is equivalent to intercept (a) in straight line equation y= a + b x The final two methods are an output of a linear regression graph treatments where the obtained (fact) results are subjected to statistical treatments, these methods can be used as an approximate indication but should not except if otherwise defined. A study was done to calculate the detection limit of atenolol- povidone iodine (8 mmol/L) system through three methods as tabulated in Table 5. Table 5. Limit of detection for atenolol at using optimum parameters 55,200 µl for cell A and cell BRespectively as an injection sample, 4.4 ml/min flow rate of carrier stream, [povidone iodine]8mmol/L. X=LOD based on slope and SB= standard deviation of blank repeated for 12 times. : Y b average response for blank= intercept (a), S b: standard deviation equal to S y/x (residual): Ŷ estimated response (mV), n: number of injection, n* number of measurement for scatter plot 6.2. Repeatability The relative standard deviation expressed as a percentage which is equally to the repeatability of the measurement. A repeated measurements for eight repeated injections were measured at steady concentrations of atenolol for three concentrations were used 5, 9, 17 mmol/L for cell A and 5, 13, 17 for cell B in optimum parameters. The obtained results is listed in Table 6 is which showed the repeatability at 5, 9, 17 mmol/L for cell A and 5, 13, 17 for cell B respectively. In addition to study of repeatability with minimum of the RSD% which equal to 1%. Table 6. Repeatability of atenolol at optimum parameters with 55 µl volume of sample for cell A and 200 µl sample volume for cell B. Response of continuous povidone iodine =1680 mV for cell A, =976 mV for cell B, response of blank = 208 mV for cell A, =96 mV for cell B, t 0.05/2, 7= 2.365, number of injection =8. Type of cell Practically based on the gradual dilution (0.03 mmol/L) (n*) (0.07 mmol/L)(n*) Theoretical based on the value of slope Theoretical based on the linear equation n Cell A 0.01 mmol/L (25 mV) 146.4848 ng/55 µL 216.7975 ng/55 µL 23.9635 µg/55 µL 20 Cell B 0.05 mmol/L (104 mV) 2.6600 µg/200 µL 2.8400 µg/200 µL 62.4600 µg/200 µL 18 [Atenolol] mmol/L Average of total quenched povidone ῩZ i (mV) Quenched povidone ( ῩZ i (mV) RSD% Confidence interval at (95%) Cell A 5 416 208 0.2668 416±0.9281 9 584 376 0.2072 584±1.0117 17 1040 832 0.2029 1280±1.7643 Cell B 5 392 296 0.2602 392±0.8529 13 544 448 0.1195 544±0.5435 17 620 524 0.2129 620±1.1037 76 7. Classical method of UV- Spectrophotometric The assessment evaluation of new developed methodology (i.e.; NAG-ADF-300-2 analyser) for the determination of atenolol using atenolol - povidone iodine (8 mmol/L) system. A new developed method was compared with the available literature method, namely UV- spectrophotometric method [40], which was based on the measurements of absorbance. Concentration range of method 0.01-6 mmol/L at λ max= 270 nm using quartz cell. Table 7. Shows the variable data treatments. The detection limit was 0.005 mmol/L (5 µmol/L) equivalent to 1.3317 µg / sample. Table 7. Different ranges for the atenolol concentration versus absorbance using spectrophotometer (Classical method). Type of mode Range of [atenolol] mmol/L(n) ŶZ i = a± Sa t+ b (Δ y/ Δxmmol/L) ± S b t [Atenolol] mmol/L at confidence level 95%,n-2 r, r 2 , R 2 % t tab at 95% ,n-2 Calculated t-value tcal=/r/√n-2 / √1-r 2 Scatter plot 0.01-6 (18) 0.3291 ±0.2033+0.3706±0.0856 [Atenolol] mmol/L 0.9167,0.8404,84.04 2.120 < 9.1785 Dynamic range or analytical range 0.01-5 (17) 0.2632±0.1750+0.4449±0.0891 [Atenolol] mmol/L 0.9396,0.8829,88.29 2.131 < 10.6344 Working range or calibration range 0.01-4 (16) 0.1926±0.1386+0.5371 ± 0.0871 [Atenolol] mmol/L 0.9623,0.9260,92.60 2.145 << 13.2375 Linear range or linear dynamic