Reverse Flow Injection Analysis Research Articles

A Novel Flow Injection Method with Chemiluminescence Detection for the Determination of Carmoisine in Gelatin Desserts.

Carmoisine dye, a red azo food colorant commonly utilized to impart a red color to synthetic food products, is the subject of this study. Here, we present a novel reversed flow injection analysis with a chemiluminescence detection (FIA-CL) method employing a newly developed homemade flow cell to determine carmoisine dye. This developed method is based on the inhibition effect of the dye on the chemiluminescence light (CL) emission generated from a luminal-hypochlorite system, whereby the reduction in CL intensity correlates directly with the concentration of carmoisine dye. Investigations into various analytical parameters were conducted to enhance method efficiency and applicability. A linear calibration graph of 4.0 to 100.0µg mL-1 was established (R² = 0.9993), with a detection limit of LOD = 2.93µg mL-1. Subsequent application of the proposed method to analyze gelatine dessert samples yielded results in reasonable agreement with those obtained using the reported HPLC method, as evidenced by student t-test and F-test analyses.

Pseudo-water-soluble Fe2O3 as Nanozyme catalyzed chemiluminescent reaction for detection of brilliant blue in gelatin and beverages

Converting solid iron oxide nanoparticles into a “pseudo-water-soluble” form before applying them to chemiluminescent reactions leads to enhance the chemiluminescence intensity. Using 8-hydroxyquinoline as a colloidal agent, a new, fast, and simple method of synthesizing pseudo-water-soluble Fe2O3 nanoparticles was developed. SEM, VSM, SAED, HRTEM, XRD, FTIR, and EDS techniques were used to characterize the synthesized Fe2O3 nanoparticles. Fe2O3 nanoparticles synthesized in this study have superior peroxidase-like activity (POD-like) and are stable under a wide range of pH and temperature. The chemiluminescence reaction of luminol-H2O2 is intensified and accelerated by a colloidal solution of Fe-nanoparticles/8-hydroxyquinoline. Reverse-flow injection analysis was employed to determine brilliant blue. A chemiluminescent sensing method based on iron oxide nanozymes was utilized for sensitive detection of the brilliant blue synthetic dye, achieving a limit of detection of 0.06 mg/L and a dynamic linear range of 0.1 to 50 mg/L. The recovery and relative standard deviations of real samples were found to be 97.83–99.93% and 0.09–3.07%, respectively. An analysis of a sample, from injection to obtaining the maximum peak, could be performed in less than one minute.

Spectrophotometric determination of amoxicillin in pharmaceutical formulations using normal and reverse flow injection analysis systems: A comparison study

The combination of flow injection technique and spectrophotometric detection can be employed for the rapid and low-cost methods for antibiotic assay. In this work, two flow injection analysis (FIA) systems, normal and reverse (nFIA and rFIA), combined with UV-Vis spectrophotometric technique were used for amoxicillin determination in its pure and pharmaceutical preparations. A colorimetric coupling reaction between amoxicillin and diazotized p-toluidine produced a bright yellow azo dye and quantified at a maximum wavelength of 426 nm. For nFIA and rFIA procedures, the amoxicillin calibration graphs had an RSD of less than 2% and were linear in the ranges of 5-200 and 1-140 µg/mL, respectively. The limits of detection and quantification were 1.41 and 4.71 µg/mL of amoxicillin for nFIA and 0.39 and 1.31 µg/mL for rFIA, respectively. The physical and chemical factors that could influence color sensitivity were investigated. Various commercial formulations containing different amounts of amoxicillin were successfully tested using the developed approaches, which demonstrated to be easy, rapid, reproducible, and interference-free.
 KEY WORDS: Amoxicillin, Normal FIA, Reverse FIA, p-Toluidine, Diazotization reaction
 Bull. Chem. Soc. Ethiop. 2024, 38(3), 577-590. 
 DOI: https://dx.doi.org/10.4314/bcse.v38i3.3

Normal and Reverse Flow Injection Analysis Methods for Estimation of Mesalazine in Pharmaceutical Dosage Forms

