A Chemical Ionization Mass Spectrometric Study of Fluorescamine and Fluorescamine - Amino Acid Derivatives

Abstract

The chemical ionization mass spectra of fluorescamine and fluorescamine - amino acid derivatives have been studied using methane and ammonia as reagent gases. Major ions in the spectra are protonated molecular ions, adduct ions and ions formed by loss of an oxygen atom.

Fluorescamine, 4-phenyl-spiro[furan-2(3H),1′-phthalan]3,3′-dione, is a powerful new fluorogenic reagent for assaying primary amines.¹ and EI² and EI³ mass spectrometric investigations of fluorescamine and its derivatives were carried out. Our present study reports the CI mass spectral analysis of fluorescamine and some of its amino acid derivatives.     

Evaluation of Fluorescamine as a Phosphogenic Derivatizing Reagent of Primary Amines

Abstract

Fluorescamine has been shown to be a promising derivatizing reagent for the room temperature phosphorescence analysis of primary amino acids. The fluorescamine concentration used for derivatization in solution was 600 μg/ml and for direct derivatization, 1160 μg/ml. For derivatized primary amino acids the limits of detection were found to be between 0.8 ng and 2.9 ng with linear dynamic ranges between 2 and 3 orders of magnitude.     

Detection and determination of N-nitrosamines by thin-layer chromatography using fluorescamine.

A novel procedure is described for the detection and determination of N-nitrosamines (NAs) on thin-layer chromatographic plates. Ultraviolet irradiation of NAs on activated plates (silica gel or aluminum oxide) yields primary or secondary amines, which after spraying with fluorescamine reagent give fluorescent or non-fluorescent products, respectively. The majority of the 24 NAs examined afforded NAs, although the volatile dimethyl-, diethyl- and pyrrolidine-N-nitrosamines gave limits of 500, 40, and 40 ng, respectively. Spectrophotometric determinations of the relative fluorescence of 0.1-40 nmoles of NAs gave rise to nearly linear calibration curves when plotted on a log-log scale.     

Fluorescence Assay of Proline: Competitive Inhibition of Fluorescamine Reaction

Abstract

The rate of reaction of fluorescamine with proline is faster than with primary amino acids. Proline competitively inhibits the fluorogenic reaction of primary amines with fluorescamine, the degree of inhibition being proportional to the amount of proline. Under the conditions described, 5×10^?7 M proline produces a 10% diminution of fluorescence. An assay of proline or hydroxyproline based on fluorescence inhibition would be more sensitive than colorimetric assays based on ninhydrin or fluorescamine.     

Detection and determination of N-nitrosamino acids by thin-layer chromatography using fluorescamine

A novel procedure is described for the detection and determination of N-nitrosamino acids (NAAs) on activated silica gel thin-layer chromatographic plates. N-Nitrososarcosine, N-nitrosoproline, and N-nitroso-4-hydroxyproline could be detected as fluorophors at the 200-pmole level (20-30 ng) after being irradiated with ultraviolet light and sprayed with fluorescamine reagent. Spectrophotometric determination of the relative fluorescence of 0.4-40 nmoles of NAAs gave rise to similar calibration curves when plotted on a log-log scale. An application of this method to the detection of NAAs in uncooked bacon is described.     

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.

     

柱后衍生高效液相色谱法测定虾中14种磺胺类药物残留量

建立了虾中14种磺胺类药物残留量的柱后衍生高效液相色谱检测方法。样品在加入内标物磺胺吡啶后用乙酸乙酯提取,提取液浓缩后用4 mL乙酸乙酯溶解残余物,用盐酸溶液反萃取,正己烷去脂,盐酸溶液经滤膜过滤后,加入乙腈、甲醇和3.5 mol/L乙酸钠溶液（体积比为5：5：20）的混合溶液混匀后,经高效液相色谱分离,用荧光胺衍生试剂进行柱后衍生,荧光检测器检测。采用基质标样添加法绘制标准曲线,内标法定量。对柱后衍生系统参数进行了优化,确定了荧光胺溶液的浓度、流速和反应温度分别为0.2 g/L、0.15 mL/min和50℃。14种磺胺类药物在5~200 μg/L范围内具有良好的线性。磺胺类药物的定量限（LOQ,S/N=10）为1.0~5.0 μg/kg。在1.0~100.0 μg/kg添加水平内,磺胺类药物的平均回收率为77.8%~103.6%,相对标准偏差（RSD）为2.9%~9.1%（n=6）。实验结果表明该方法灵敏、准确,重复性好,适用于虾中磺胺类药物的残留检测。     

Quantitative Bestimmung von Sterigmatocystin in verschimmelten pflanzlichen Nahrungsmitteln

Zusammenfassung Ein Verfahren für die quantitative Bestimmung von Sterigmatocystin in pflanzlichen Nahrungsmitteln durch zweidimensionale DC-Trennung eines Extraktes und Fluorescenzintensitätsmessung der mit AlCl₃ behandelten DC-Platte wird beschrieben. Durch die hier vorgeschlagene Auftrageweise auf der DC-Platte ist ein rasches Identifizieren und Auswerten von Probe- und Eichflecken möglich. Als Extraktionsmittel hat sich Acetonitril-KCl-Lösung am besten bewährt. Versuche mit zugesetztem Toxin zeigten Verluste von weniger als 5 %.

