263 © 2025 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License A New Green CFIA/MZT System for Spectrophotometric Determination of Anthranilic Acid Jehan Sh. Hussien 1* and Bushra B. Qassim 2 1,2 Department of Chemistry, College of Sciences, University of Baghdad, Baghdad, Iraq. *Corresponding Author. Received:24 April 2025 Accepted: 21 July 2025 Published: 20 October 2025 doi.org/10.30526/38.4.4155 Abstract Anthranilic acid (AA) was determined in pure material via a new system of the CFIA method that involves less chemical consumption (μL), is rapid, semiautomated, simple, and accurate. Two reactions of the AA were conducted; the first, with 4-amino antipyrine and potassium periodate as the oxidizing agent, yielded a pinkish-violet colored product, which was measured at λmax 550 nm. The second reaction, using 2,2`-bipyridine with FeCl3, produced a Pinkish-red product, which was measured at λmax 521 nm. Other chemical and physical factors that affect the stability of the complex products in the suggested process include flow rate, reagent concentration, sample volume, dispersion, and reaction coil. The linearity ranges were (10-400) and (5-150) µg.mL -1 , the average recovery percentages were 100.995 and 100.974, while the error percentages were 0.995 and 0.974, respectively. The detection limit values were 1.63 and 0.69 μg.mL -1 , respectively. A relative standard deviation of 0.28% and 0.65% was observed, and the sample throughput was 80 and 90 samples/hour, respectively. The devised approach was tested against the conventional method and was shown to be more sensitive. Moreover, a t-test was used to contrast "true value" with "practical value". At the 95% confidence level, it was found that the values were not significantly different. Keywords: Anthranilic acid, 4-Amino antipyrine, 2,2`-Bipyridine, Continuous flow-injection analysis merging zones technique, Potassium periodate. 1. Introduction Anthranilic acid (AA), synonymous with O-aminobenzoic acid, formula C6H4(NH2) (COOH), the M.wt was 137.14 g mol -1 , D was 1.412 g cm -3 , and (m.p.) was 146-148⁰ C and a b.p. of 200ºC (1). The compound is classified as aromatic due to its two neighboring functional groups: a carboxylic acid and an amine. This classification is further supported by the 'ortho' (a replaced benzene ring) structure, which renders the substance amphoteric. The AA derivatives are regarded as an inexpensive and effective starting point for the synthesis and manufacturing of several commercially available medications. A variety of AA analogues have been shown to have biological activity as well as possible anticancer, antibacterial, insecticidal, antiviral, and anti-inflammatory properties (2-4). An essential component of almost 70 medicines, 50 clinical medications, 20 agrochemicals, and a variety of fine chemicals is AA. Every year, more than 400,000 tons are generated for the https://orcid.org/0009-0007-2369-9132 mailto:jeehan.shojaa1105d@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3876-3161 mailto:bushrabqassim@gmail.com https://orcid.org/0009-0007-2369-9132 mailto:jeehan.shojaa1105d@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3876-3161 mailto:bushrabqassim@gmail.com https://orcid.org/0009-0007-2369-9132 mailto:jeehan.shojaa1105d@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3876-3161 mailto:bushrabqassim@gmail.com https://orcid.org/0009-0007-2369-9132 mailto:jeehan.shojaa1105d@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3876-3161 mailto:bushrabqassim@gmail.com https://orcid.org/0009-0007-2369-9132 mailto:jeehan.shojaa1105d@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3876-3161 mailto:bushrabqassim@gmail.com https://orcid.org/0009-0007-2369-9132 mailto:jeehan.shojaa1105d@sc.uobaghdad.edu.iq https://orcid.org/0000-0003-3876-3161 mailto:bushrabqassim@gmail.com IHJPAS. 