Iodine-azide Reaction Research Articles

Application of the TLC image analysis technique for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement

A simple and sensitive thin-layer chromatography (TLC) method coupled with an image analysis technique was developed for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement with good precision and accuracy. Separation was performed on silica gel plates using ethanol‒toluene (2:3, V/V) as the mobile phase. The visualization of chromatograms was based on iodine–azide reaction; therefore, pre-chromatographic derivatization reaction of amino acids with phenyl isothiocyanate was performed. Digital images of TLC plate chromatograms were converted into peak chromatograms, and quantitative analysis was conducted using TLSee software.

Application of image analysis technique for the determination of thiophanate methyl by thin-layer chromatography

ABSTRACTA simple and convenient thin-layer chromatography (TLC) method combined with image analysis technique was developed to determine thiophanate methyl. The detection of pesticide was based on iodine–azide reaction. Digital images of TLC plate chromatograms were analysed using TLSee software, and quantitative analysis was conducted. The linearity (0.3–3.0 µg per spot), sensitivity, accuracy and precision of the system were investigated.

Application of image analysis technique for the determination of organophosphorus pesticides by thin-layer chromatography

A simple and convenient thin-layer chromatography (TLC) method combined with image analysis technique was developed to determine malathion and fenthion in apple and orange juices. The detection of pesticides was based on iodine—azide reaction. Digital images of TLC plate chromatograms were analyzed using TLSee® software, and quantitative analysis was conducted. The linearity, sensitivity, accuracy, and precision of the system were investigated.

Determination of Urinary 6-Mercaptopurine and Three of its Metabolites by HPLC–UV Coupled with The Iodine–Azide Reaction

The presented method is able to determine 6-mercaptopurine (6-MP), 6-thioguanine, 6-mercaptopurine riboside and 6-thioguanine riboside in urine, and is thereby dedicated to control of thiopurine therapy of children with acute lymphoblastic leukemia. Good separation of the mentioned compounds was achieved on a C18 stationary phase with a sodium azide and sodium heptane sulfonate solution, acetonitrile and water at ratio of 50:1:49 (v/v/v). Coefficient of regression is >0.99 for all linearity ranges. LOD and LOQ are 0.3, 0.4, 0.3, 0.8 and 0.4, 0.6, 0.5 and 0.9 nmol/ml of urine for 6-MP, 6-thioguanine, 6-mercaptopurine riboside and 6-thioguanine riboside, respectively. Intra- and inter-day recovery and RSD are close to 100% and less than 10%, respectively, for all investigated thiopurines. The elaborated method was successfully applied for detection and quantitation of 6-MP and its selected metabolites in patients' urine samples.

Rp-Hplc–Uv Method Coupled With Post-Column Iodine-Azide Reaction For Determination of Free Captopril in Urine Samples

A free urinary captopril is measured indirectly employing the iodine-azide reaction in post-column mode. The pre-clean-up and/or derivatization step is needless, so that the method is adequate for rapid captopril determination in the urine samples and its monitoring at clinical trial. Captopril is separated on a C4 column by the eluate composed of sodium azide solution (4% [w/v], pH 5.8), acetonitrile and water at a ratio of 50:5:45 (v/v/v). The linearity exists in the range from 0.06 to 2.25 µmol/ml of urine. LOD and LOQ receive 0.03 and 0.06 µmol/ml of urine, respectively. Inter-day precision and accuracy of measurements of the captopril-spiked urine samples were 9, 8 and 5%, and 104, 107 and 105%, respectively, for 0.060 (low level), 0.50 (medium level) and 1.25 (high level) µmol/ml of urine. Captopril content was determined in real urine samples delivered from patients treated with this drug.

Determination of thiouracils in high-performance thin-layer chromatography with combination of iodine-azide reaction

The paper presents the application of post-chromatographic iodine-azide reaction for the determination of three thiouracils (6-benzyl-, 6-methyl-, and 6-propyl-2-thiouracil) in high-performance thin-layer chromatography (HPTLC). The HPTLC plates developed by methanol were sprayed with a freshly prepared mixture of sodium azide, potassium iodide, and starch solution adjusted to pH 5.5, and exposed to iodine vapour for 5 s. Because of the induction properties of the C-S and C=S bonds of thiouracil molecule in the iodine-azide reaction, the spots became visible as white spots on a violet-brown background.Scanning of the HPTLC plates was performed on a PC scanner and analysed by TLSee software. Determination range was 7–16 pmol per spot, 80–160 nmol per mL of urine, or 133–266 nmol per 1 mL of serum.

