A mixed-bed, multi-derivatization approach using polymeric reagents for derivatizations of amines in high performance liquid chromatographic detection

Immobilized, polymeric reagents containing covalently attached tagging groups have been synthesized and reacted individually, off-line or on-line, pre-column in high performance liquid chromatographic (HPLC) detection. These reagents have also been combined into a single, mixed-bed reactor, useful for simultaneously preparing several derivatives from a single analyte, all at the same time. Each derivative possesses different chromatographic and detection properties, dependent on the nature of the original polymeric reagent containing the immobilized, tagging species. These particular reagents were designed to impart Ultraviolet/fluorescence, Ultraviolet/electrochemistry (oxidative/reductive or oxidative-hv-electrochemistry) to the final derivatives. Variations in the amounts/ratios of polymeric reagents contained in a single mixed-bed reactor will lead to varying ratios of the final derivatives. These can be predicted knowing the approximate reactivity of each polymeric reagent, percent derivatizations, and overall rates for each reagent towards a given substrate. In this first example of mixed-bed, polymeric reagents for improved derivatization approaches in chromatography, emphasis has been placed on simple amines or amine-like analytes. Multiple derivatives can be effectively used to improve the identification of an unknown analyte in a complex sample matrix, as well as to improve the detectability of that analyte. As one real world application, amphetamine in human urine was quantitated via on-line derivatizations with a mixed-bed reactor. With the least sampling work-up, the resulting sample solutions were directly injected into the on-line derivatization HPLC system for quantitation. The method was validated by single blind spiking experiments. The precision and accuracy were acceptable.     

Mixed-bed polymeric o-nitrobenzophenone reagents for the on-line derivatization of amines in high performance liquid chromatography.

Several polymer-bound o-nitrobenzophenone reagents containing different detector-sensitive tags have been combined in the same reactor for the on-line derivatization of amine samples. The formation of multiple derivatives allows numerous opportunities for quantitation from the same injection, and also allows improved identification from the retention times of the multiple derivatives. Changing the reaction conditions changes the ratio of the products formed, so that changes in the ratio of peak heights and areas can also be used for analyte identification. In this work, propylamine was derivatized in acetonitrile on-line, precolumn. Changing the reaction conditions, of reaction time, temperature, solvent, presence of catalyst and the components of the reactor, changed the ratio of the derivatives formed. These changes in product formation with changing reaction conditions were applied to the identification and quantitation of amphetamine and methamphetamine in urine. The drugs were identified by the retention times of their derivatives, the ratio of the peak areas of the derivatives and the change in the ratios after changing reaction conditions. Each derivative was also used for quantitation of levels of spiked concentrations of amphetamine and methamphetamine, with relative errors less than 8%.     

苯骈三氮唑的胺烷基化: 二级胺的合成

在干苯溶液中, 用4-N, N-二甲胺基苯甲醛, 苯骈三氮唑和芳胺或杂环芳胺回流,生成N-取代-1-苯骈三氮唑基-P-1N,N-二甲胺基)苄胺, 然后利用NaBH~4还原, 结果得到N-取代-4-N,N-二甲胺基苄胺, 反应条件温和、产率高、后处理方便, 一般只需一次重结晶, 即可得到纯产物。因此, 是合成二级胺的较好方法。     

Kinetics of the addition of electrophilic vinyl reagents on protonated tertiary amines supported by a polymeric chain

Starting from the mechanism of the addition of vinyl methyl ketone to protonated tertiary polyamine, we examined the kinetics of addition of acrylic acid, acrylamide and methyl acrylate to P4VP in the presence of HBr. The reactivities depend on the electrophilic character of the double bond; the kinetics of addition of vinyl methyl ketone to poly(2-methyl-5-vinylpyridine), poly(2-vinylpyridine), poly(2-vinylquinoline) and poly(dimethylaminostyrene) in the presence of HBr depend essentially on the steric hindrance of the tertiary amine.     

Polymer-supported organotin reagents for regioselective halogenation of aromatic amines

[reaction: see text] Polymer-supported triorganotin halides were used in the halogenation reaction of aromatic amines. Treatment of aromatic amines with n-butyllithium and polymer-supported organotin halides gave the corresponding polymer-bound N-triorganostannylamines, which by treatment with bromine or iodine monochloride gave the para-halogenated aromatic amines with high yields and high selectivities. The polymer-supported organotin halides reagents regenerated during the course of the halogenation reaction can be reused without loss of efficiency. The presence of tin residues in halogenated aromatic amines was also investigated and evaluated at under 20 ppm after three runs.     