2025, 38(4) 264 manufacturing of agrochemicals (5). Industrially, AA is an intermediate in the production of azo dyes and saccharin. Besides, AA and its analogues form a functional pharmacophore in the Glafenine, Floctafenine, and Fenamates. They are classified as anti-inflammatory drugs and antibiotics, and serve as the basis for the synthesis of medicinal products like Methaqualone, which is available under the trade name Mandrax (6). Several analytical methods for the determination of AA in various forms, including the spectrophotometric method (7), potentiometric titration (8), fluorometric (9), liquid chromatography (10), and high-performance liquid chromatography (HPLC) (11), are delicate. Still, they all require expensive and time-consuming equipment. Conversely, continuous flow-injection analysis merging zones (CFIA/MZ) is a green analytical method and environmentally friendly because it offers several advantages in analytical chemistry, including being economical, having reduced chemical consumption, being semiautomatic, and enabling high sampling (12-16). Many studies have worked in this field using a different FIA system (17-21) that has one channel manifolds/MZT. This study aims to determine of AA in pure material via the developed green CFIA systems through a new oxidative coupling reaction with a suitable coupling agent. 2. Materials and Methods A Shimadzu UV-1800 UV-VIS A spectrometer (Japan) was employed for determining all of the absorption in the batch process. A modified detection device has an F.C. composed of quartz silica (1 cm) with a volume of 80 μL. Scheme 1 shows the suggested FIA/MZT in this technique. Scheme 1. The manifold module of the developed CFIA/MZ system for determination of AA. A single-channel system that was developed using distilled water (D.W.) as a carrier at a rate of mL/min through the seven-three-way injection valve. A peristaltic pump (Master Flex C/L, USA) was used to squeeze the D.W., which travels at 90°, containing (3) loops made of Teflon (I.D. 0.5 mm), and was used for loading the organic acid AA or its derivative in L1, the coupling reagent in L2, and the oxidizing agent in L3. The substances are mixed using a glass R.C. (2 mm, I.D.). Each measurement and absorbance spectrum made during the developed FIA investigation was performed by the improved Optima photometer (301, the defendant), a single-beam VIS-Spectrophotometer, Japan. The absorbance can be measured with the Kompensograph C1032 (Siemens) or an optical multimeter (DT9205A, OVA, China). It was calculated as the average peak height of responses (n=3) in mV. The analytical class contributed all of the experimental materials, reagents, and solvents used in this IHJPAS. 