Development and validation of a reversed-phase HPLC method with post-column iodine-azide reaction for the determination of thioguanine

A high-performance liquid chromatographic method of reversed-phase with a post-column iodineazide reaction has been developed and validated for the determination of thioguanine. Isocratic elution was performed on a column of C18 using acetonitrile- water-sodium azide solution (1.5%; pH 6.5) 16: 34: 50 (v/v/v) as a mobile phase with flow-rate of 0.5 mL/min. Monitoring of unreacted iodine in post-column iodine-azide reaction induced by thioguanine resulted in its detection at 350 nm. The method applied to thioguanine was linear within the scope of values 8–100 nM (r 2 > 0.9988). The relative standard deviation (RSD 96%) prove that the intra-day precision and the accuracy were satisfactory. The lower limits of detection (LLD) and quantification (LLQ) of thioguanine were established at the levels of 6 and 8 nM, respectively. The elaborated method was validated and applied to thioguanine determination in tablets.

High Performance Liquid Chromatography Coupled with Post-Column Iodine-Azide Reaction for the Determination of Benzylthiouracil in Urine

The reaction between iodine and azide ions induced by benzylthiouracil was applied as a post-column detection system for determination of benzylthiouracil in urine. Neither extraction, nor preconcentration of the sample are necessary. The reproducibility, linearity, and recovery were evaluated under the optimum conditions. The benzylthiouracil standards added to normal urine show that the response of the detector, set at 350 nm (corresponding to unreacted iodine in the post-column iodine-azide reaction), was linear within the concentration range of 0.4-4.5 nmol · mL−1 urine. Recovery and the relative standard deviation values for precision within the calibration range were from 94 to 104% and from 1 to 4.5%, respectively. Lower limits of detection (LLD) and quantitation (LLQ) were 0.3 and 0.4 nmol · mL−1 urine, respectively.

Postcolumn Iodine–Azide Reaction as a Detection System in HPLC for the Determination of Methylthiouracil in Urine

This article describes a system for determining methylthiouracil in urine based on the sensitizing induction of methylthiouracil on the reaction between iodine and azide ions and combined techniques of high-performance liquid chromatography and simple postcolumn detection. The optimal conditions for iodine–azide reaction and high-performance liquid chromatographic separation were determined. The reproducibility, linearity, and recovery were evaluated under the optimal conditions. The methylthiouracil standards added to normal urine show that the response of the detector, set at 350 nm (corresponding to unreacted iodine in the postcolumn iodine–azide reaction), is linear within the concentration range of 0.4–5.0 nmol mL−1 urine. The relative standard deviation values for precision and recovery within the calibration range varied from 0.9 to 4.8% and from 94 to 105%, respectively. Limits of detection (LOD) and quantitation (LOQ) were 0.3 and 0.4 nmol mL−1 urine, respectively.

Determination of propylthiouracil in pharmaceutical formulation by high-performance liquid-chromatography with a post-column iodine-azide reaction as a detection system

A high-performance liquid chromatographic method with a post-column iodine-azide reaction has been chosen and tested for validity in quantitative determination of propylthiouracil in tablets. A mobile phase with a flow rate of 1.4 ml/min was conducted in the form of isocratic chromatography on a C18 column with acetonitrile-water-sodium azide solution (2.5%; pH 5.5) 24:26:50 (v/v/v). Unreacted iodine from post-column iodine-azide induced by reaction was monitored with visible detection at lambda=350 nm. The method proved both its linearity within the range of 8-100 nM (r2>0.9988) and satisfactory results of inter-day precision (RSD<4.2%) and accuracy (recovery>91%). The limits of detection (DDL) and quantification (DQL) reached the levels of 5 and 8 nM, respectively. The validation of the method comprised also its specificity. The results obtained proved the suitability and appropriateness of the suggested method for intended use.