Determining the actual amounts of active ingredients in various pharmaceutical commercial forms is still receiving a lot of attention. Two flow injection analysis (FIA) methods were suggested for determination of mesalazine (MES) in pharmaceutical forms. Normal and reverse FIA systems (nFIA and rFIA) combined with UV-Vis spectrophotometric technique were used for the analysis. The methods involved using two mods of FIA systems for measuring a colored product result from coupling of MES with 2,2'-dihydroxybiphenyl after oxidized with sodium periodate in alkaline medium. The absorbance of the red colored dye was measured at maximum wavelength of 500 nm. The calibration graphs for MES were linear in the ranges 2.5-200 and 0.5-60 µg/mL with RSD of best than 3% for both methods respectively. Also the limits of detection were 1.2 and 0.2 µg/mL and limits of quantitation were 3.6 and 0.7 µg/mL of MSL for nFIA and rFIA systems respectively. All physical and chemical conditions of flow systems such as flow rate, reaction coil length and reagent concentrations were carefully studied. The proposed methods were applied for determining MES in four commercial tablets without any interferences.

Live synthesis of selective carbon dots as fluorescent probes for cobalt determination in water with an automatic microanalyzer

A new strategy integrating the straight synthesis of carbon dots (CDs) and their direct use for the determination of heavy metals by means of fluorescence quenching is presented. The proposal consists of a modular analyzer, which includes a low temperature co-fired ceramics (LTCC) microreactor for the synthesis of CDs and a cyclic olefin copolymer (COC) microfluidic platform, which automatically performs a reverse flow injection analysis (rFIA) protocol for the determination of heavy metal ions in water by CD fluorescence quenching. As a proof of concept, nitrogen-doped CDs were synthesized from acrylic acid and ethylenediamine (ED) with quantum yields (QYs) of up to 44%, which are selective to cobalt. With the described system, we synthesized homogeneous CDs without the need for further purification and with the minimum consumption of reagents, and optimized fluorescence measurements can be performed with freshly obtained luminescent nanomaterials that have not undergone decomposition processes. They have an average hydrodynamic diameter of 4.2 ± 0.9 nm and maximum excitation and emission wavelengths at 358 nm and 452 nm, respectively. The system allows the automatic dilution and buffering of the synthesized CDs and the sample prior to the determination of cobalt. The concentration of cobalt was determined with good sensitivity and a limit of detection of 7 μg·L−1 with a linear range of 0.02–1 mg·L−1 of Co2+. Spiked tap water and river water samples were analyzed, obtaining recovery from 98 to 104%. This demonstrates the potential of the equipment as an efficient on-site control system for heavy metal monitoring in water.Graphical abstract

High-throughput determination of lipopolysaccharide by flow injection analysis using dipicolylamine-type fluorescent probe

Endotoxin (lipopolysaccharide: LPS), a bacterial toxin, is currently quantified by batch analysis using a reagent made from Limulus amebocyte lysate (LAL). Although the LAL method is sensitive, a long measurement time and gelation have hindered its application to online water quality monitoring. In this paper, we report a novel high-throughput analytical technique that combines flow injection analysis (FIA) and a dipicolylamine-type fluorescent probe, which was previously reported to exhibit strong fluorescence emission in response to LPS. A comparison of normal and reverse FIA systems revealed that the normal FIA offers more rapid detection with throughput of 30 samples per hour, which is the highest among known LAL methods and alternative LPS detection methods that have been recently reported. On the other hand, the reverse FIA requires much less reagent (480 μL per sample). Continuous online monitoring of water quality and analysis of real samples spiked with LPS were realized by this technique.

An improved reverse flow injection analysis (rFIA) technique for determination of nanomolar concentrations of ammonium in natural waters with automatic background fluorescence detection: Ammonification during a Karenia brevis bloom in Tampa Bay

