Sequential Injection Analysis Method Research Articles

Development of a sequential injection analysis method for the automatic speciation of inorganic selenium in water samples

Selenium is an important micronutrient for the human body, but biological concentrations on either side of its narrow nutritional window can lead to serious health impacts. Furthermore, Se(IV) has been reported to be more toxic than Se(VI). Therefore, prudent control and monitoring of Se intake should involve speciation of this element. Herein, the development and validation of an automatic sequential injection analysis (SIA) system and method for the speciation of Se(IV) and Se(VI) in water samples is reported for the first time. This method is based on the starch-iodide reaction using Se(IV) as the oxidant, which subsequently forms a blue compound with starch. Total selenium is determined after an online pre-reduction of Se(VI) to Se(IV) with HCl at 75 °C. The concentration of Se(VI) is then calculated as the difference between the total Se and Se(IV) concentrations. The calibration curves for Se(IV) and total Se are characterised by linear ranges of 0 – 1.5 mg L-1 and 0 – 2.5 mg L-1, limits of detection (LODs) of 0.02 mg L-1 and 0.07 mg L-1, and averaged relative standard deviations (RSDs) (n = 3) of 1.33 % and 2.95 % respectively, with a sampling rate of 10 h−1. The newly developed SIA method has been successfully applied to drinking water samples spiked with both Se species and good recoveries in the range 94 – 109 % have been achieved.

Automated approach for the evaluation of glutathione-S-transferase P1-1 inhibition by organometallic anticancer compounds

A novel automated method based on sequential injection analysis (SIA), a non-segmented flow injection technique, was developed to evaluate glutathione S-transferase P1-1 (GST P1-1) activity in the presence of organometallic complexes with putative anticancer activity. The assay is based on the reaction of L-glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) in the presence of GST P1-1 to afford the GS-DNB conjugate and the reaction may be monitored by an increase in absorbance at 340 nm. A series of ruthenium, iron, osmium and iridium complexes were evaluated as GST P1-1 inhibitors by evaluating their half-maximal inhibitory concentration (IC50). An iridium compound displays the lowest IC50 value of 6.7 ± 0.7 µM and an iron compound displays the highest IC50 value of 275 ± 9 µM. The SIA method is simple to use, robust, reliable, and efficient and uses fewer reagents than batch methods and each analysis takes only 5 minutes.

Automatic Identification of Myeloperoxidase Natural Inhibitors in Plant Extracts.

The aim of this study is the development of an automated method for myeloperoxidase activity evaluation and its application in testing the inhibitory action of different plant extracts on the activity of the enzyme. This enzyme has its concentration increased in inflammatory and infectious processes, so it is a possible target to limit these processes. Therefore, an automatic sequential in-jection analysis (SIA) system was optimized and demonstrated that it is possible to obtain results with satisfactory accuracy and precision. With the developed method, plant extracts were studied, as promising candidates for MPO inhibition. In the group of selected plant extracts, IC50 values from 0.029 ± 0.002 mg/mL to 35.4 ± 3.5 mg/mL were obtained. Arbutus unedo L. proved to be the most inhibitory extract for MPO based on its phenolic compound content. The coupling of an automatic SIA method to MPO inhibition assays is a good alternative to other conventional methods, due to its simplicity and speed. This work also supports the pharmacological use of these species that inhibit MPO, and exhibit activity that may be related to the treatment of infection and inflammation.

Sequential injection spectrophotometric method for screening bromate produced as an ozonation water disinfection by-product.

The aim of this work was the development of an automatic sequential injection analysis method to monitor the ozonation process for water disinfection. The determination was based on the reaction between bromate and o-dianisidine in the presence of bromide in acidic medium. The determination parameters were studied and adjusted to enable bromate quantification in the range 0.35-4.0mg BrO3-/L with a limit of detection of 20μg BrO3-/L. The choice of a sequential injection procedure enabled a minimal consumption of reagents and no need for sample pre-treatment. The developed sequential injection proved to be accurate with < 5% relative deviation when compared to ICP-MS and an average of 101% in recovery percentages studies. It was effectively applied to monitor an ozonation process enabling the follow-up of the process with real-time quantification of the bromate content.