range 0.05-2.5 (13) 0.0864±0.0599+0.7157± 0.0548 [Atenolol] mmol/L 0.9934,0.9869,98.69 2.201 28.7747 8. Assessment of NAG -ADF-300–2 Analyser Using Two Cell And Multi Solar Cells For The Determination of Atenolol In Drugs The newly developed methodology (NAG-ADF-300-2) was used for the determination of atenolol in three different samples of drugs from three different of companies (Atenolol, Bristol, UK, 100 mg),(Vascoten, medochemie, Cyprus, 100 mg) and (Novaten, Ajanta, India, 100 mg). The continuous flow injection analysis used of homemade NAG-ADF-300-2. Which that mean a long distance chasing photometer for 300 mm length with 2mm path length to chase and accumulate output response from attenuation of incident light at 0-180 0 via the use of two cells of 110 mm (cell A) and 60 mm length (cell B). The newly developed methodology comparison with UV-Spectrophotometric method via measurement at λ max =270 nm. A series of solution were provided of each drug (20 mmol/L) by transferring of 0.5 mL to each of the five volumetric flask (10 mL) followed by the addition of 0.0, 0.2, 0.4, 0.6, 0.8 mL from 50 mmol/L of standard solution to obtain 0,1,2,3,4 mmol/L for developed method ,while classical method (20 mmol/L) by transferring of 0.5 mL to each of five volumetric flask (10 mL) followed by addition of 0.0, 0.04, 0.06, 0.08, 0.1 mL from 50 mmol/L of standard solution of atenolol to obtain 0,0.2,0.3,0.4,0.5 mmol/L. Taking into a consideration that the first flask is for the sample. The measurements were conducted by both methods. Results were mathematically treated for method of standard addition. Table 8 A, B. have shown a practical consist of active ingredient at 95% confidence level & efficiency of evaluation in addition to paired t-test which shows a comparison at two difference paths [41, 42].First test: Comparison of newly developed method (NAG-ADF-300-2) analyser with official quoted value B.P [43]. (100 mg) as shown in Table 8 B. (column 5) by calculated t-values of each individual company and these compare with tabulated t-value. A hypothesis can be estimated as follow null hypothesis: There is no important 77 difference between the means obtained from three source of three different companies (ѿ i) and quoted value (µ) i.e.; Ho: ѿ i = µ For: Atenolol (Bristol, 100 mg, UK), Vascoten (Medochemie, 100 mg, Cyprus) and Nova ten (Ajanta, 100 mg, India) companies. Against: Alternative hypothesis: there is an important difference between the means and quoted value i.e.; ѿ o ≠ µ for: different three companies Some value obtained tcal > t tab (4.303) confidence level at 95% and degree of freedom =2; Null hypothesis will be reject and accepting the alternative hypothesis; these mean that there is an important difference between the quoted active ingredient value and the measured value. One this base; the newly developed method can be used equally well as standard reference methods. Another obtained tcal –value indicated that there was no significant different between the newly developed method and claimed method by the company as calculated t – value is less than tabulated t – value. So, the newly method capable was used as an alternative analysis method for the evaluation of atenolol in different drugs. Second test: Using paired t – test at α = 0.05 (2-tailed) for using developed method NAG- ADF-300-2 analyser and the compare with classical method using shimadzu (UV-1800 double beam) spectrophotometer as shown in table 8 B (column 6). Taking into the consideration that all drugs from different companies are the same population i.e.; neglecting individual differences between one manufacturer and another. Assumption null hypothesis Ho: µ NAG-ADF-300-2 analyser =µ UV-SP. There is no significant difference between the mean of different two