Ammonium is the most energetically favorable form of inorganic nitrogen that phytoplankton can take up, and its availability often limits phytoplankton biomass. Recently, the majority of methods for quantification of ammonium at nanomolar concentrations have used the reaction between ammonium and o-phthaldialdehyde (OPA). Two different approaches have been employed. One involves gas diffusion of ammonia across a membrane. The other entails a more direct approach and utilizes sulfite as a reducing agent to form a fluorescent product. A benefit of the separation technique is that natural fluorescence of the samples does not yield a false positive signal, however membrane failure and clogging are challenging analytical problems. The direct reaction between the analyte and OPA, although devoid of problems associated with membranes, requires a correction for background fluorescence. This work presents the development of a reverse flow injection analysis (rFIA) method with automatic background fluorescence correction and a low detection limit, along with its application to measure underway ammonium concentrations in Tampa Bay during a bloom of the toxic dinoflagellate Karenia brevis with concurrent fish kills in June of 2021. During field testing elevated concentrations of ammonium were found to coincide with the largest number of observed floating dead fish and lethal K. brevis concentrations. The method employs a sulfite-formaldehyde reagent mixed with the sample into which an OPA reagent is injected and heated to activate fluorescence. Fluorescence of the sample is measured before the injection of OPA and at the peak of OPA injection, allowing for differentiation of the background fluorescence from the signal proportional to analyte. The detection limit and the limit of quantitation for this technique are 2.3 nM and 7.5 nM, respectively, and the coefficient of variation is 0.6% for replicate deionized water blanks (N = 10). Advantages of this method compared to techniques capable of both nanomolar detection of ammonium and improved accuracy via background fluorescence correction include a greater sample throughput, an increase in reported useful reagent lifetime, and the ability to quantify ammonium in coastal waters.

Ferrozine colorimetry and reverse flow injection analysis (rFIA) based method for the determination of total iron in aqueous solutions at nanomolar concentrations

Iron is one of the most microbiologically and chemically important metals in natural waters. The biogeochemical cycling of iron is significantly influenced by the redox cycling of Fe(II) and Fe(III). Because of the unique chemistry of iron, it is often needed to analyze iron at nano-molar concentrations. This article describes a reverse flow injection analysis (rFIA) based method with ferrozine spectrophotometric detection to quantify total iron concentration in stream water at nanomolar concentrations. The rFIA system has a 0.65 nM detection limit and a linear dynamic range up to 1.40 μM for the total iron analysis. The detection limit was achieved using a 1.0 m long liquid waveguide capillary flow cell, 1.50 m long knotted reaction coil, 87.50 μL injection loop and a miniature fiber optics spectrophotometer. The optimized colorimetric reagent has 1.0 mM ferrozine, 0.1 M ascorbic acid, 1.0 mM citric acid and 0.10 M acetate buffer adjusted to pH 4.0. The best sample flow rate is 2.1 mL min−1 providing a sample throughput of more than 15 samples h−1. The linear dynamic range of the method can be adjusted by changing the volume of the injection loop. The rFIA manifold was assembled exclusively from commercially available components.

A shipboard method for catalytic kinetic spectrophotometric determination of trace Cu(II) concentrations in seawater using reverse flow injection analysis coupled with a long path length liquid waveguide capillary cell

By using reverse flow injection analysis (rFIA) and a 1.0 m liquid waveguide capillary cell (LWCC), an automatic and sensitive catalytic kinetic spectrophotometric method for on-line monitoring of Cu(II) concentrations in estuarine and coastal waters was established. Cu(II) detection was based on its catalytic effect on glutathione (GSH) oxidation by potassium ferricyanide under acidic conditions. The change in absorbance of potassium ferricyanide at 420 nm was used to monitor the reaction spectrophotometrically. Experimental parameters were optimized using a univariate experimental design approach and it was ensured that the method was free of interference from co-occurring ions such as Fe(Ⅱ), Fe(III), Mn, Zn, Cd, Al, Cr, Ni and Co. The proposed method was shown to have high sensitivity with a detection limit of 0.15 nmol L-1 in a pure water matrix and 0.23 nmol L-1 in a seawater matrix. Linearity was achieved within the range of 0.45–15 nmol L-1 in a pure water matrix and 0.69–10 nmol L-1 in a seawater matrix using a 200 cm reaction coil, while the upper limits can be extended to 40 and 25 nmol L-1, respectively, by using a shorter (100 cm) reaction coil. A high level of Cu(II) recovery was maintained, ranging between 95.2% and 101%. The relative standard deviations (RSDs) of blanks and samples spiked with 5 nmol L-1 Cu(II) standard were 1.52% (n = 13) and 1.10% (n = 13), respectively. The method was confirmed to be free of carry-over effect and a sample throughput of 26 h−1 was achieved. The method was successfully applied to underway and on-board determination of Cu(II) concentrations from a surface transect in the Jiulong Estuary and a vertical profile in the Yangtze Estuary, China, respectively.