Employing sequential injection analysis technique and chemometric optimization approach for developing diltiazem assay method

Sequential injection analysis (SIA) technique and chemometric optimization were employed, for the first time, to develop a new method for the assay of diltiazem in pharmaceutical formulations. The method was based on the spectrophotometric detection of permanganate reduction by diltiazem in acidic media at 526 nm. The 23 full factorial design and response surface plot were adopted to optimize permanganate concentration, acid concentration and flow rate. The method was validated as per the International Union of Pure and Applied Chemistry (IUPAC) guidelines. Relatively, wide Beer's law limit (10-180 mg/L) with satisfactory linearity (correlation coefficient = 0.9996) and recovery (95.4-98.1%) were obtained. The method also recorded good repeatability (RSD = 1.19-1.64%, n = 10) and intermediate-precision (2.84%, n = 5, once per a day). The method is sensitive enough for determining diltiazem in pharmaceutical formulations with limits of detection (1.30 mg/L) and quantification (3.94 mg/L). The method is rapid with sample frequency of 32 sample/h. The method was applied to real pharmaceutical samples and the results obtained were realized by parallel analysis by another validated method. The method enjoys the potentials of SIA and chemometric optimization with respect to good accuracy, precision, sensitivity and rapidity. The method is also inexpensive in terms of instrumentation as well as the consumption of reagents and samples. Besides good safety for handling solutions, the SIA technique offers significant waste minimization and manpower reduction to the proposed method. Therefore, the SIA method is suitable in pharmaceutical laboratory for quality control purpose.

Exploitation of reaction mechanisms for sensitivity enhancement in the determination of phosphorus by sequential injection analysis

The molybdenum blue method using antimony and ascorbic acid was studied for the determination of phosphorus (as orthophosphate) by means of sequential injection analysis (SIA). In order to avoid the interference of the Schlieren effect in the photometric measurements a stopped-flow kinetic approach was adopted monitoring the absorbance of the reaction bolus inside the flow cell. Aiming at enhancing the sensitivity of the method, the effect of the order of addition of the reactants was studied. It was found that the best sensitivity was attained by adding separate reagents and acidifying only after the phosphate, molybdate and antimony solutions were already mixed; the reductant (ascorbic acid) was then added. In this way a sensitivity enhancement in excess of 10 times was obtained when compared to the addition of the phosphate solution to the acidified mixture of molybdate and antimony.It is proposed that the difference in sensitivities could be explained by the existence of different mechanisms for the formation of the intermediate phosphoantimonyl molybdic acid (PMA). Thus the selected sequence in the order of addition, where sulphuric acid is added to the mixture of the other reactants would lead to higher production of PMA in turn conducting to a faster reduction reaction.The resulting SIA method was validated finding limits of detection (3s/m) and quantification (10s/m) of 0.0077 and 0.026 mg-P L−1 respectively. Linearity was confirmed in the range up to 2 mg-P L−1. Precisions (sr, n = 10) were in the range 1.8%–4.0%. 32 water samples of different types and origins were analysed by the proposed method and by ion chromatography, obtaining a regression curve y = 0.990× - 0.0019, with a determination coefficient R2 = 0.973.

Determination of Formaldehyde in Tofu by Sequential Injection Analysis Using Acetoacetanilide as Hantzsch Reaction Reagent

Formaldehyde is a toxic and dangerous compound that is still often used freely by irresponsible traders or producers as food additives. At low concentrations, formaldehyde can cause eye, nose, throat and skin irritations. Exposure to large amounts of formaldehyde can cause severe pain, vomiting, coma, and even death. According to the International Program on Chemical Safety (IPCS), the threshold for formaldehyde in food that can still be tolerated in adult bodies is 1.5 mg to 14 mg per day while in a drinking water is 0.1 ppm. Determination of formaldehyde generally employ a titration method, which requires a large amount of sample and reagent, and long analysis time. This work aims to determine the formaldehyde in tofu commercially available in traditional markets of Malang City by Sequential Injection Analysis method using acetoacetanilide (AAA) at optimum conditions. Determination of formaldehyde is based on the Hantzsch reaction, which involves a cyclization between AAA and formaldehyde in the presence of ammonia so that it will produce a yellow dihydropyridine derivative product detected at a wavelength of 359 nm. The optimum condition for the formation of the reaction product occurred at an AAA concentration of 0.03 M, an ammonium acetate concentration of 1.5 M, a reaction time of 50 s and a flow rate of 75 µL/s. Excellent sensitivity and accuracy could be attributed to our developed formaldehyde detection method as the detection limit of 4.5 ppb and the recovery values of > 95 were achieved. Low reagent consumption, less waste production, and high throughput analysis are other advantages of our proposed method.