2025, 38(4) 265 research, and all concentrations previously generated. Standard AA 98% (M.wt= 137 g.mol -1 BDH): 1000 µg. mL -1 . A stock solution of AA was prepared by weighing 0.0500 g of pure substance, dissolving it in 5 mL of methanol, and completing the volume to 50 mL in a volumetric flask with D.W. Reagent's stock; 4-amino antipyrine (4-AAP ≥ 97%) M.wt= 203 g.mol -1 , Merck Germany 1×10 -3 M, 0.0203 g of the reagent was dissolved in 100 mL volumetric flask with D.W, and 3×10 -3 M of 2,2-bipyridine (BPY 98%, M.wt=156.19 g.mol -1 , BDH) dissolved 0.0468 g in 5mL of ethanol, then completed with D.W. in a 100 mL volumetric flask. Oxidizing agent's stock solution; Potassium periodate (KIO4 99.8%) M.wt 230 g.mol -1 , Merck Germany 1×10 -4 M, was preparing by dissolved 0.0023 g in 100 mL vol. flask with D.W., FeCl3 37.5%) (M.wt= 162.2 g.mol -1 , Merck) 1×10 -4 M. A 0.0016 g of FeCl3 was dissolved in 0.5 mL of HCl 35%, w/w, 1.19 g.mL -1 , BDH, 0.1 mol.L -1 and completed to100 mL with D.W. Interference preparation: 0.05 g of each of the interfering substances (sucrose 99.9%, cellulose 99.8%, lactose 99%, glucose 99.9% and sodium citrate 99%) was dissolved in 50 mL of D.W. 3. Results 3.1. Suggested reactions of anthranilic acid with 4-amino antipyrine and bipyridine Determination of 25 μg.mL -1 AA based on coupling with 3×10 -3 M of 4-AAP in 5×10 -4 M of KIO4 as oxidizing agents in a 10 mL volumetric flask to create a colored complex (pink- violet) using a spectrophotometric approach, measured at λmax 550 nm, as shown in Figure 1 and Scheme 2. Figure 1. A- Absorption spectrum of 25 μg.mL -1 AA; (S) Pink-violet colored product against blank solution. b. Blank solution (4-AAP+KIO4). B- Linear calibration curve estimation for AA organic acid using the classical method. Spectrophotometric determination of 25 µg. mL -1 AA based on coupling with 2×10 -3 M BPY in the presence of 3×10 -4 M of FeCl3 as oxidizing reagent in a 10 mL volumetric flask to yield (pink-red) compound was scanned at λmax 521 nm as shown in Figure 2 and Scheme 3. y = 0.015x + 0.1047 R² = 0.999 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 15 20 25 30 35 40 45 50 A b s. [AA] (µg.mL-1) IHJPAS. 2025, 38(4) 266 Scheme 2. Proposed reaction of AA and 4-AAP. Figure 2. The Absorption spectrum of 25 μg. mL -1 AA; A- Pink-red colored product against blank solution BYP, FeCl3. B- Linear calibration curve for the determination of AA using the classical method. Scheme 3. Propose a mechanism of the reaction for AA with BPY using the batch method. 3.2. The manifold of the suggested CFIA/MZ system Chemical optimal conditions were examined for the organic acids (AA). A first experiment was determining the optimal concentration of the reagent (4-AAP), it’s found 3×10 -3 M while for the second reagent BPY optimal concentration was 4×10 -3 M, as seen in Figures 3 (A, B), other investigate was ideal concentration of the oxidative agents, KIO4, FeCl3 were 5×10 -4 M, 2×10 -4 M consequently, as seen in Figure 4 A and B. The better order added was examined, which was (O.A in L1, Ox in L2, and R in L3) for the 1st system and (O.A in L1, R in L2, and Ox in L3) for the 2nd system, as shown in Figures 5A and B. y = 0.0215x + 0.1497 R² = 0.9979 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 5 10 15 20 25 30 A b s. [AA] (µg.mL-1) B IHJPAS. 2025, 38(4) 267 Figure 3. Influence the concentration of reagents A- (AA, 4-AAP), B- (AA, BPY) in the FIA/MZ system. Figure 4. Best concentration of oxidizing agent for two CFIA systems. A- AA with KIO4. B- AA with FeCl3 Figure 5. Sequence of chemicals addition which loaded in L1, L2, L3. A- (AA+4-AAP), B- (AA+BPY). 