Flow-injection determination of amine contaminants in cyclamate samples based on temperature for controlling selectivity

This paper describes a flow-injection (FI) method for the simultaneous determination of aniline and cyclohexylamine impurities in cyclamate products. The method consists of the derivatization of amines with 1,2-naphthoquinone-4-sulfonate under selective and non-selective conditions. Here, the selectivity is achieved by working at 20 degree C, at which only aniline reacts, whilst higher temperatures (80 degree C) lead to a non-selective reaction of the two analytes. The FI manifold is composed of two flow cells for the spectrophotometric detection of derivatives at 480 nm. Experimental conditions have been optimized by factorial design and multicriteria making approach. Quantification is accomplished by differential analysis of the analyte contributions in the double peaks generated when the sample reaches cell 1 and cell 2. Results obtained with the proposed method are in satisfactory agreement with those provided by the standard method for the analysis of cyclamate samples.     

丹磺酰氯柱前衍生-超高效液相色谱-串联质谱法测定人体尿样中的环己胺

建立丹磺酰氯柱前衍生-超高效液相色谱-串联质谱法测定人体尿样中环己胺的方法。冷冻样品经解冻、离心后,用丹磺酰氯衍生,固相萃取小柱净化。目标化合物采用 Waters ACQUITY CSHTM C18色谱柱(50 mm×2.1 mm,1.7μm)分离,以甲醇和0.002 mol/L乙酸铵溶液为流动相梯度洗脱,采用电喷雾离子源电离、正离子多反应监测模式质谱检测。环己胺在2.5~200μg/L浓度范围内有较好的线性关系,相关系数大于0.999,回收率为98.7%~102.3%,精密度为3.1%~5.2%,检出限和定量限分别为1.0和3.0μg/L。结果表明,本方法操作简单、准确可靠,可适用于人体尿液中环己胺的定量分析。应用本方法测定200份学生尿液样品,环己胺检出率为34.5%。     

Derivatization of HPLC/Fluorescence Quantitation of Maduramicin Ammonium in Feed and Premixes at Levels Down to 5 ppm

Abstract

A reverse phase HPLC method with fluorescence detection and pre-column derivatization is described for determination of maduramicin (and possibly other non-fluorigenic ionophores containing a hemiketal ring) in feeds, premixes, and technical material. Dansyl hydrazine derivatization together with Florisil Sep-Pak clean-up is described. Isocratic HPLC with external standard is used. Method development, specificity and the chemistry of derivatization is discussed.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

Quantification of the bisphosphonate alendronate using capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing

A quantitative method was developed for the direct identity confirmation and quantification of alendronate using CE-MS combined with a pH-assisted focusing technique, dynamic pH barrage junction focusing. A pH-induced variation in electrophoretic mobility led to online focusing of alendronate at the sample/pH barrage boundary, significantly improving the detection sensitivity. In addition, the use of a flow-through microvial CE electrospray interface and the multiple reaction monitoring mode of MS further improved the specificity and quantification capability of this technology. This quantitative method presented a wide linear dynamic range over 8–2000 ng/mL and an LOD of 2 ng/mL. A 460-fold improvement in sensitivity was obtained when pH barrage junction focusing was applied during the CE process, in comparison to when normal CE was conducted without online sample stacking. The superior detection sensitivity over previously reported methods enables direct analysis of bisphosphonate compounds, eliminating tedious pre-column sample enrichment and derivatization. Validation of alendronate content in a commercial drug tablet further proved the reliability and power of this method. This simple method with no sample derivatization, superior sensitivity, and short run time (<8 min) is a promising alternative for accurate quantification of alendronate and other types of bisphosphonate compounds in both drug formulations and plasma samples.     

Silica Based 3,5-Dinitrobenzoyl (Dnb) Reagent for Off-Line Derivatization of Amine Nucleophiles in HPLC

Abstract

We describe here a new silica based derivatization reagent, containing the 3,5-dinitrobenzoyl tag, for solid phase derivatization of amines. It can be used for the off-line derivatization of primary and secondary amines. the amide derivatives can be easily detected under conventional UV detection modes. the entire synthetic method, structural characterization, and optimization of derivatization conditions of this solid phase derivatization reagent are described, Also, the reagent was tested in the on-line, pre-column derivatization mode for reversed phase HPLC, as well as for histamine analysis in fish samples     

9-Fluoreneacetyl-tagged, solid-phase reagent for derivatization in direct plasma injection.

We describe here a resin-based derivatization reagent, containing a 9-fluoreneacetyl tag on a controlled-pore substrate, for direct injection analysis of amphetamine in plasma. On-line, pre-column derivatization was performed by direction injection of diluted plasma sample into an sodium dodecyl sulfate-containing mobile phase. Amphetamine was trapped in the hydrophobic derivatization column and derivatized at elevated temperature by the activated solid-phase reagent. The derivatized 9-fluoreneacetyl amphetamide was separated by reversed-phase high-performance liquid chromatography with a step gradient and determined by fluorescence detection. The synthesis scheme, characterization, and optimization of the derivatization conditions for the solid-phase reagent are described. The method was evaluated by reproducibility tests and single blind spiking analysis. This solid-phase reagent combined with a surfactant containing mobile phase provided a sensitive and simple procedure for on-line derivatization in direct injection analysis of biological fluids.     