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Quantitative determination of sterigmatocystin in mouldy vegetable foods

Summary The quantitative determination of sterigmatocystin is achieved by two-dimensional thin-layer chromatography (TLC) of extracts from vegetable foods and measurement of the fluorescence of the spots on AlCl₃-treated TLC plates. Samples and controls (references) are applied to the TLC plates in a way that permits their quick identification and assay. Acetonitrile-KCl solution has proved to be the most advantageous extracting agent. Losses of less than 5 % were observed in tests with added toxin.

     

Identification and interaction of amino acids with leucine-anthracene reagent by TLC and spectrophotometry: experimental and theoretical studies

A new reagent has been synthesized by coupling anthracene moiety to L-leucine. The reagent is characterized by different analytical techniques. It is capable for easy identification of various amino acids on thin-layer chromatography plates by developing distinguishable colors (detection limit between 0.1-0.5 μg at cold condition and 0.1-0.4 μg after heating). This reagent also binds with different amino acids very strongly in solution (methanol). Estimation of equilibrium binding constants of this new reagent with different amino acids has also been carried out. The values of the binding constants are lowest for L-Tyrosine (6.86 × 103 dm3 mol(-1)) and highest for L-Arginine monohydrochloride (8.86 × 10? dm3 mol(-1)) at 25°C. A theoretical study (Hartree-Fock) has been performed to investigate the interaction of the reagent with a representative amino acid, glycine.     

HPLC Determination of Amino And Carbonyl Compounds with Dabsyl Chloride and Dabsyl Hydrazine

The new chromophoric reagent, 4-dimethylaminoazobenzene-4′-sulfonyl chloride (dabsyl chloride) was synthesized by reaction of sodium 4-dimethylaminoazobenzene 4′-sulfonate with phosphorus pentachloride. Dabsyl chloride reacted rapidly with all amino acids, aliphatic amines, and polyamines to form the corresponding dabsyl derivatives. The dabsyl amino acids (10^?11 mol) were visualized on thin layer plates and found to be photo-stable. During the last 12 years, in combination with HPLC, this newly developed labeling reagent has been shown to be very promising for microdetermination of amino acids, aliphatic amines and polyamines. Recently, another new chromophoric reagent, dabsylhydrazine, was synthesized from the reaction of dabsyl chloride with hydrazine. This reagent reacted readily with monosaccharides and carbonyl compounds to form the corresponding chromophoric dabsylhydrazones with strong absorbance at 425 nm. The application of this new reagent in HPLC analysis of monosaccharides was discribed. By use of Nova-PAK C₁₈ reverse-phase column and a concave gradient system of water and acetonitrile as eluent, the detection limits of monosaccharides in the range of 10 pmol have been reached.     

2,3-Diphenylquinolizinium bromide as a fluorescent derivatization reagent for amines

2,3-Diphenylquinolizinium bromide (2,3-DPQ) is proposed as a fluorogenic reagent for amino compounds. A spectrofluorimetric method based on its use is described which allows the determination of μg ml^?1 to ng ml^?1 levels of primary and secondary amines, including aromatic and cyclic compounds. The precision of the method was 4–8% (relative standard deviation) (n=10). The influence of several external factors on the derivatization reaction was studied using piperidine as a model compound. The analytical reaction can be effected at room temperature, which avoids the degradation of labile sample amines, e.g., catecholamines, and simplifies the experimental procedure. The presence of an excess of a basic catalyst (triethylamine) was critical for the reaction to develop satisfactorily. Fluorescence due to the reaction product was detected 5 min after the start of the analytical reaction.     

Cation analysis by thin-layer chromatography and reflectance spectroscopy : Part I. The rapid identificaiion of cations resolved on thin-layer plates

A rapid method has been devised whereby 14 cations — Al³⁺, Bi³⁺, Cd²⁺, Cr³⁺, Co²⁺, Cu²⁺, Fe³⁺, Pb²⁺, Mn²⁺, Hg²⁺, Ni²⁺, Ag⁺, Sn²⁺ and Zn²⁺ —. can be separated on one-dimensional, cellulose thin-layer plates and then identified by means of their reflectance spectra The procedure requires micro-amounts of sample and only one spray reagent.     

Studies on Fluorescamine. Part II - Thin-Layer Chromatographic Mobilities of Fluorescamine Positive Drugs

Abstract

Fluorescamine reacts with primary amines, it does not react with secondary or tertiary amines, to yield bright acquamarine fluorescent products. This reaction has been developed into a spot test useful for differentiation of amphetamine from methamphetamine; previous spot tests did not have this degree of specificity. The fluorescent products of several primary amines have been demonstrated to have sufficient stability to permit thin-layer chromatography comparisons with authentic materials. The fluorescent derivative of amphetamine has been demonstrated to be capable of differentiation from other fluorescent products formed from fluorescamine based on its unique migration in three different TLC systems.     