3.3. Physical variables A flow rate was studied for both reactions, so the optimum flow rate was found to be 7-8 mL/min, as shown in Figure 6 A, B. While the optimum length of the R.C. of new CFIA systems was (100,50) cm consequently for the two reactions, as shown in Figure 7 A and B. 0 100 200 300 400 500 600 700 800 0 1 2 3 4 5 6 7 8 9 10A ve ra ge p e ak h e ig h t as m V ( n = 3 ) [4-AAP] × 10-3 (M) 0 50 100 150 200 250 300 350 400 450 500 0 1 2 3 4 5 6 7 8 A ve ra ge p e ak h e ig h t as m V ( n = 3 ) [BPY] × 10-3 (M) 0 100 200 300 400 500 600 700 800 0 1 2 3 4 5 6 7 8 9 10 A v e r a g e p e a k h e ig h t a s m V ( n = 3 ) [KIO4] × 10-4 , (M) 0 100 200 300 400 500 600 0 1 2 3 4 5 6 7 8 9 10 A v e r a g e p e a k h e ig h t a s m V ( n = 3 ) [FeCI3] × 10-4, (M) 0 100 200 300 400 500 600 700 O.A+R+Ox. O.A+Ox.+R R+Ox.+O.A O.A+Ox. & R Carrier A v er a g e p ea k h ei g h t a s m V ( n = 3 ) Sequence of addition in injection valve A 0 50 100 150 200 250 300 350 400 450 500 O.A+R+O O.A+O+R R+O+O.A O.A+O & R Carrier A v er a g e p ea k h ei g h t a s m V ( n = 3 ) Sequence of addition in injection valve B IHJPAS. 2025, 38(4) 268 Figure 6. Influence of flow rate A- AA with 4-AAP, B- AA with BPY. Figure 7. Effect of reaction coil of A- AA (4-AAP), B- AA (BPY) of CFIA systems. Therefore, the sample throughput is about 80,90 samples/hour, for the first and second reaction, respectively, which is calculated by finding the time required to inject the solutions into the seven three-way injection valve loops. It took nearly 15 seconds to find the time needed to reach the maximum peak height. It was nearly 30.25 seconds. The optimal size loops for the AA tow reaction were 40-40-40 cm; (78.50-78.50-78.50) µL and 30-30-40 cm; (58.88-58.88-78.50) µL, as shown in Figure 8 A and B. Figure 8. Effect of injected volume in CFIA systems AA A- with 4-AAP. B- with BPY. 0 100 200 300 400 500 600 700 800 5.0 6.0 7.1 8.0 9.1 10 A v a r a g e p e a k h ig h t a s m V ( n = 3 ) Flow rate (mL.min-1) A 0 50 100 150 200 250 300 350 400 450 500 6.0 7.0 8.0 9.0 10.0 11 A v a r a g e p e a k h ig h t a s m V ( n = 3 ) Flow rate (mL.min-1) B 0 100 200 300 400 500 600 700 50 100 200 A v er a g e p ea k h ei g h t a s m V ( n = 3 ) Length of R.C (cm) A 0 50 100 150 200 250 300 350 400 50 100 200 A v er a g e p ea k h ei g h t a s m V ( n = 3 ) Length of R.C (cm) B 0 100 200 300 400 500 600 700 800 20-40-40 30-40-40 40-40-40 60-40-40 30-20-40 30-30-40 30-60-40 30-30-20 30-30-30 30-30-60 A v e r a g e p e a k h e ig h t a s m V ( n = 3 ) * Length of loop (cm) for [AA (L1) KIO4 (L2)4-AAP (L3)] A 0 100 200 300 400 500 600 700 20-40-40 30-40-40 40-40-40 60-40-40 30-20-40 30-30-40 30-60-40 30-30-20 30-30-30 30-30-60 A v e r a g e p e a k h e ig h t a s m V (n = 3 ) Length of loop (cm) for [AA (L1)-BPY (L2)-FeCI3(L3)] B IHJPAS. 2025, 38(4) 269 3.4. Dispersion The estimated dispersion of AA is calculated using the equation, mD= Co/C, as shown in Table 1. Table 1. Dispersion value of the two reactions of [AA- 4-AAP], [AA-BPY] via flow injection. [AA-4-AAP] µg.mL -1 *Cₒ(cm) *Cmax(cm) *D [AA+BPY] µg.mL -1 *Cₒ(cm) *Cmax(cm) *D 50 5.8 0.083 1.261 25 4.0 3.3 1.212 200 10.8 0.1 1.286 75 8.0 6.2 1.290 *D; Dispersion, Co; The peak without dilution, C; The peak after dilution. 