methods. An alternative hypothesis: There is an important difference between the mean of classical method and NAG- ADF-300-2 analyser i.e.; Alternative H1: µ NAG-ADF-300-2 analyser ≠ µ UV-SP. The obtained results indicated clearly that there was no significant differences between newly developed method and UV-spectrophotometric (classical method) at 95% (α = 0.05) confidence level as the calculated tcal (0.2107 and /-0.3551/) is less than t tab (4.303) for each cell (i.e.; cell A & cell B) for the evaluation of atenolol in pharmaceutical drugs as shown in Table 8 B. (column 6). 78 Table 8. A: Standard addition results for the determination of atenolol in three samples of drugs using NAG-ADF-300-2 analyser for cell A, cell B and classical methods. Ŷ: Estimated response in mV for developed method and absorbance for UV-Sp. method, r: correlation coefficient, r 2 : coefficient of determination, R 2 %: percentage capital R square, UV –Sp.: UV –Spectrophotometric method, t0.05/2,ꞵ=1.96 at 95%,t0.05/2,3=3.182 for n=5. No. of sample Commercial name , Company Content Country Type of method Newly developed methodology Cell A Cell B UV-Sp. Classical method Absorbance measurement at λ max =270 nm Confidence interval for the average Weight of Tablet ѿ i ± 1.96σn-1/√n at 95% (g) Weight of sample equivalent to 1.33168 g(20 mmol/L)of the active ingredient Wi (g) Theoretical content for the active ingredient at 95% (mg) Wi ±1.96σn-1 /√n Atenolol mmol/L Equation of standard addition at 95% for n-2 r , r 2 , R 2 % 0 0.20ml 0.40ml 0.60ml 0.80ml ŶZ i (mV)=a mV ± Sa t+ b(Δ y mV /Δxmmol/)± S b t[atenolol]mmol/L 0 1.00 2.00 3.00 4.00 0 0.04ml 0.06ml 0.08ml 0.10ml ŶZ i =a± Sat+ b (Δ y / Δxmmol/L )± S b t[atenolol]mmol/L 0 0.20 0.30 0.40 0.50 1 Atenolol Bristol 100 mg UK 0.4190±0.00204 5.5801 100±0.4868 90 130 240 310 379 78.2±41.3514+75.8±16.8815 [Atenolol]mmol/L 0.9927,0.9855,98.55 65 120 190 240 309 63.2±13.0732+60.8±5.3372 [Atenolol]mmol/L 0.9988,0.9977,99.77 0.392 0.511 0.518 0.551 0.612 0.4024±0.0528+0.4086±0.1604[Atenolol]mmol/L 0.9780,0.9564,95.64 2 Vascoten Medochemie 100 mg Cyprus 0.4030±0.0015 5.3661 100±0.3723 95 210 308 398 489 104.8±22.7863+97.6±9.3026[Atenolol]mmol/L 0.9986,0.9973,99.73 85 150 235 320 400 78±16.2250+80±6.6240 [Atenolol] mmol/L 0.9990,0.9980,99.80 0.592 0.681 0.751 0.812 0.856 0.5860±0.0274+0.5442±0.0834 [Atenolol]mmol/L 0.9965,0.9931,99.31 3 Novaten Ajanta 100 mg India 0.4028±0.0031 5.3643 100±0.7696 110 220 340 450 580 106±14.9249+117±6.0932[Atenolol]mmol/L 0.9996,0.9992,99.92 78 166 250 330 420 79.2±6.7350+84.8±2.7496 [Atenolol] mmol/L 0.9998,0.9997,99.97 0.351 0.423 0.459 0.511 0.532 0.3504±0.0204+0.3745±0.0620 [Atenolol]mmol/L 0.9960,0.9920,99.20 79 Table 8. B: Summary of results for practical content, efficiency (Rec %) for determination of atenolol in three samples of drugs and t -test for comparison two methods. µ: quoted value, x d: average of difference between two type of method (developed& classical), n (no. of sample) = 3, σn-1: standard deviation of different, ѿ i: practically weight in mg,t0.05/2,2=4.303. No. of sample Type of method Newly developed methodology Cell A Cell B UV-Sp. Classical method Absorbance measurement at λ max =270 nm Practical concentration ( mmol/L) in 10 ml in 250 ml Practical weight of atenolol ѿ i(g) ±4.303 σn-1/√n Weight of atenolol in tablet ѿ i (mg) ±4.303 σn-1/√n Efficiency of determination Rec.