Development and application of a shipboard method for spectrophotometric determination of nanomolar dissolved sulfide in estuarine surface waters using reverse flow injection analysis coupled with a long path length liquid waveguide capillary cell

An automated and sensitive colorimetric method was established for on-line determination of trace sulfide concentrations in estuarine surface waters using reverse flow injection analysis coupled with a liquid waveguide capillary cell (LWCC). The mixed reagent was injected into the sample stream, with detection performed using a 1 m path length LWCC with a detection wavelength of 665 nm. Experimental parameters were optimized using an orthogonal and univariate experimental design. Interference from co-occurring ions such as NO2−, NO3−, SO32−, S2O32−, and HCO3− was also investigated. The proposed method exhibited high sensitivity with a detection limit of 1.7 nmol L−1. Linearity was achieved within the range of 10–1200 nmol L−1, with the upper limit extended to 32 μmol L−1 by replacing the LWCC with a 3 cm flow cell. The recovery rates ranged between 96.2% and 106%, with a relative standard deviation (RSD) of 2.01% (n = 14) for samples spiked with 100 nmol L−1 sulfide standard. The proposed method exhibits various advantages, including no effect from salinity or carry-over, a wide linear range (10–1200 nmol L−1 with LWCC and 0.25–32 μmol L−1 with a 3 cm flow cell), low detection limit (1.7 nmol L−1), low reagent consumption (0.095 mg N, N-dimethyl-p-phenylenediamine dihydrochloride and 0.063 g Fe(NH4)(SO4)2 per sample), and high sample throughput rates (24 h−1). This method was successfully applied in a shipboard environment for the analysis of dissolved sulfide in surface waters of the Jiulong Estuary (China).

Toward a versatile flow technique: Development and application of reverse flow dual-injection analysis (rFDIA) for determining dissolved iron redox species and soluble reactive phosphorus in seawater

A versatile flow analyzer that extended the features of reverse flow injection analysis (rFIA) was developed in this study and named reverse flow dual-injection analysis (rFDIA). Compared with typical rFIA, the analyzer requires less reagent and is more environmentally friendly, which has two injection valves and two reagent loops for the accurate and successive injection of two reagents. With a 2-m long liquid waveguide capillary cell (LWCC) and a spectrophotometer, the analyzer was applied to underway determination of dissolved iron redox species in estuarine and coastal waters. Detection limits of 0.18 and 0.20 nmol L−1 were achieved for Fe(II) and Fe(II + III), respectively and a linear dynamic range of 0.5–450 nmol L−1 was obtained for both Fe(II) and Fe(II + III). The sample throughput for the simultaneous measurement of Fe(II) and Fe(II + III) was 12 h−1, and each analysis consumed only 8 mL sample, 520 μL ferrozine solution, and 260 μL ascorbic acid solution. The analyzer was also used to measure nanomolar amounts of soluble reactive phosphorus (SRP) in seawater. The detection limit and the linear dynamic range for the SRP assay were 0.5 nmol L−1 and 1.5–850 nmol L−1. For SRP determination, the sample throughput was 20 h−1, and each analysis required 9 mL of sample, 130 μL of mixed reagent solution and 260 μL of ascorbic acid. The analytical results were reproducible, with a relative standard deviation of 1.4% (2.5 nmol L−1, n = 10), 2.1% (2.5 nmol L−1, n = 10), and 2.1% (10 nmol L−1, n = 11) for Fe(II), Fe(II + III), and SRP, respectively.

A New Charge Transfer Reaction for Spectrophotometric Determination of Nitrazepam Using Reverse Flow Injection Analysis

A new sensitive spectrophotometric method conjugated with reverse flow-injection analysis has been developed for the assay of nitrazepam (NZP) in the bulk drug and dosage form. The proposed method is based on the formation of charge-transfer complex between reduced NZP (donor) with a new reagent (acceptor) namely, 4,4'-tetramethyl-diaminodiphenyl-methane in the presence of NaIO4 as an oxidant. A violet-colored product was quantified spectrophotometrically at 560 nm. All the chemical and physical conditions of the reaction have been studied carefully. Under the selected optimum conditions, Beer’s law was obeyed in the concentration range of 5–300 µg/mL. The limits of detection and quantification were 5 and 16 µg/mL, respectively, with a sampling frequency of 62 sample/h. The proposed method was further successfully applied for the assay of NZP in commercial dosage forms and compared statistically with the reference method using t- and F-tests. The method was characterized by its accuracy and simplicity.