3.5. Calibration curve Preparation of a sequence of AA solutions from 1-1000 μg.mL -1 , followed by injection into the developed unit with reagent 4-AAP and the oxidative agent KIO4 (10-400 μg). mL -1 , the linear range of AA, as shown in Table 2 and Figure 9 A and B. Preparation of a sequence of AA solutions from 1-1000 μg.mL -1 , then injection on the developed unit with the reagent BPY and the oxidative agent FeCl3, the linear range of AA (5-150) μg.mL -1 , as shown in Figure 10 and Table 3. Table 2. Linear calibration curve for determination of AA with 4-AAP via FIAT using KIO4. Figure 9. A- Linear calibration curve for detection of AA, 4-AAP via the suggested CFIAT. B- Response variation with increasing of concentrations of (AA,4-AAP) via the developing CFIAT. y = 2.2627x + 231.61 R² = 0.9974 0 200 400 600 800 1000 1200 0 50 100 150 200 250 300 350 400 A v e r a g e p e a k h e ig h t a s m V ( n = 3 ) [AA] µg.mL-1 A [AA-4AAP] (μg.mL -1 ) Average response (y̅) (mV) RSD% S.E.M *E/y % 10 228 0.26 227.93±1.48 0.65 25 289 0.28 289.43 ±2 0.69 50 349 0.13 348.99 ±1.13 0.32 100 483 0.19 483.17 ±2.26 0.47 200 680 0.03 680.26 ±0.47 0.07 300 925 0.10 925.33±2.29 0.25 400 1121 0.52 1121 ±14.41 1.29 IHJPAS. 2025, 38(4) 270 Figure 10. A- Linear calibration curve for determination of AA via the suggested CFIAT.B- Response variation with increasing of concentrations of (AA, BPY) via the CFIA. Table 3. Linear calibration curve for evaluations of AA-BPY in FIAT using FeCl3.. * Average peak height (n=3) expressed as mV, * bx a at different concentration (x), * S.E.M ȳ ± t0.05 (𝛔𝐧−𝟏√𝐧) *𝐸/𝑦% =𝑡𝑡𝑎𝑏 𝑆𝐷/ √𝑛×100 %/ 𝑦. The repeatability was calculated for the first and second reactions by choosing two different concentrations of AA (50,200) μg and (25,75) μg.mL -1 ; consequently, it is included in the calibration curve using the developed CFIAT(n=8) as shown in Tables 4 and 5. Table 4. Repeatability of successive measurement of AA-4-AA, (n=8) using the FIA system. [AA-4-AAP] Found Error Rec% Erel% RSD% 50 51.293 1.293 102.586 2.586 0.398 200 197.827 2.173 98.913 -1.087 0.272 Table 5. Repeatability of successive measurement of AA-BPY, (n=8) using FIA system. [AA-BPY] Found Error Rec% Erel% RSD% 25 25.406 0.406 101.626 1.626 0.550 75 75.242 0.242 100.322 0.322 0.744 3.6. Analysis of variance Determination of the sum of squares (SS) of the variance values ( i) based on the regression's average value. The value (F) is obtained by dividing the result by the square root of the degrees of freedom (1), as shown in Tables 6 and 7. Table 6. Analysis of variance for the suggested CFIA system for AA-4-AAP. Source of Variation SS Df MS *Fcal. F crit *Between Groups (error) 1237303.1428 1 1237303.1428 21.4124 4.7472 *Within Groups (regression) 693412.57142 12 7784.380952 Total 193071557 13 y = 5.3936x + 113.24 R² = 0.9992 0 160 320 480 640 800 960 1120 0 25 50 75 100 125 150 A v e r a g e p e a k h e ig h t a s m V ( n = 3 ) [AA] µg.mL-1 A [AA-BPY] (μg.mL -1 ) Average response (y̅) (mV) RSD% S.E.M *E/y % 5 141 0.40 140.78±1.41 1.00 10 173 0.45 289.43 ±1.94 1.12 25 267 0.61 348.99 ±4.01 1.50 50 380 0.22 483.17 ±2.07 0.54 75 502 0.12 680 ±1.52 0.30 100 651 0.27 500.89±1.52 0.25 150 909 0.09 909.45 ±1.95 0.21 IHJPAS. 2025, 38(4) 271 Table 7. Analysis of variance for the suggested CFIA system for AA-BPY. Source of Variation SS Df MS *F cal. F crit *Between groups (error) 656211.50 1 656211.50 16.1153 4.7472 *Within groups (regression) 488636.00 12 40719.67 Total 11448475 13 *Between groups (error) ni (y i- y GM) 2 . *Within groups (regression) (ni-1) S1 2 , *F cal=F(S1) 2 / F(S2) 2 . 