Screening of quinolone residues in pig muscle by planar chromatography

Summary A high-performance thin-layer chromatographic method based on solid-phase extraction has been developed for the qualitative determination of seven quinolones (enrofloxacin, ciprofloxacin, danofloxacin, norfloxacin, flumequine, oxolinic acid and nalidixic acid) in pork muscle. After preparation of the samples by extraction and clean-up by solid-phase extraction on reversed-phase cartridges, extracts were spotted and eluted on silica gel plates. The plate is first inspected under UV illumination at 312 nm, then sprayed with terbium chloride solution and again monitored under 312 nm UV. The method has been validated to a level of 15 μg kg⁻¹ for enrofloxacin, ciprofloxacin, danofloxacin, norfloxacin and 5 μg kg⁻¹ for flumequin, oxolinic acid and nalidixic acid.     

Electrolytic preparation of a reduced ninhydrin reagent for amino acid analysis

Summary A flow-through electrolysis using a column electrode was applied to the preparation of a reduced ninhydrin reagent suitable for a highly reproducible and sensitive determination of amino acids. Non-reduced ninhydrin solution, which could be stably stored for long duration even in contact with atmospheric oxygen, was fed at a constant flow rate of 0.25 ml min^–1 into the flow-through electrolysis cell, whereby ninhydrin was reduced to hydrindantin. The hydrindantin content in the reagent (presumably 1.61 mol%) was sufficiently constant for the subsequent quantitative reaction of the amino acids being eluted from the HPLC system. The electrolytically prepared reagent was practically identical with those prepared by chemical reduction.

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Elektrolytische Herstellung von reduziertem Ninhydrinreagens für die AminosÄureanalyse

     

Adsorption–Photometric Determination of Cationic Surfactant Traces

A procedure has been developed for the adsorption–photometric determination of cationic surfactants in natural water. The procedure is based on the adsorption preconcentration of the cationic surfactants on silica gel, the reaction of the concentrate with the anionic reagent bromothymol blue to form ion pairs on a solid surface, and the photometric determination of excess bromothymol blue in solution. The analytical range is (0.5–5) × 10^–5 M for a 50-mL sample.     

Spectrophotometric determination of beryllium with carminic acid

Summary A new reagent has been proposed for the determination of beryllium. The method is based on the reaction between beryllium and carminic acid, forming a Be-carminic acid complex at pH 4. The absorption is measured at 580 nm. The molar absorptivity is 2.25×10³ mol^–1 cm^–1. Beer's law is obeyed from 0.4 ppm to 1.6 ppm of beryllium. The effects of pH, reagent concentration and interferences from foreign ions have been studied. The method has been applied to the determination of beryllium in polluted water and blood samples.

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Spektralphotometrische Bestimmung von Beryllium mit CarminsÄure

     

Determination of Caffeine in Small Plasma Samples by Gas-Liquid Chromatography with Thin-Layer Chromatographic Sample Clean-Up

Abstract

A sensitive method for the determination of caffeine in small plasma samples (50 μ1) is described. The samples are extracted with chloroform and the extrects are freed from interfering compounds by thin-layer chronatography. The spots of caffeine and the internal standard, 7-ethyl theophylline, are scraped off the plates and both compounds are eluted from the scrapings by means of a glass capillary collector. The caffeine in the eluate is quantitated by gas-     

Postcolumn derivatization of amino acids using reaction flow chromatography columns with fluorescence detection: A fast new approach to selective derivatization techniques

Reaction flow (RF) chromatography with fluorescamine reagent and fluorescence detection (FLD) was used for the analysis of amino acids. The performance of RF chromatography was tested against several optimized conventional postcolumn derivatization (PCD) methods. RF columns achieved greater sensitivity compared to conventional PCD methods, without the need for reaction loops, which resulted in more efficient separations. The RF-PCD method also achieved limits of detection (LOD) from the low picomole to subnanomole range. The calibration data of the RF-PCD technique yielded R²?≥?0.99 and % relative standard deviation in peak areas ranging from 0.34% to 5%. Through reaction flow chromatography, multiplexed detection was also achieved allowing the monitoring and analysis of derivatized and nonderivatized flow streams simultaneously.     

Quantitative TLC Analysis of Sterol (24β-Ethylcholesta-5,22E,25-triene-3β-ol) in Agnimantha (Clerodendrum phlomidis Linn)

A quantitative method using silica gel 60F₂₅₄ high performance thin layer chromatography plates, automated bandwise sample application, and automated visible mode densitometric method has been developed for the determination of 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO) in the aerial part of Clerodendrum phlomidis. ECTO was used as a chemical marker for the standardization of C. phlomidis plant extracts. The separation was performed on silica gel 60F₂₅₄ TLC plates using chloroform-methanol (98.5: 1.5, v/v) as mobile phase. The quantitation of ECTO was carried out using the densitometric reflection/absorption mode at 650 nm after post chromatographic derivatization with anisaldehyde reagent. A precise and accurate quantification can be performed in the linear working concentration range of 150–400 ng band⁻¹ with good correlation (r ² = 0.996). The method was validated for peak purities, precision, robustness, limit of detection (LOD) and quantitation (LOQ), etc. as per ICH guidelines.