% Individual t-test for compared between quoted value &practical value (ѿ i -µ)√n /σn-1 cell A and cell B Paired t –test Compared between two methods tcal= X d √n/σn-1 t tab at 95% confidence level(n-1) 1 1.0317 20.6332 1.3738±0.6112 103.1645 ±45.8976 103.1645% 0.2967 < 4.303 cellA X d=0.4725 σn-1=3.8841 0.2107 < 4.303 cell B X d= - 1.6243 σn-1=7.9219 /-0.3551/ < 4.303 1.0395/ 20.7895 1.3842 ±0.3928 103.9469 ±29.4974 103.9469% 0.5757 < 4.303 0.9848 19.6960 1.3114 ±0.0270 98.4788 ±2.0275 98.4788% 2 1.0738 21.4754 1.4299 ±0.3982 107.3764 ±29.9023 107.3764% 1.0615 < 4.303 0.9750 19.5000 1.2984 ±0.2341 97.4995 ±17.5790 97.4995% /-0.6121/ < 4.303 1.0768 21.5360 1.4340 ±0.0780 107.6792 ±5.8570 107.6792% 3 0.9060 18.1196 1.2065 ±0.3582 90.5978 ±26.8977 90.5978% /-1.5041/ < 4.303 0.9340 18.6792 1.2437 ±0.1982 93.3958 ±14.8839 93.3958% /-1.9093/ < 4.303 0.9356 18.7128 1.2460 ±0.083 93.5633 ±6.2325 93.5633% 80 9. Conclusion The assessment of long distance chasing photometer (NAG-ADF-300-2) through this research work was applied using comparison between NAG-ADF-300-2 analyser with classical UV- spectrophotometric method using atenolol with povidone iodine in aqueous medium. Chemical and physical parameters were studied in this research work. It was recognized that a narrower range is obtained with UV-spectrophotometric, while a wider range was the characteristic of NAG-ADF-300-2 analyser. A long distance chasing photometer (NAG-ADF-300-2) is the choice with excellent extended detection and a wider applicability. In the future using a new long distance chasing photometer as a flow cell will have 300 mm as a distance with 2 mm as a path length to chaise and to accumulate the output resulted from Attenuation and the Diverged or Fluorescence light at 0-90 via two flow cells of 110 mm and 60 mm length (NAG-ADF-300-2) for study and determination of some selected drugs. References 1. Tulia, R.G.; Gowrj, S.D.; Kadgapathi, P.; Satyanarayana, B. A validated RP-HPLC method for simultaneous estimation of atenolol and indapamide in pharmaceutical formulations. E-Journal of Chemistry. 2011, 8, 3, 1238-1245. 2. Esam, B.; Mohamed, G.; Ahmed, A.; Waleed, E.B. Spectrofluorometry method for atenolol determination based on gold nanoparticles. Acta Pharm.2018, 68, 243–250. 3. Agolkar, B.B.; Chavan, L.D.; Chondhekar, T.K.; Shankarwar, S.G. Kinetics and mechanistic Study of oxidation of atenolol drug in acidic medium by 12-tungstocobaltate (III). Journal of Chemistry and Chemical Sciences.2016, 6, 1, 1-8. 4. Bilal, Y.; Sakir, A. Determination of atenolol in human urine by Gas Chromatography - Mass Spectrometry methods. Journal of Chromatographic science.2011, 49, 365-369. 5. Samin, H.; Abolghasem, J. Solubility of atenolol in ethanol + water mixtures at various temperatures. J. Serb. Chem. Soc.2015, 80, 5, 695–704. 6. Genaro, Chairm, A. R. Remington pharmaceutical sciences. 17th ed. St .printing in the United States of America by Mark printing company Easton, Pennsylvania.1985, 904. 7. Jan, K.; Anna, K. Stability of atenolol, acebutolol and propranolol in acidic environment depending on its diversified polarity. Pharmaceutical development and technology.2006, 11, 409-416. 8. Belal, F.; Al-Shaboury, S.; Al-Tamrah, A. Spectrofluorometric determination of labetalol in pharmaceutical preparations and spiked human urine through the formation of coumarin derivative. J. Pharm. Biomed. Anal.2002, 30, 1191-1196. 9. Bavili-Tabrizi, A.; Bahrami, F.; Badrouj, H. Avery simple and sensitive Spectrofluorometric method based on the oxidation with cerium (IV) for the determination of four different drugs in their pharmaceutical formulation. Pharm. Sci.2017, 23, 50-58. 10. Chiu, F.C.K.; Zhang, J.N.; Li, R.C.; Raymond, K. Efficient assay for the determination of atenolol in human plasma and urine by high performance liquid chromatography with fluorescence detection. Journal of ChromatographyB.1997, 691, 2, 473-477. 11. Bilal, Y.; Sakir, A. Determination of atenolol in human urine by using HPLC, Sep. Sci .plus .2018, 1, 4–10. 12. Anelise,W.; Daniele CDO, Janine, D.M.; Karin, G.; Clarice, M.B. Validation of UV spectrophotometric and HPLC methods for Quantitative determination of atenolol in pharmaceutical preparations. Lat.Am.J.Pharm.2007, 26, 5,765-770. 