3.7. Analytical characteristic of the developed methods Calibration curve as S.E.M for determination of AA with (4-AAP,BPY) using a new CFIA/MZT is shown in Table 8. Table 8. Calibration curve as S.E.M for determination of AA with (4-AAP, BPY) using a new CFIA/MZT. Parameters [AA-4-AAP-KIO4] [AA-BPY-FeCl3] λmax (nm) 550 521 Regression equation; y bx a y 2.2627x 231.61 y 5.3936 x 113.24 Linear range (µg mL -1 ) 10- 400 5-150 ( ̅) 100.995 100.974 ( ̅) ( ) ( ) 0.995 0.974 0.280 0.647 Intercept (a); (a y– b x) 231.61 113.2409 Slope (b); (mL. µg -1 ) b Σi [ (xi –x̅) (yi – ӯ)]/Σi(xi –x̅) 2 ) 2 2.2627 5.3936 Linearity (R 2 %) 0.9974 0.9992 (r): r Σi [ (xi –x̅) (yi – ӯ)] [(Σi (xi –x̅) 2 ) (Σi (yi – ӯ) 2 )] 0.5 0.9987 0.9996 (Sb)Sb Sy/x /[ Σi( xi –x̅ ) 2 ] 0.5 0.0521 0.0685 (Sa) 𝑺𝒂= 𝑺𝒚𝒙 √𝒏Σ(𝒙𝒊−𝒙 ̅) 𝒊𝟐 10.8375 5.2644 (LOD) 1.6341 0.6855 (LOQ) 5.4469 2.2850 Through put (Sample .h -1 ) 80 90 Sy/x [ Σi (yi – i) 2 / (n – 2)] 0.5 ; i bxi a 19.1356 8.8677 C.l. slope (b) b ± tSb 2231.61±t 0.05 5.3936 ± t 0.0685 C.l intercept (a) a ± tSa 2.2627±10.837 113.24 ± t 5.2644 3.8. Interferences The interferences can be examined, including glucose, sucrose, lactose, cellulose, and sodium citrate, to evaluate the efficacy of the suggested CFIA/MZT, using 100 and 50 μg.mL -1 for the two reactions of AA, respectively, of the pure samples of the organic acid. No interferences were found when determining the organic acid AA using the CFIA technique, as shown in Table 9. The interference effect on the reaction of organic acids via the developed FI system is demonstrated in Table 10. IHJPAS. 2025, 38(4) 272 Table 9. Interference effect on the reaction of organic acids via the developed FI system. [AA-4-AAP-KIO4] [AA-BPY-FeCl3] Interference type Conc. of interferences (µg.mL -1 ) Average response (y̅) (mV) *Erel % *Rec % Conc. of Interferences (µg.mL -1 ) Average response (y̅) (mV) *Erel % *Rec % Standard Sucrose ---- 50 100 200 300 298 300 301 0.98 1.22 -0.52 0.98 100.98 101.48 99.48 100.98 ---- 25 50 100 361 542 539 536 0.38 0.12 -1.42 0.05 100.38 100.12 98.58 100.05 Lactose 50 100 200 461 457 456 1.29 -0.24 -0.78 101.29 99.76 99.22 25 50 100 531 533 536 0.63 -0.05 -1.14 100.63 99.95 98.86 Glucose 50 100 200 456 460 456 -0.66 -0.95 -0.08 99.22 100.95 99.92 25 50 100 362 361 360 1.54 0.78 -0.44 101.54 100.78 99.56 Cellulose 50 100 200 460 456 461 0.98 -0.66 1.22 100.98 99.34 101.22 25 50 100 361 361 361 0.88 0.40 0.69 100.88 100.40 100.69 Sodium Citrate 50 100 200 460 458 461 0.98 0.00 1.22 100.98 100.00 101.48 25 50 100 362 359 360 1.43 -1.26 -0.31 101.43 98.74 99.69 Table 10. Comptonization of the developed CFIA/MZT method for the determination of AA with other methods. 4. Discussion The dispersion value in this study was 1.2 for both reactions. A carrier was D.W. in the FIA/ MZT, which is in agreement with previous studies (22). The analysis of variance test is a way to discover if significant findings are obtained, which aid in determining the calibration curve required to accept the alternative hypothesis or reject the null hypothesis. To estimate (yi- i) 2 for (n-2), compute the sum of squares of the difference between the appraiser's yi values (S2) or the response's (yi) values, the anticipated error, and the called-for regression. The analytical characteristics of both methods (batch and FIA/ MZT) for determination of AA by two reactions [AA-4-AAP-KIO4] and [AA-BPY-FeCl3] such as (Rec%), (Erel%), (LOD) (20), (LOQ), (Ɛ), Sandals sensitivity (S), (Sy/x), Confidence limit of slope (CLb) and (CLa) were calculated (23). The confidence limit was 95% and the degrees of freedom (n-2). It is clear from the