Thin Layer Chromatography with Post-Chromatographic Iodine-Azide Reaction for Thiuram Analysis in Food Samples

A simple, sensitive, precise, and rapid planar chromatographic method for thiuram analysis was elaborated. Reaction between iodine and azide ions induced by thiuram was applied as a detection system. The developed plates were sprayed with freshly prepared mixtures of sodium azide and starch solution adjusted to pH 6.0, and exposed to iodine vapor. The spots became visible as white spots on a violet-grey background. The iodine-azide detection system was proved to be the most favorable and enabled detection of quantities at the level 0.5 pmol per spot. The iodine-azide test was compared with other visualizing techniques commonly used in planar chromatography: iodine vapor, UV254, Cu(II). Certain factors that influence detection with iodine-azide reaction as a detection system were checked. The developed method was linear over the concentration range of 2–9 pmol per spot. The proposed technique was applied to detect thiuram in food samples.

Application of the iodine–azide postcolumn reaction in RP‐HPLC for the determination of thioguanine in urine

The study focuses on the analysis of a sensitive, selective, and simple postcolumn detection method for thioguanine determination, based on the sensitizing induction of thioguanine on iodine-azide reaction and the combination technique of HPLC. The analysis was accomplished in the optimum conditions for iodine-azide detection system and HPLC separation. The values for the linear range, the LOD, and DOQ amounted to 0.8-1.7, 0.4, and 0.5 nmol/mL urine, respectively.

Determination of methimazole in urine with the iodine-azide detection system following its separation by reversed-phase high-performance liquid chromatography

The iodine-azide detection system to determine methimazole following its separation by RP-HPLC is described in this paper. The reaction between iodine and azide ions induced by methimazole was applied as a post-column reaction detection system. Neither extraction nor preconcentration of the sample was necessary. The methimazole standards added to normal urine show that the response of the detector, set at 350 nm (corresponding to unreacted iodine in the post-column iodine-azide reaction), was linear within the concentration range 2–10 nmol/mL of urine. The relative standard deviation values for precision and recovery within the calibration range were from 0.3 to 3.2% and from 97 to 102%, respectively. Limits of detection (LOD) and quantitation (LOQ) were 1 and 2 nmol/mL of urine, respectively. The method was applied to the separation and determination of patient urine samples and the analytical results were satisfactory.

High-performance liquid chromatographic determination of propylthiouracil in human urine by post-column iodine–azide reaction

A sensitive, selective and simple post-column detection method for the determination of propylthiouracil (PTU) based on its sensitizing induction on iodine–azide reaction and the combination technique of high-performance liquid chromatography has been presented. The analysis was conducted in the optimum conditions for iodine–azide detection system and HPLC separation. The linear range, the lower limit of detection and quantification for PTU in urine were established at the levels of 0.4–1.0 nmol/ml urine, 0.3 nmol/ml urine and 0.4 nmol/ml urine, respectively.

Determination of Methimazole in Pharmaceutical Preparations using an HPLC Method Coupled with an Iodine-Azide Post-Column Reaction

A simple and reliable high performance liquid chromatographic (HPLC) method with iodine-azide post-column reaction as a detection system has been developed and validated for determination of methimazole in tablets. A chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile, water, and 2.5% sodium azide solution (pH 5.5) with isocratic elution (20:30:50, v/v/v). The detection is based on spectrophotometric measurement of the residual iodine (λ = 350 nm) from the post-column iodine-azide reaction induced by methimazole, after mixing an iodine solution containing iodide ions with the column effluent containing azide ions and methimazole. Obtained chromatograms for methimazole showed negative peaks as a result of the decrease in background absorbance. The method developed for methimazole was linear over the concentration range of 0.2–2 µmol L−1. The method is accurate and precise (relative standard deviation, R.S.D. < 4%). The detection limit (defined as S/N = 3) was 0.18 µmol L−1 (3.6 pmol injected amount) for the described compound. The elaborated method was applied to determine methimazole in tablets.