Liquid chromatographic determination of aniline in table-top sweeteners based on pre-column derivatization with 1,2-naphthoquinone-4-sulfonate

A liquid chromatographic method for the determination of aniline in cyclamate sweeteners based on a pre-column derivatization with 1,2-naphthoquinone-4-sulfonate (NQS) is proposed. Aniline traces were extracted from the cyclamate samples using dichloromethane. After solvent evaporation, the dry residue was derivatized with NQS at pH 9.5 and 85 degrees C for 1 min. The aniline derivative, which was extracted from the reacting mixture, was redissolved in the eluent solution and injected into the chromatographic system. The separation of aniline derivative from other amine impurities was carried out in a C18 column using a 2% acetic acid-methanol (40:60, v/v) mobile phase. Results from the analysis of aniline in the sweetener samples with the proposed method were compared with those from the standard method. A good concordance between the two methods was observed.     

Rapid analysis of amino acids using pre-column derivatization

A new approach to the pre-column derivatization and analysis of amino acids is described. The method is based upon formation of a phenylthiocarbamyl derivative of the amino acids. The derivatization method is rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The liquid chromatographic system allows for the rapid, bonded-phase separation with ultraviolet detection of the common amino acids with 12-min analysis time and a 1-pmol sensitivity.     

Measurement of plasma and urine amino acids by high-performance liquid chromatography with electrochemical detection using phenylisothiocyanate derivatization

The use of reversed-phase liquid chromatography (LC) with pre-column derivatization for the analysis of amino acid mixtures is becoming established as a possible cheaper alternative to commercial amino acid analysers. The available derivatization procedures all have disadvantages when applied to clinical samples, partly due to the interferences found with body fluids when ultraviolet or fluorescence detection is used. An LC method is described for the separation of amino acids in blood or urine, using pre-column derivatization with phenylisothiocyanate (PITC), gradient elution and electrochemical detection. The use of electrochemical detection of PITC derivatives virtually eliminates interferences and enables the secondary amino acids to be measured. Examples are shown of normal urine and plasma and samples from patients with cystinuria and maple syrup urine disease.     

HPLC analysis of polyamines and their acetylated derivatives in the picomole range using benzoyl chloride and 3,5-dinitrobenzoyl chloride as derivatizing agent

Analytical methods are described for the quantitative determination of putrescine, cadaverine, spermidine, spermine and the acetylated derivatives of spermidine and spermine in biological fluids using pre-column derivatization with either benzoyl chloride or 3,5-dinitrobenzoyl chloride, which were added to each sample as solutions in diethyl ether. Putrescine, spermidine and spermine can be analysed in seminal plasma at nanogram levels when benzoyl chloride is used as derivatizing agent. In the analysis of putrescine, cadaverine, spermidine and acetyl derivatives of spermidine and spermine, higher sensitivity is obtained with 3,5-dinitrobenzoyl-chloride. This method can readily be used in the determination of acetylated polyamines in urine samples.     

在线自动化柱前衍生-高效液相色谱法测定食品中组胺的研究

建立了在线自动化柱前衍生.高效液相色谱法测定食品中组胺的新方法.通过对测定过程中各个影响因素进行优化,如自动化衍生程序的设定,衍生试剂的用量,衍生体系pH影响等,确立了适宜的测定条件.在该条件下,对于组胺的检出限为0.01 μg/mL,在0.05～100 λg/mL范围内,线性关系良好(r2＞0.999).通过对样品基质进行加标,检出限为0.20 mg/kg.将所建立的方法应用于金枪鱼罐头,烟熏鲣鱼,冻鲭鱼等样品中组胺的测定,测得的组胺含量为0.59～167 mg/kg,加标回收率均大于97%,测定值的相对标准偏差均小于5%.所建立的方法适用于大量样品的常规分析测定.     

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10-10,000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17 degrees C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.     

Sensitive method of detection, quantitation and purification of peptides using pre-column derivatization with phenyl isothiocyanate

A sensitive method for the detection, quantitation and purification of peptides is described. The method is based on pre-column derivatization of peptides with phenyl isothiocyanate to form phenylthiocarbamoyl derivatives (PTC peptides). The derivatized peptides are analysed by reversed-phase high-performance liquid chromatography on a Zorbax ODS column (5 micron) and detected at 269 nm with a sensitivity limit of 1-5 pmol. The technique was utilized for the separation of a mixture of closely related synthetic peptides. The eluted PTC peptides were collected with an average recovery yield of 75% as determined by amino acid analysis. This method of separation of PTC peptides was also combined with the determination of the complete structure of recovered PTC-dynorphin A-(1-13) using the solid-phase sequenator (Sequemat). The advantages of the derivatization method are the rapidity and completeness of the reaction, the stability of the product, the sensitivity and specificity of the detection of derivatized peptides and the compatibility of the technique with subsequent analytical procedures. A particular application of this method was exemplified by the dosage of enkephalins secreted from perfused bovine adrenal glands.     