81 13. Rafik, K.; Alaa, Q.; Khulod, K.D.; Saleh, A.L. Design, Synthesis, and in vitro kinetics study of atenolol prodrugs for the use in aqueous formulations. The Scientific Word Journal.2014, 1-13. 14. Arindam, B.; Bidyut, D.; Krishnendu, B.; Mithun, Ch.; Somnath, B. Development & validation of stability indicating high performance liquid chromatographic method for simultaneous estimation of atenolol & indapamide in tablet dosage form. Journal of Pharmacy Research.2011, 4, 6, 1677-1680. 15. Bilal, Y. Determination of Atenolol in Pharmaceutical Preparation by Zero-, First-, Second- and Third Order Derivative Spectrophotometric Methods, Fabad. J. Pharm. Sci.2008, 33,119–129. 16. Fadnis, G.A.; Agarwal, R. Kinetic and mechanistic study of oxidation of atenolol by cerium (IV) in sulphuric acidic medium. Agarwal Rashmi ET al.IRJP.2012, 3, 3, 268-270. 17. Sayyed, H.; Katapalle, R.; Salim, S.; Mazhar, F. Permanganate study of oxidation of atenolol in acidic medium .World Journal of pharmacy and pharmaceutical Sciences.2017, 6, 2, 1281-1289. 18. Hiremath, G.C.; Kulkarni, R.M.; Nandibewoor, S.T. Kinetics of oxidative degradation and deamination of atenolol by aqueous alkaline permanganate. Indian Journal of Chemistry.2005, 44, 245-250. 19. Seema, Monika D. In vitro sustained delivery of atenolol, an antihypertensive drug using naturally occurring clay mineral montmorillonite as a carrier.Eur.Chem.Bull.2013, 2 , 11, 942-951. 20. Gotardo, M.A.; Sequinel, R.; Pezza, L.; Pezza, H.R. Determination of atenolol in pharmaceutical formulations by diffuse reflectance spectroscopy. ECL. Quím, São Paulo.2008, 33, 4, 7-12. 21. Prashanth, K.N.; Basavaiah, K.; Raghu, M.S.; Vinay, K.B. Determination of Atenolol and its preparations by Acid-Base Titration in Non-aqueous Medium .Der Pharmacia Lettre.2012, 4, 5, 1534-1540. 22. Turkey, N. S. New system for successive irradiation and spectral signal transfer with (Twelve) optical Fiber using continuous flow injection for colored and turbid solutions, patent, no.4859. Central organization for Standardization and Quality Control, H01L31/0232, 38, Baghdad, Iraq classification, 2017. 23. Turkey, N.S. Multi snow- white light emitting diode analyser with twin detectors to Distinguish and Determine transparent (clear) and turbid solutions, patent, no.5605. Central Organization for Standardization and quality control, H05B37/02, 4, Baghdad, Iraq classification, 2018. 24. Shaker, I.M.A.; Turkey, N.S. Junction cell for On-line fast chemiluminescence reaction and light energy Transfer by optical fiber, patent, no.5330. Central Organization for Standardization and quality control,G01N2021/754, G01N21/76, 4, Baghdad, Iraq classification, 2018. 25. Shaker, I.M.A.; Turkey, N.S.; Hussein, F. A. The use of atomic forces microscopy in explaining and distinguish between Precipitated drugs using instrumental methods, patent, no. 5421. Central organization for Standardization and quality control, G01N33/0013, 6, Baghdad, Iraq classification, 2018. 82 26. Shaker, I.M.A.; Turkey, N.S. Differential mass accumulation by the effect of Neodymium in photometric Analysis ISNAG-Nd-10S-3D, patent, no. 5604. Central Organization for Standardization and quality control, B22F3/03, H01F41/02, 4, Baghdad, Iraq classification, 2018. 27. Shaker, I.M.A.; Turkey, N.S. Fixed magnetic field photometer for attenuated light measurements of incident irradiation of white snow LED (6500K colour temperature) array of six “NAG-MAG-A”, patent, no. 5777. Central Organization for Standardization and quality control, G01N21/62, 3, Baghdad, Iraq Classification, 2019. 