Thermogravimetric studies of polymeric reagents: a polymeric o-benzyne precursor

Thermogravimetry for the study of polymeric reagents is described and used to demonstrate the thermic behavior of a polymeric o-benzyne precursor     

Porous copolymer-based cation exchanger for the off-line preconcentration of aromatic amines from water

Summary A weakly polar porous copolymer and the sulfonic acid cation exchanger based on this copolymer were tested as sorbents for off-line preconcentration of aromatic amines from water. Minicolumns packed with the 1,4-di(methacryloyloxymethyl)naphthalene—divinylbenzene copolymer and the cation exchanger were used for the solid-phase extraction of polar amines. In order to study the sorption properties of these polymeric materials, the recoveries and breakthrough volumes ofp-aminophenol,o, m andp-phenylenediamine, aniline,o andp-anisidine,p-nitroaniline, ando-toluidine were determined.     

New procedure of selected biogenic amines determination in wine samples by HPLC

A new procedure for determination of biogenic amines (BA): histamine, phenethylamine, tyramine and tryptamine, based on the derivatization reaction with 2-chloro-1,3-dinitro-5-(trifluoromethyl)-benzene (CNBF), is proposed. The amines derivatives with CNBF were isolated and characterized by X-ray crystallography and ¹H, ¹³C, ¹⁹F NMR spectroscopy in solution. The novelty of the procedure is based on the pure and well-characterized products of the amines derivatization reaction. The method was applied for the simultaneous analysis of the above mentioned biogenic amines in wine samples by the reversed phase-high performance liquid chromatography. The procedure revealed correlation coefficients (R²) between 0.9997 and 0.9999, and linear range: 0.10–9.00 mg L⁻¹ (histamine); 0.10–9.36 mg L^-1 (tyramine); 0.09–8.64 mg L⁻¹ (tryptamine) and 0.10–8.64 mg L⁻¹ (phenethylamine), whereas accuracy was 97%–102% (recovery test). Detection limit of biogenic amines in wine samples was 0.02–0.03 mg L⁻¹, whereas quantification limit ranged 0.05–0.10 mg L⁻¹. The variation coefficients for the analyzed amines ranged between 0.49% and 3.92%. Obtained BA derivatives enhanced separation the analytes on chromatograms due to the inhibition of hydrolysis reaction and the reduction of by-products formation.     

Phenyl esters, preferred reagents for mono-acylation of polyamines in the presence of water

In the presence of water, several diamines and one triamine were mono-acylated at ambient to moderate temperatures using phenyl esters and a phenyl carbonate as acylation agents in good to excellent isolated yields. Both linear and cyclic polyamines were suitable substrates, and the acylating agents can be aryl and alkyl carboxylic acid esters.     

HPLC determination of polyamines in the femtomole range

A new method for the chromatographic determination of polyamines is presented. The procedure consists of the reaction of 9-fluorenylmethyl chloroformate (FMOC-Cl) with polyamines at 50 +/- 1 degrees C for 10 min, followed by stepwise elution chromatography. The reaction is simple and the derivatives are stable. All the polyamine-FMOC derivatives can be separated within 13 min. The linearity of this method is satisfactory in the range of 0.5-100 pmol. The detection limits for putrescine, cadaverine, spermidine and spermine are 83.3 fmol, 109 fmol, 25.5 fmol and 22.7 fmol respectively.     

An HPLC fluorescence detection system for amines

A detection system is described which is based on fluorescent ion-pair formation between tertiary amine drugs and dimethoxyanthracene sulphonate. A dynamic micro-extraction principle is then used to isolate the ion-pairs from the excess reagent. The band broadening of this extraction detector was kept below 20% using standard auto-analyser equipment. With the enhanced selectivity and sensitivity of this approach it was possible to analyse chloropheniramine in urine at ppb concentrations using a pre-column clean-up trace enrichment step.     

Chiral amines as reagents for HPLC-MS enantioseparation of chiral carboxylic acids

Mass spectrometry (MS) has become a popular analytical technique because of its high sensitivity and specificity. Therefore, the use of a chiral derivatization reagent for the MS detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis is very limited. The applicability of commercially available chiral amines as the derivatization reagents for the enantiomeric separation of chiral carboxylic acids is reported in this paper by using non-steroidal anti-inflammatory drugs (NSAIDs), i.e. ibuprofen, flurbiprofen, and loxoprofen. The efficiency of the chiral reagents was evaluated in terms of tagging easiness, separation by reversed-phase chromatography, and detection sensitivity by electrospray ionization (ESI)-MS/MS. Among the tested eight chiral amines, i.e. (R)-(+)-4-(3-aminopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-APy), (S)-(+)-1-(2-pyrrolidinylmethyl)-pyrrolidine (PMP), L-prolinamide, (3R)-(-)-1-benzyl-3-aminopyrrolidine, (S)-(+)-1-cyclohexyl-ethylamine, (3R)-(+)-3-(trifluoroacetamido)-pyrrolidine (TFAP), (R)-(-)-1-aminoindan (AI), and (S)-(+)-tetrahydrofurfuryl-amine, DBD-APy, PMP, AI, and TFAP could be used as the chiral reagents for the enantiomeric separation of the NSAIDs. The Rs values and the detection limits of the derivatives were in the range of 1.29-3.85 and 0.57-0.96 fmol, respectively. These four reagents were applied for the determination of the NSAIDs in rat plasma.     