Sequential Injection Analysis for Determination of Ammonia in Livestock Wastewater Using Acetoacetanilide as Hantzsch Reaction Reagent

Ammonia is a dangerous chemical compound in high concentrations. About 80-90% of ammonia total emissions come from animal fertilizer that produced from livestock, such as cattle farming. Based on the Minister of Environment Regulation Number 5 of 2014, the ammonia level in waste water standards for livestock activities is 25 mg/L. Some of the most common methods that used for determining ammonia are selective spectrophotometry and selective electrode, but the method has a disadvantage such as time consuming and low sensitivity. To overcome this problem, the aim of this study was to determine the dissolved ammonia levels in the liquid waste stream of cattle farms using the Sequential Injection Analysis (SIA) method by UV-Vis spectrophotometry at a wavelength of 307 nm based on the Hantzsch reaction using acetoacetanilide and formaldehyde reagents in acetate solution to form Dihydropyridine derivatives. The optimum condition of product formation occured in the order of reagents and sample segmentation of formaldehyde (R)-ammonia (S)-AAA(R) acetate with the volume ratio 75μL: 50μL: 100μL, respectively. The optimum conditions of the proposed method are 0.06 M of AAA concentration, 15% of formaldehyde concentration, reaction time of 40 s, and flow rate of 175 μL. High sensitivity (LOD:0.122 mg/L), fast analysis (175 s/sample) and high accuracy (recovery test > 94%), less and harmless waste could be attributed as advantages of the proposed SIA method.

Automatic methodologies to perform loading and release assays of anticancer drugs from mesoporous silicon nanoparticles

Two automatic methodologies were developed to perform the loading and release assays of drugs from porous silicon (PSi) nanoparticles. 5-Fluorouracil (5-FU) was selected as a model drug, and different functionalized PSi nanoparticles were used. While for drug loading studies the reproducible characteristics of flow systems were explored regarding mixture and volumes taken, in the drug release flow methodology versatility in accommodating different devices around the valve were tested. Fluorescent properties of 5-FU were used with detection limit of 9 × 10−4 mg L−1 and a linear response up to 5 mg L−1 The drug loading and release procedures were optimized in sequential injection analysis (SIA) systems obtaining a huge economy regarding the time spent (4 min towards 2 h). Batch and SIA methods were tested and compared for the different behaviours of the PSi nanoparticles towards both methodologies and no noteworthy differences were obtained with Student's t-test for loading with a calculated t value of 2.04 showing the absence of statistical differences. All the results present a RSD less than 10%. So, the developed automatic methodologies are a viable alternative to load drugs and to study the release profiles from PSi nanoparticles.

Sequential injection analysis for the determination of fluoroquinolone antibacterial drug residues by using eosin Y as complexing agent

A sequential injection analysis (SIA) method was developed for the rapid and sensitive determination of fluoroquinolone residues, including norfloxacin, ciprofloxacin and enrofloxacin, in fish samples. The method is based on the reaction between fluoroquinolone drug and eosin Y in Britton-Robinson buffer (pH 2.0), forming pink colored complexs with maximum absorptions at 522, 525 and 527 nm for norfloxacin, ciprofloxacin and enrofloxacin, respectively. Linearity ranges were found to be 0.05–10.0 mg L−1 (r2 = 0.9996), 0.1–10.0 mg L−1 (r2 = 0.9995) and 0.05–10.0 mg L−1 (r2 = 0.9997) for norfloxacin, ciprofloxacin and enrofloxacin, respectively. The detection limit was found to be in the range of 0.013–0.019 mg L−1. The method was tested and validated for various parameters according to main guidelines. The proposed SIA method was successfully applied for the determination of fluoroquinolone drug residues in fish samples with the sampling rate of 47 h−1. The results demonstrated that the method is accurate, precise and reproducible, while being simple, rapid, economical and less time consuming. It can be suitably applied for the estimation of fluoroquinolone drug residues in routine quality control.