Charge Transfer Spectrophotometric Determination of Metronidazole in Pharmaceutical Formulations by Normal and Reverse Flow Injection Analysis Coupled with Solid-Phase Reactor Containing Immobilized FePO4

Two rapid, simpleand sensitive flow injection methods were developed for the estimation of metronidazole (MRZ) in pharmaceutical formulations. The proposed methods were based on charge transfer reaction between metol (N-methyl-p-aminophenol sulfate) as a π-acceptorand reduced MRZ as an n-donor to produce a blue colored chargetransfer complex. Method A depends on the reaction of reduced MRZ with metol (MT) in the presence of NaIO4 using two lines manifold to form blue colored product exhibiting absorption maxima at 700 nm.While method B depends on charge transfer reaction of reduced MRZ with MT in presence of a solid phase reactorcontainingfixedFePO4 on cellulose acetateusing reverse flow injection manifold to form a blue colored productwhich was measured spectrophotometrically at690 nm.Various experimental parameters for both methods were studied. Beer's law was obeyed in the ranges of2.5-200 and 2.5-150 μg mL-1,with r2 of 0.9995 and 0.9972;whilethe detection limit values were2.53 and 2.12μg mL-1 for methods A and B, respectively. Both of the suggested methods were successfully applied for the estimation of MRZ in commercial formulations. The results of the developed methods were compared with those obtained by the British pharmacopeia method, showinghigh accuracy and precision.

Spectrophotometric and Reverse Flow Injection Method Determination of Nitrazepam in Pharmaceuticals Using O-Coumaric Acid as a New Chromogenic Reagent

A spectrophotometric- reverse flow injection analysis (rFIA) method has been proposed for the determination of Nitrazepam (NIT) in pure and pharmaceutical preparations. The method is based upon the coupling reaction of NIT with a new reagent O-Coumaric acid (OCA) in the presence of sodium periodate in an aqueous solution. The blue color product was measured at 632 nm. The variation (chemical and physical parameters) related with reverse flow system were estimated. The linearity was over the range 15 - 450 µg/mL of NIT with detection limits and limit of quantification of 3.425 and 11.417 µg mL-1 NIT,respectively. The sample throughput of 28 samples per hour was achieved. Suggested method was successfully applied for the determination of NIT in its dosage forms.

Spectrophotometic determination of Lorazepam in pharmaceutical tablets using batch and reverse flow injection methods

A reverse flow injection analysis (RFIA) spectrophotometric method have been developed and compared with batch method for the determination of lorazepam (LOR) at the microgram level in pure and pharmaceutical dosage forms. The methods are based on the reaction of LOR with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) in the presence of ferric chloride in aqueous medium. The water soluble green color product was measured at λmax 659 nm. Under the optimum conditions, linearity was observed from 2 to 40 and 25 to 400 µg/mL LOR with detection limits of 0.61 and 2.29 µg/mL LOR by batch and RFIA procedures respectively. All the chemical and physical parameters related with reverse flow system were carefully considered and both methods were successfully applied for the determination of LOR in its dosage forms. These simple and high throughput methods could be utilized for pharmaceutical analysis of LOR.

Automated determination of ammonium in natural waters with reverse flow injection analysis based on the indophenol blue method with o-phenylphenol

An automated colorimetric method for the determination of ammonium in natural water was established using an integrated reverse flow injection analysis (rFIA) system based on the indophenol blue method with o-phenylphenol, which is a less toxic phenolic compound. The system was operated with laboratory-programmed software written in LabVIEW. The experimental parameters for ammonium determination were optimized based on a univariate experimental design. With the optimized conditions, the detection limit of the proposed method was 0.07μmol·L−1 and 0.08μmol·L−1, and the linearity was up to 50μmol·L−1 and 35μmol·L−1 for fresh water and seawater, respectively. The relative standard deviations were below 1.3% (n=8) and the recoveries varied from 89.8±1.0% to 106.5±2.9% for spiked natural water samples. The sample throughput was about 30h−1. The salinity effect from seawater was carefully investigated. The system has been successfully applied in-field in the Jiulong River for 24h on-line monitoring of ammonium concentration in surface water, and also used in the laboratory for analysis of ammonium in estuarine and coastal water samples. The analytical results obtained with the proposed method shown good agreement with those using reference methods. All results indicated that the newly developed automated rFIA method for ammonium determination was well suitable for laboratory analysis as well as for on-line monitoring.