28. Hayder, Q. M.; Nagam, S.T. Fluorimetric determination of cefotaxime sodium in pharmaceuticals preparation via the quenching of the continuous fluorescence of calcein using a homemade ISNAG-continuous flow injection analyser. International Eurasian Conference on Biological and Chemical sciences.2018, 26, 27, 259 -270. 29. Nagam, S.T.; Hayder, Q.M. Assessment of ISNAG fluorimeter (Total fluorescence measurements at +90 0 & -90 0 using four solar cell on each side for 100 mm distance at 2 mm path length) with well-known fluorescent molecules via CFIA. Iraq Journal of Science.2018, 59, 1B, 240-250. 30. Nagam, S.T.; Hayder, Q.M. New approach for the determination of ciprofloxacin hydrochloride using fluorescence resonance energy transfer (FTER) and continuous flow injection analysis via ISNAG-fluorimeter. J. pharm. sci. & Res.2019, 11, 4, 1563-1570. 31. Nagam, S.T.; Hussein, F.A. Continuous flow injection analysis- precipitation reaction of Ibuprofen with sodium nitro pros side using low pressure mercury lamp tube (UV-light) and detection of diverged Scattered lights (Visible light) at 2 x 90 0 using multi solar cells that covers 2 x 100 mm distance 2 mm path Length. Journal of Pharmacy and Biological Sciences.2018, 13, 1, 60-75. 32. Nagam, S.T.; Hussein, F.A. Newly developed method for determination indomethacin using phosphotungstic acid by continue flow injection analysis via homemade ISNAG- fluorimeter. Journal of Applied Chemistry.2018, 11, 1, 25-39. 33. Nagam, S.T.; Mustafa, K.K. Determination of mefenamic acid using C e (IV) sulfate as an oxidant reagent Via the use of the new mode of irradiation (array of six identical LEDS) and detection (twin solar cells)Through turbidity measurement by CFIA. I. J. R. P.C.2016, 6, 2, 271-290. 34. Retno, D.; Suswardana, Arief, B.; Windodo, W. The effect povidone iodine on the wound healing process: A study on fibroblast populated collagen lattice (FPCL) model. J. Med. Sci. 2014, 46, 3, 103-107. 35. Hubner, N.O.; Kramer, A. Review on the efficacy safety and clinical applications of polihexanide a modern wound antiseptic. Skin. Pharmacal. Physical.2010, 23, 17-27. 36. Shaker, I.M. A.; Turkey, N.S. Long distance chasing photometer for 300 mm length with 2 mm path length to chase and to accumulate output resulted from Attenuation incident light 0-180 0 and the Diverged of fluorescence light at 0-90 0 via two flow cells of 110 mm and 60 mm length ( NAG-ADF-300-2), patent, no.5776. Central Organization for Standardization and Quality Control, G01N2021/0328, 3, Baghdad, Iraq classification, 2019. 83 37. Zholt, K.; Tanya, S.; Yaroslavl, B.; Nataliya, K.; Svitlana, Z. Potentiometric sensor for the determination of povidone-iodine. Analytical &Bioanalytical Electrochemistry.2014, 6, 3, 367-378. 38. Anongtip, S.A.; Somchai, S.; Narubodee, P.; Wilaiporn, B.; Titpawan, N.; Rutthapol, S.; Teerapol, S. Quantitative analysis of povidone - iodine thin films by X - ray photoelectron spectroscopy and their physicochemical properties. Acta pharm.2017, 67,169-186. 39. Hiremath, G.C.; Kulkarni, R.M.; Nandibewoor, S.T. Kinetics of oxidative degradation and deamination of atenolol by aqueous alkaline permanganate. Indian Journal of Chemistry.2005, 44, 245-250. 40. Fernandes, N.; Nimdeo, M.S.; Choudhari, V.P.; Kulkarni; R.R.; Pande, V.V.; Nikalje, A.G. Dual Wavelength and simultaneous equation spectrophotometric methods for estimation of atenolol and indapamide in their combined dosage form. Int. J. Chem. Sci.2008, 6, 1, 29-35. 41. Bluman, A.G. Elementary statistics .3th ed.St .WCB/MC Graw– Hill, New York, 1997. 42. Miller, J.M.; Miller, J.C. Statistical and chemo metric for analytical chemistry. 5th ed. St. Person education limited, 2005. 43. The British Pharmacopoeia Commission Secretariat. Part of the Medicines and Healthcare products Regulatory Agency (MHRA). British Pharmacopoeia, Her Majesty's Stationery Office, London, UK, 2009.