Simultaneous SIA analysis of pH and total acidity measurements in milk

The development of a sequential injection analysis method for automatic determination of pH and total acidity in milk is proposed. For this purpose, firstly, three micro-sized composite pH electrodes were prepared and tested. A microliter dead volume flow-cell was designed for composite pH and reference electrodes. Potentiometric performance characteristics of the pH electrodes were examined by a computer-controlled potentiometric system in both stagnant and flowing environments. Three different micro-columns with the best working pH electrode were applied on the flowing stream line in the sequential injection analysis system to fulfill the titration efficiency for total acidity. The sequential injection analysis system was optimized for two parameters (pH and total acidity) by testing all the variables. Mobile phase concentration, volume and flow-rate together with sample concentration, volume and sample flow-rate were fixed for the best stable values by arranging the experimental programming steps. Total acidity of ten different milk samples was analyzed using the sequential injection analysis system developed. The same samples were also titrated in the classical way using phenolphthalein indicator for the comparison. The results of both methods analyzed statistically by SPSS software were found to be compatible with each other. pH analysis of ten different milk samples carried out by using the sequential injection analysis system were also measured with a glass electrode. The results obtained by both methods analyzed by SPSS were also found to be compatible with each other.

Determination of dasatinib in the tablet dosage form by ultra high performance liquid chromatography, capillary zone electrophoresis, and sequential injection analysis.

Dasatinib is a novel oral prescription drug proposed for treating adult patients with chronic myeloid leukemia. Three analytical methods, namely ultra high performance liquid chromatography, capillary zone electrophoresis, and sequential injection analysis, were developed, validated, and compared for determination of the drug in the tablet dosage form. The total analysis time of optimized ultra high performance liquid chromatography and capillary zone electrophoresis methods was 2.0 and 2.2 min, respectively. Direct ultraviolet detection with detection wavelength of 322 nm was employed in both cases. The optimized sequential injection analysis method was based on spectrophotometric detection of dasatinib after a simple colorimetric reaction with folin ciocalteau reagent forming a blue-colored complex with an absorbance maximum at 745 nm. The total analysis time was 2.5 min. The ultra high performance liquid chromatography method provided the lowest detection and quantitation limits and the most precise and accurate results. All three newly developed methods were demonstrated to be specific, linear, sensitive, precise, and accurate, providing results satisfactorily meeting the requirements of the pharmaceutical industry, and can be employed for the routine determination of the active pharmaceutical ingredient in the tablet dosage form.

Miniaturizing and automation of free acidity measurements for uranium (VI)-HNO3 solutions: Development of a new sequential injection analysis for a sustainable radio-analytical chemistry

A miniaturized and automated approach for the determination of free acidity in solutions containing uranium (VI) is presented. The measurement technique is based on the concept of sequential injection analysis with on-line spectroscopic detection. The proposed methodology relies on the complexation and alkalimetric titration of nitric acid using a pH 5.6 sodium oxalate solution. The titration process is followed by UV/VIS detection at 650nm thanks to addition of Congo red as universal pH indicator. Mixing sequence as well as method validity was investigated by numerical simulation. This new analytical design allows fast (2.3min), reliable and accurate free acidity determination of low volume samples (10µL) containing uranium/[H+] moles ratio of 1:3 with relative standard deviation of <7.0% (n=11). The linearity range of the free nitric acid measurement is excellent up to 2.77molL−1 with a correlation coefficient (R2) of 0.995. The method is specific, presence of actinide ions up to 0.54molL−1 does not interfere on the determination of free nitric acid. In addition to automation, the developed sequential injection analysis method greatly improves the standard off-line oxalate complexation and alkalimetric titration method by reducing thousand fold the required sample volume, forty times the nuclear waste per analysis as well as the analysis time by eight fold. These analytical parameters are important especially in nuclear-related applications to improve laboratory safety, personnel exposure to radioactive samples and to drastically reduce environmental impacts or analytical radioactive waste.

A Sequential Injection Analysis Method for the Determination of Glyphosate and Aminomethylphosphonic Acid in Water Samples

A conventional laboratory method for the determination of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) is high performance liquid chromatography (HPLC) followed by post column derivatization with o-phthaldialdehyde (OPA). However, AMPA is partly decomposed in the process causing a deviation in the AMPA detection or even making a simultaneous detection of glyphosate and AMPA with the described postcolumn procedure impossible. We used a compact sequential injection analysis system and optimized the process conditions for both analytes independently from each other. The process conditions were adjusted to the different chemical characteristics of AMPA (primary amine) and glyphosate (secondary amine), which needs to be oxidized to a primary amine prior to the derivatization. An ion exchange column was included in the system to eliminate amines interfering with the method. Limits of detection of 16 and 9 µg L -1 for glyphosate and AMPA, respectively are similar to those achieved with HPLC methods reported in literature, but compared to conventional HPLC methods only small amounts of reagents are consumed.