Stopped-flow determination of 2-mercaptopyrimidines as inductors of the iodine-azide reaction

A stopped-flow method for the determination of 2-mercaptopyrimidine and its 14 derivatives by the iodine-azide has been elaborated. Spectrophotometric detection was applied and the decrease in the absorbance of the iodine–starch complex was monitored at 595 nm within 5 s. The effect of the concentration of the reagents on the rate of the reaction was investigated and a kinetic method for determination of compounds is proposed. 2-Mercaptopyrimidines can be determined at 10 −6 to 10 −5 mol L −1 level and the relative standard deviation is below 1%. The elaborated method has been applied to the determination of 6-methyl- and 6-propyl-2-thiouracil in drugs.

Planar chromatography of heterocyclic thiols with detection by use of the iodine-azide reaction

Reaction between iodine and the azide ion induced by heterocyclic thiols has been used for detection in TLC and HPTLC. The developed plates were sprayed with a freshly prepared solution of sodium azide and starch, adjusted to a pH within the range 5.5–6.0, and then exposed to iodine vapor. The thiols became visible as white spots on a violet-gray background. The iodine-azide reagent enabled detection of quantities in the range 1–80 pmol per spot. The iodine-azide test was compared with other visualizing techniques commonly used in planar chromatography (iodine vapor and UV 254 ). The method was applied to detection of 2-thioguanine and 6-mercaptopurine in biological samples.

Separation of amino acids as phenyl thiocarbamyl derivatives by normal and reversed-phase thin-layer chromatography

To separate groups of amino acids we have used a process of derivatization involving formation of phenyl thiocarbamyl derivatives. Phenyl isothiocyanate was used as derivatization reagent. This particular reagent was chosen because the proposed method of detection entailed use of the iodine-azide reaction, which can be applied to sulfur compounds because their properties induce this reaction. Because amino acids do not belong to this group, it was necessary to perform a suitable transformation. The derivatization process was performed directly on the plate or in a tube, depending on the stationary phase chosen. After normal-phase (NP) TLC development with appropriate mobile phases, the plate was sprayed with a freshly prepared mixture of sodium azide and starch solution and exposed to iodine vapor. In reversed-phase TLC the plates were developed with mobile phase containing sodium azide and starch solution and then exposed to iodine vapor without being dried. In both NP and RP-TLC the catalytic effect of ...

Determination of thiopental in urine sample with high-performance liquid chromatography using iodine–azide reaction as a postcolumn detection system

The reaction between iodine and azide ions induced by thiopental was utilized as a postcolumn reaction for chromatographic determination of thiopental. The method is based on the separation of thiopental on an Nova-Pak CN HP column with an acetonitrile-aqueous solution of sodium azide as a mobile phase, followed by spectrophotometric measurement of the residual iodine (lambda=350 nm) from the postcolumn iodine-azide reaction induced by thiopental after mixing an iodine solution containing iodide ions with the column effluent containing azide ions and thiopental. Chromatograms obtained for thiopental showed negative peaks as a result of the decrease in background absorbance. The detection limit (defined as S/N=3) was 20 nM (0.4 pmol injected amount) for thiopental. Calibration graphs, plotted as peak area versus concentrations, were linear from 40 nM. The elaborated method was applied to determine thiopental in urine samples. The detection limit (defined as S/N=3) was 0.025 nmol/ml urine. Calibration graphs, plotted as peak area versus concentrations, were linear from 0.05 nmol/ml urine. Authentic urine samples were analyzed, thiopental was determined at nmol/ml urine level.

Detection of mercaptopyridines and mercaptopyrimidines in planar chromatography with iodine–azide reaction as a detection system

Reaction between iodine and azide ion induced by mercaptopyridines and mercaptopyrimidines was utilized as a detection system in TLC and HPTLC. The developed plates were sprayed with a freshly prepared mixtures of sodium azide and starch solution adjusted to pH 5.5, and exposed to iodine vapour. The spots became visible as white spots on violet-grey background. The iodine-azide detection system has been proved to be the most favourable and enabled to detect quantities per spot in the range of 1-20 pmol (HPTLC) and 1-60 pmol (TLC). The iodine-azide tests were compared with other visualizing techniques commonly used in planar chromatography (iodine vapour and UV254). The developed method was applied to detection of thiopental in biological samples.