A catalytic spectrophotometric method for determination of nanomolar manganese in seawater using reverse flow injection analysis and a long path length liquid waveguide capillary cell

A sensitive and precise method for determination of nanomolar manganese in seawater was developed, using reverse flow injection analysis, a long path length liquid waveguide capillary cell, and spectrophotometric detection. The reaction was based on manganese catalyzed oxidation of leucomalachite green with sodium periodate. Various experimental parameters were investigated and optimized. Foreign trace metal ions of iron, copper, zinc, nickel and aluminum did not cause obvious interference with manganese detection. Low manganese seawater was prepared and used as the blank and standards’ matrix, to eliminate the seawater matrix effect. The method detection limit was 0.20nmolL−1, and the quantification range was 0.50–10.00nmolL−1, which should be sensitive enough and suitable for open ocean seawater analysis. The seawater certified reference material NASS-6 was used to test the accuracy, and good agreement was obtained. The proposed method was applied to analyze seawater samples collected at the SEATS station in the South China Sea. The vertical profile of the total dissolvable manganese is reported and discussed.

Determination of clonazepam in pharmaceutical preparations using simple high-throughput flow injection system

This study was aimed at examining two flow injection-spectrophotometric systems (normal and reverse) for the determination of clonazepam (CLO) at the microgram level in pure and pharmaceutical dosage forms. The estimation of CLO has been developed by conjugating a normal (or reverse) flow injection analysis (nFIA or rFIA) and spectrophotometric detection with phloroglucinol as a coupling reagent. Beer’s law was obeyed over a range of 50–400 and 30–400 μg/mL. The limits of detection were 11 and 8 μg/mL and the sampling rates were 51 and 28 samples per hour for nFIA and rFIA respectively. Both systems were successfully applied for the determination of CLO in its commercially available dosage forms. A comparison between the proposed flow systems was also done. These simple and high throughput methods could be utilized for pharmaceutical analysis of CLO.

Sequential determination of multi-nutrient elements in natural water samples with a reverse flow injection system

An integrated system was developed for automatic and sequential determination of NO2−, NO3−, PO43−, Fe2+, Fe3+ and Mn2+ in natural waters based on reverse flow injection analysis combined with spectrophotometric detection. The system operation was controlled by a single chip microcomputer and laboratory-programmed software written in LabVIEW. The experimental parameters for each nutrient element analysis were optimized based on a univariate experimental design, and interferences from common ions were evaluated. The upper limits of the linear range (along with detection limit, µmolL−1) of the proposed method was 20 (0.03), 200 (0.7), 12 (0.3), 5 (0.03), 5 (0.03), 9 (0.2) µmolL−1, for NO2−, NO3−, PO43−, Fe2+, Fe3+ and Mn2+, respectively. The relative standard deviations were below 5% (n=9–13) and the recoveries varied from 88.0±1.0% to 104.5±1.0% for spiked water samples. The sample throughput was about 20h−1. This system has been successfully applied for the determination of multi-nutrient elements in different kinds of water samples and showed good agreement with reference methods (slope 1.0260±0.0043, R2=0.9991, n=50).

Determination of Doxycycline Hyclate by Batch and Reverse Flow Injection Analysis Based on the Oxidative Coupling Reaction with 3-Methyl-2-benzothiazolinone Hydrazone hydrochloride (MBTH)

New, simple and sensitive batch and reverse FIA spectrophotometric methods for the determination of doxycycline hyclate in pure form and in pharmaceutical preparations were proposed. These methods based on oxidative coupling reaction between doxycycline hyclate and 3-methylbenzothiazolinone-2-hydrazone hydrochloride (MBTH) in the presence ammonium ceric sulfate in acidic medium, to form green water-soluble dye that is stable and has a maximum absorbance at 626 nm. A calibration graph shows that a Beer's law is obeyed over the concentration range of 1-80 and 0.5-110 ?g.mL-1 of DCH for the batch and rFIA respectively with detection limit of 0.325 ?g.mL-1 of DCH for r-FIA methods. All different chemicals and physical experimental parameters affecting the development and stability of the colored product were carefully studied. The proposed methods were successfully applied for the determination of DCH in pharmaceutical preparations.