Sequential Injection Analysis with Square Wave Voltammetry (SI-SWV) Detection for Investigation of Adsorption of Picloram on a Clay Soil

This paper describes a sequential injection analysis method, with detection by square wave voltammetry using a hanging mercury drop electrode, to determine the herbicide picloram in soil extracts (0.010 mol L-1 CaCl2). The method was applied for determination of the Freundlich parameters related to adsorption of picloram on a tropical soil sample. The major advantage of the proposed method over the conventional chromatographic methods is the high sampling throughput of 190 analyses per hour, with detection and quantification limits of 0.036 and 0.12 mg L-1, respectively. The Kf (0.0016 ± 0.0005 µmol1-1/n L1/n kg-1) and 1/n (0.85 ± 0.06) obtained by the proposed method did not differ from those determined by liquid chromatography with UV detection.

High-throughput sequential injection assay method for chlorpromazine

This manuscript reports the optimization and validation of a new, rapid, reagent-saving and environmentally-safe method for chlorpromazine assay in pharmaceutical formulations utilizing sequential injection analysis (SIA) technique. Despite its benefits over univariate approach, chemometrics has been rarely exploited for optimizing SIA methods. Moreover, in those methods, some conditions have not been considered in optimization process. In the current study, more conditions, namely volumes and concentrations of reagents, have been optimized by chemometrics for the first time. A developing oxidation reaction of chlorpromazine by permanganate in acidic media was adopted. A spectrophotometric detection of the reduction of permanganate was applied at 526 nm using miniaturized fiber optic devices. The 24 full-factorial design and response surface approaches were exploited for method optimization. The method was validated and realized by a British Pharmacopoeia method. Experimental conditions and analytical aspects of the current SIA method were compared with those of a previous SIA method. Some analytical aspects were comparable while other aspects demonstrated better results from the current method than those from the previous one. The current method is rapid with a sample frequency of 51.4 samples/h. It is also reagent-saving with a total volume of consumed reagents and sample of 260 μL as well as environmental-safety with a total volume of waste production of 1260 μL. The method is therefore efficient for industrial-scale pharmaceutical analysis.

Screening of conditions controlling spectrophotometric sequential injection analysis

BackgroundDespite its potential benefits over univariate, chemometrics is rarely utilized for optimizing sequential injection analysis (SIA) methods. Specifically, in previous vis-spectrophotometric SIA methods, chemometrically optimized conditions were confined within flow rate and reagent concentrations while other conditions were ignored.ResultsThe current manuscript reports, for the first time, a comprehensive screening of conditions controlling vis-spectrophotometric SIA. A new diclofenac assay method was adopted. The method was based on oxidizing diclofenac by permanganate (a major reagent) with sulfuric acid (a minor reagent). The reaction produced a spectrophotometrically detectable diclofenac form. The 26 full-factorial design was utilized to study the effect of volumes of reagents and sample, in addition to flow rate and concentrations of reagents. The main effects and all interaction order effects on method performance, i.e. namely sensitivity, rapidity and reagent consumption, were determined. The method was validated and applied to pharmaceutical formulations (tablets, injection and gel).ConclusionsDespite 64 experiments those conducted in the current study were cumbersome, the results obtained would reduce effort and time when developing similar SIA methods in the future. It is recommended to critically optimize effective and interacting conditions using other such optimization tools as fractional-factorial design, response surface and simplex, rather than full-factorial design that used at an initial optimization stage. In vis-spectrophotometric SIA methods those involve developing reactions with two reagents (major and minor), conditions affecting method performance are in the following order: sample volume > flow rate ≈ major reagent concentration >> major reagent volume ≈ minor reagent concentration >> minor reagent volume.