current findings, that the methods proposed in the research manuscript are new and have not been previously addressed in the field of flow injection analysis and have many advantages: simple, fast, sensitive, and highly modeled with analysis Analytical method Comment LOD Linear range Ref Spectrofluorometric Coupling reaction of the antibiotic with diazotized AA to form a yellow azo dye was shown absorption at 419 nm. 0.2813 μg.mL -1 0.5-60 μg.mL -1 27 Potentiometric titration The acid-base equilibrium of AA have been characterized micro dissociation constants ------- -------- 28 Electrochemical sensor Determine of AA has emerged as a possible moderator for use in co-polymer systems. 2.13 nM 1.0 ×10 -8 - 4.0×10 −4 M. 29 HPLC The separation was performed on a C18 column using the mobile phase-buffer (potassium dihydrogen ortho( 10 -50 μg/mL 0.99 μg.mL -1 30 Voltammetry Voltametric techniques in pH 5.0 phosphate buffer solutions with ionic strength 0.2M. 1.94 nM 1.0×10 -8 - 3.0×10 - 6 M 31 IHJPAS. 2025, 38(4) 273 per hour without the need for separation or extraction processes and the use of solvents or expensive equipment, especially since AA has great importance as it is used in industry as an intermediate material for the production of azo dyes and saccharin, and in the preparation of perfumes to imitate the scents of jasmine and orange, and in pharmaceutical preparations as a diuretic, which is in agreement with previous data (24-27). The AA spiked with a half, equal, and double increments of interfering concentration. Absolute error and increase in the interference concentration do not affect the response intensity value with high organic acid recoveries (28, 29) 5. Conclusion The proposed method in this study is classified as green chemistry for the determination of aromatic organic acid (AA), which has a sweetish taste, using a new type of flow injection analysis technology (friendly to the environment) by consuming small amounts of microliter volumes of the organic acid. Toxic organic reagents were used to complete the reactions. Following an examination and evaluation of the scientific literature within the CFIA systems, it was found that there is currently no published work for the determination of the AA in pure material and estimation of its biological activities. So, the unique and novel study concept was to use the CFIA/MZT for the spectrophotometric determination of AA. The advantages of the proposed CFIA method include simplicity, sensitivity, a wide operating range, semi- automated determination of analyte without the need for a pretreatment step or extraction, high recovery, and its highest applications in analytical chemistry. Acknowledgment The authors thank their colleagues at the College of Sciences, University of Baghdad, for their cooperation in completing this work, and they appreciate everyone who participated in the study. Conflict of Interest The authors declare that they have no conflicts of interest. Funding None. Ethical Clearance The Scientific Committee at the Department of Chemistry, College of Science, University of Baghdad, has approved this work. References 1. Ahmed AA, Musa SA, Fugu MB, Mohammed AI, Adam HB, Wakil IM. A comprehensive review on anthranilic acid-derived Schiff bases and their metal chelates: Structures and applications. Chem Rev Lett. 2023; 6(3):350-389. https://doi.org/10.22034/crl.2023.401158.1227. 2. 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