Development of an automated on-line pre-column derivatization procedure for sensitive determination of histamine in food with high-performance liquid chromatography-fluorescence detection

An improved sensitive method was developed and validated for the determination of histamine in food samples by using automated on-line pre-column derivatization coupled with high performance liquid chromatography and fluorescence detection (HPLC-FLD). o-Phthaldialdehyde (OPA) was adopted as derivatization reagent, and a "sandwich" (OPA+histamine+OPA) aspiration mode for the automated on-line pre-column derivatization was found to efficiently enhance the method sensitivity and precision. Histamine in food samples was efficiently extracted with a methanol-phosphate buffer solution (50:50, v/v) at 60 degrees C for 30 min, and purified with Waters Oasis MCX solid-phase extraction (SPE) cartridge. The limit of quantification for this method is 0.2 mg/kg, which is very sensitive for histamine determination. With the "sandwich" injection program, 3.7% of relative standard deviation (RSD) was achieved by five replicative determinations of a sample blank spiked with 0.25 mg/kg histamine standard. Histamine in food samples such as fumitory skipjack and mackerel was analyzed with relative recoveries over 95% at spiking level of 150 mg/kg, as well as canned tuna fish and cheese with relative recoveries up to 98% at spiking levels of 0.50 and 5.0 mg/kg, respectively. The proposed method was validated with a sample from the Food Analysis Performance Assessment Scheme (FAPAS) as a standard certified material; and the results (140+/-6 mg/kg) agreed well with the assigned value (139 mg/kg).     

High-performance liquid chromatographic determination of valproic acid in plasma using a micelle-mediated pre-column derivatization

A method for the determination of valproic acid (2-propylpentanoic acid) in plasma by high-performance liquid chromatography (HPLC) after pre-column derivatization is described. The derivatization of valproic acid with a fluorophore and UV label, 4-bromomethyl-7-methoxycoumarin, is performed in plasma diluted with an aqueous micellar system. No extraction or solvent evaporation steps are required. The mechanism of the derivatization of the carboxylic acid is based on phase-transfer catalysis. The sample preparation, including the derivatization step, is rapid and very simple. The proposed HPLC-method was evaluated and compared with a standard immunological assay used for the determination of valproic acid in plasma.     

The Determination of Cyclohexylamine in Aqueous Solutions of Sodium Cyclamate by Electron-Capture Gas Chromatography

Abstract

A sensitive primary amine assay, capable of detecting 10^?11 g. and utilizing the determination of the amine N-2, 4-dinitrophenyl derivative by electron-capture gas chromatography is described. The method is exemplified by the determination of cyclohexylamine in sodium cyclamate.     

A new and simple method to determine trace levels of sulfonamides in honey by high performance liquid chromatography with fluorescence detection

A novel method for the simultaneous analysis at trace level of sulfonamides (sulfaguanidine, sulfanilamide, sulfacetamide, sulfathiazole, sulfapyridine, sulfachloropyridazine, sulfamerazine, sulfameter, sulfamethazine, sulfadoxine, sulfadiazine, sulfamonomethoxine, sulfadimethoxine) in honey is described. Methanol has been used in the sample treatment step to avoid the emulsion formation and to break the N-glycosidic bond between sugars and sulfonamides. The determination is carried out by liquid chromatography in gradient elution mode, with fluorescence detection after the on-line pre-column derivatization with fluorescamine. The influence of parameters such as the mobile phase composition, column temperature, pH or injection volume, on the separation has been taken into account and the derivatization step has also been optimized. Recoveries of the compounds on spiked honey samples ranged from 56% for sulfadoxine to 96% for sulfacetamide, with relative standard deviations below 10%. The quantitation limits are between 4 and 15 ng g⁻¹.     

Trimethylsilyldiazomethane derivatization coupled with solid-phase extraction for the determination of alendronate in human plasma by LC-MS/MS

Alendronate is an important representative of bisphosphonates, strongly polar compounds that lack chromophores. With rare exceptions, derivatization of the analytes is necessary for bioanalysis. In this study, a rapid liquid chromatography–tandem mass spectrometry method employing pre-column derivatization was developed and validated for the determination of alendronate concentrations in human plasma. The procedure was based on derivatization with trimethylsilyldiazomethane during solid-phase extraction on a weak anion-exchange solid-phase cartridge, which integrated sample purification and derivatization into one step. The alendronate derivative was eluted with methanol. Chromatographic separation was performed on a Capcell PAK-C18 column. The total run time was 6.5 min. The calibration curve was linear in the range 1.00–1,000 ng/mL using d6-alendronate as the internal standard. The lower limit of quantification was 1.00 ng/mL. The intra- and inter-assay precision (in RSD) calculated from quality control samples was less than 15%, and the accuracy was between 98.1% and 100.2%. The validated method was successfully applied to characterize the pharmacokinetic profiles of alendronate following the intravenous infusion of 5 or 10 mg alendronate sodium to healthy volunteers.