New off-line aircraft instrumentation for non-methane hydrocarbon measurements

New off-line instrumentation was developed to implement measurements of non-methane hydrocarbons (NMHC) on (French) research aircraft. NMHC are collected on multisorbent tubes by AMOVOC (Airborne Measurements Of Volatile Organic Compounds), a new automatic sampler. AMOVOC is a versatile and portable sampler targeting a wide range of NMHC at high frequency (sampling time of 10 min). Multisorbent tubes are analyzed on the ground by short-path thermal desorption coupled with gas chromatography and mass spectrometry. The development and optimization of both NMHC sampling and analysis are reported here. On the one hand, the paper points out technical choices that were made according to aircraft constraints and avoiding sample loss or contamination. On the other hand, it describes analytical optimization, tube storage stability, and moisture removal. The method shows high selectivity, sensitivity (limit of detection less than 10 ppt) and precision (less than 24%). Finally, NMHC data collected on French aircraft during the African Monsoon Multidisciplinary Analysis campaign are reported for the first time. The results highlight instrumentation validity and protocol efficiency for NMHC measurements in the lower and upper troposphere.     

New reagents for determination of thorium

Summary O-, m- and p-hydroxy, 3-methoxy, 4-hydroxy, p-methoxy and 3,4-dimethoxy derivatives of cinnamic acid have been investigated for the separation and precipitation of thorium either when present alone or along with trivalent rare-earths. Except for somewhat low results with o-Coumaric acid at low concentration of thorium, all of these acids are satisfactory. However, from the point of speed and expediency, p-hydroxy cinnamic acid (p-coumaric acid) gives a granular precipitate and is to be recommended. Methoxy derivatives of cinnamic acid readily reduce Ce^IV to Ce^III state and therefore offer an advantage when precipitation of thorium is required to be carried out in presence of quadrivalent cerium.     

Iron-catalyzed oxidative monoarylation of primary amines with organozinc reagents

The reaction of a primary zinc amide with a diorganozinc reagent gives a secondary amine in the presence of Fe(acac)(3) as a catalyst and 1,2-dichloroisobutane (DCIB) as an oxidant. Halogen groups such as F, Cl, Br, and I are tolerated well. The dichloride oxidant and heat are essential to achieve the C-N bond formation presumably from a catalytic iron intermediate species bearing aryl and amido groups.     

New phosphorylating reagents for deoxyribonucleosides and oligonucleotides

New phosphorylating reagents 1 and 2 were prepared in three steps from 4-hydroxybenzaldehyde. They showed good efficiency in the solid phase synthesis of 5′-phosphate monoester nucleosides. End-phosphate DNA sequence synthesis demonstrated the efficiency of the new reagents (1 and 2) according to the general procedure of automated DNA synthesis. The oxidation of P(III) to P(V) and the removal of benzyl protecting groups were achieved in a single step by treatment with a 0.02 M I₂/pyridine/H₂O solution. Due to this one-pot treatment, it is possible to use the phosphorylating reagents (1 and 2) for the synthesis of base-sensitive ODNs. The reagents 1 and 2 are unique among phosphorylating reagents.     

Sensitive ferrocene reagents for derivatization of amines for high-performance liquid chromatography with electrochemical detection

Six reagents possessing ferrocene as an electrophore were prepared and evaluated for pre-column derivatization of amino compounds for their determination by high-performance liquid chromatography with electrochemical detection. The utility of these reagents was investigated employing phenethylamine as a model compound. Among these six, N-succinimidyl 3-ferrocenylpropionate was the best with respect to reactivity, stability and electrochemical properties. The developed method was applied to the determination of putrescine formed from ornithine by ornithine decarboxylase.     

Posttranslational modifications of collagen studied by off-line coupling of HPLC and CE

A typical example of non-enzymatic change of collagen is glycation (the Maillard reaction, formation of advanced glycation end products) resulting from the reaction of sugars with the epsilon-amino group of lysine. Posttranslational non-enzymatic modifications of collagen by sugars were studied. Collagenous tissues were incubated as a test protein separately with both glucose and ribose. The collagen mixture was digested by bacterial collagenase and separated by reversed-phase HPLC (in a Jupiter Proteo 90 A column). The eluate from this HPLC separation was collected as seven fractions and consecutively analysed by CE in a bare fused silica capillary (57/50 cm x 75 mm id) using 100 mM sodium 1-heptanesulfonate in 100 mM phosphate buffer, pH 2.5 (NaH2PO4 adjusted to pH by phosphoric acid). The chromatographic and electromigration behaviour of individual peptides varied considerably. This off-line HPLC-CE coupling made it possible to discover minor changes in the structure of collagen caused by posttranslational modifications. A new HPLC-CE technique for peptide analysis was developed, and applied to the identification of posttranslational modifications in slowly metabolised test proteins.