11-Molybdobismuthophosphate—A new reagent for the determination of ascorbic acid in batch and sequential injection systems

The guanidinium salt of the new heteropolymolybdate 11-molybdobismuthophosphate Gua(6)PBiMo(11)O(40) (11-MBP) was synthesized, characterized and used as a reagent for batch spectrophotometric (SP) and sequential injection determination of ascorbic acid (AsA). When compared to other Keggin's heteropolyanions, the reduction of 11-MBP with AsA is both fast and maximal within a pH range of 1.6-2.0. The stoichiometry of the reaction was determined using molar ratio and continuous variation methods and was shown to be 1:1. The molar absorptivity of the reduced form of 11-MBP was 6.0 x 10(3)L mol(-1)cm(-1) at 720 nm. The reaction is also specific for AsA. Only cysteine, hydroquinone and hydroxyacids were found to interfere with the reaction, while no interference was observed with the common reducing agents, including reducing sugars, catecholamines, nitrite, sulfite and iron(II) ions. Batch SP and sequential injection analysis (SIA) systems were developed for the determination of AsA, with calibration ranges of the SP methods at 2x10(-6)-8 x 10(-5)M for a 10mm cell and 5 x 10(-7)-3 x 10(-5)M for a 50mm cell and a limit of detection at 3 x 10(-7)M. The linear range of the SIA method was 6 x 10(-6)-5 x 10(-4)M, with a detection limit of 2 x 10(-6)M and a sample throughput of 15 h(-1). The proposed methods were successfully used for the determination of AsA in both pharmaceuticals and fruit juices, and the results were consistent with those provided by the 2,6-dichlorophenolindophenol method.

Exploiting sequential injection analysis technique to automate on-line sample treatment and quantitative determination of morphine in human urine

A simple uni-stream sequential injection analysis (SIA) manifold was developed to automate a method for the assay of morphine in human sample. The proposed SIA method includes on-line sample treatment, coupling reaction and spectrophotometric measurement. A rapid algorithm controlled the adopted procedure was critically programmed. For sample treatment, solid-phase extraction (SPE) was carried out into a homemade microcolumn, installed in the SIA manifold. Sufficient sample clean-up, extraction and preconcentration were obtained by SPE. A coupling reaction of morphine with diazonium salt of aniline hydrochloride was adapted to SIA. The product of the reaction, an azo-morphine derivative, was spectrophotometrically detected at 390 nm. Parameters that influenced the efficiency of the proposed method, including solution volumes, diazonium concentration, flow rate and residence time, were optimized. The proposed method was linear in a range of 0.10–2.5 μg ml −1. The limits of detection and quantification were 0.023 and 0.076 μg ml −1, respectively. The detectability of the method was enhanced by the preconcentration and the use of an extended pathlength (50 mm) of a flow cell. The method was validated by an HPLC method. Comparable results with respect to accuracy (recovery 96.3–97.1), repeatability (R.S.D. < 2.4%) and intermediate precision (R.S.D. < 3.1) were gained. The full-automation and miniaturization of the utilized technique offer rapidity, safety in handling urine sample and reagents as well as reduction of reagent and sample volumes. The method is suitable for the application in forensic cases as an initial test and clinical analysis to prevent overdose-induced toxicity.

DPPH Radical Scavenging Activity of Several Naturally Occurring Coumarins and Their Synthesized Analogs Measured by the SIA Method

ABSTRACTCoumarins, naturally occurring compounds derived from benzopyran, have recently been studied extensively for their antioxidant properties. A lot of coumarins have been isolated and identified from natural sources and many others have been synthesized. It is also known that pharmacological and biochemical properties and thus also therapeutic application of simple coumarins depend upon the pattern of their substitution.As a part of studies of biological effects, four naturally occurring coumarins and 18 synthesized analogs of several compounds were assayed for 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity. For this purpose the highly reliable DPPH test modified to be performed by sequential injection analysis (SIA) system was used. This in our laboratory-developed method was originally proposed for antioxidant screening of large series of plant extracts. In this assay, the DPPH test using the SIA method was used for fast and sensitive evaluation of EC50 of coumarins. The evaluation of EC50 of a single compound takes only 15 to 30 min. The structure-activity relationships of tested compounds are also established. The results verified 7,8-dihydroxy-4-methylcoumarins as excellent DPPH radical scavengers. Obtained results correspond with those of other studies and suggest the SIA procedure as a suitable method for fast and sensitive antioxidant analysis of various types of compounds.