Derivatisation for liquid chromatography/electrospray mass spectrometry: synthesis of pyridinium compounds and their amine and carboxylic acid derivatives

A simple method has been developed for the pre-column derivatisation of low molecular weight primary and secondary amines and carboxylic acids using quaternary nitrogen compounds to enhance their detection by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS). The synthesis of seven novel quaternary nitrogen reagents is described. The derivatives are designed to be relatively small molecules to avoid some of the steric hindrance problems that may be associated with larger derivatisation reagents. The compounds have amine and carboxylic acid functional groups with which to derivatise carboxylic acids and amines, respectively. Two of the compounds contain a bromine atom in order to assess the advantages of a bromine isotope pattern in the mass spectra. This acts as a simple marker for derivatisation and enables data processing by cluster analysis.Activation of the carboxylic acid group was achieved by the use of either 1-chloro-4-methylpyridinium iodide (CMPI) or the more reactive 1-fluoro-4-methylpyridinium p-toluenesulphonate (FMP).1 Using both of these active reagents, the degree of nucleophilic substitution was investigated for the derivatisation of a variety of small molecules. Whilst giving some increase in the ESI-MS response for the derivatised compounds, the FMP itself acted as a derivatising reagent in a competing reaction. In the light of this finding, FMP was reacted with the test compounds separately and gave positive results as a derivatising reagent. Detection of the 'pre-charged' derivatives of amines and carboxylic acids by LC/ESI-MS was investigated with respect to their ESI response and chromatography.     

Derivatization for liquid chromatography/electrospray mass spectrometry: synthesis of tris(trimethoxyphenyl)phosphonium compounds and their derivatives of amine and carboxylic acids

A simple method for the derivatization of primary amines and carboxylic acids with tris(trimethoxyphenyl)phosphonium (TMPP) reagents to enhance their detection by electrospray mass spectrometry (ESI-MS) has been developed. The synthesis of novel TMPP reagents and their stable isotopically labelled analogues is described. Through the use of stable isotopically labelled TMPP "tags", incorporation of a doublet (1:1, (1)H/(2)H or (12)C/(13)C) into the target molecule can be achieved, enabling the use of isotopic target analysis to detect compounds of unknown molecular weight but with a characteristic isotope pattern and accurate mass difference.     

A carbonyl oxygen migration in electrospray ionization mass spectrometry and its application in differentiating alpha- and beta-alanyl peptides

A novel rearrangement reaction with a carbonyl oxygen migration was observed in the electrospray ionization tandem mass spectra of N-diisopropyloxyphosphoryl dipeptides and their analogues. A possible mechanism was proposed and supported by the MS/MS study, derivatization of different functional groups and deuterium labeling experiments. It was found that metal ions could catalyze the rearrangement through a five-membered ring intermediate. A strong affinity between the phosphoryl group and oxygen atom in the gas phase was proposed to result in this kind of rearrangement reaction, which might provide some basic information on the nature of phosphorylation in biochemistry. The replacement of N-terminal alpha-alanine by beta-alanine stopped the migration, which provides a simple method for differentiating the alpha- and beta-alanine residues at the N-terminus of peptides.     

Desorption electrospray ionization (DESI) mass spectrometry and tandem mass spectrometry (MS/MS) of phospholipids and sphingolipids: Ionization, adduct formation, and fragmentation

Desorption electrospray ionization (DESI) mass spectrometry was evaluated for the characterization of glycerophospholipid standards, including glycerophosphocholine (GPCho), glycerophosphoglycerol (GPGro), glycerophosphoethanolamine (GPEtn), glycerophosphoserine (GPSer), glycerophosphoinositol (GPIns), cardiolipin (CL), and sphingolipid standards, including sulfatides (ST) and sphingomyelin (SM). Of specific interest were the effects of surface and solvent composition on signal stability and intensity, along with the ions observed in the full scan mode and the fragmentations seen upon collisional activation for each of the above classes. These experiments were performed without the addition of matrix compounds to the sample and were conducted in the free ambient environment at atmospheric pressure. The compounds GPSer, GPGro, GPIns, ST, and CL were best analyzed in the negative ion mode while PE was ionized efficiently in both positive and negative ion modes. SM and GPCho, which typically generate more abundant ions in the positive ion mode, could be analyzed in the negative ion mode by the addition of anionic reagents such as acetate to the spray solvent. Full scan DESI mass spectra and tandem (MS/MS) spectra for this representative set of physiological phospho/sphingolipids are presented. Similarities with other ionization methods in terms of fragmentation behavior were strong, although ambient ionization of untreated samples is only available with DESI. The effect of surface and solvent properties on signal intensity and stability were determined by depositing standard compounds on several different surfaces and analyzing with various proportions of methanol in the aqueous spray. Analysis was extended to complex mixtures of phospholipids and sphingolipids by examining the total lipid extract of porcine brain and by direct analysis of rat brain cryotome sections. These types of mixture analyses and molecular imaging studies are likely to represent major areas of application of DESI.     

Compounds having thiourea moiety as derivatization reagents in liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS): synthesis of derivatization reagents for carboxylic acids

The derivatization reagents for carboxylic acids, N‐(Pyridin‐3‐yl)hydrazinecarbothioamide, N‐[4‐(dimethylamino)phenyl]hydrazinecarbothioamide, 1‐(2‐aminoethyl)‐3‐(pyridin‐3‐yl)thiourea, 1‐(2‐aminoethyl)‐3‐[4‐(dimethylamino)phenyl]thiourea and 4‐(2‐aminoethyl)‐N‐phenylpiperazine‐1‐carbothioamide were synthesized. These reagents reacted with carboxylic acids at 60°C for 45 min in the presence of the condensation reagents. The generated derivatives were favorably separated on the reversed‐phase column and sensitively detected by electrospray ionization tandem mass spectrometry. These reagents enhanced the electrospray ionization response of the analyte and generated a particular product ion efficiently by collision‐induced dissociation, and thus they were suitable for MS/MS detection. Copyright © 2010 John Wiley & Sons, Ltd.     

Polarization induced electrospray ionization mass spectrometry for the analysis of liquid,viscous and solid samples

In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.     

Chemical ionization mass spectrometry of derivatives of L-dopa and L-tryptophan and their detection in tumour samples

Conditions have been established for the detection by chemical ionization mass spectrometry (CIMS) of compounds related to L -dopa (3-hydroxy-L -tyrosine) and L -tryptophan without derivatization. The least fragmentation of the parent ion occurs in positive ion CIMS when methylamine is used as reagent gas. The compounds can also be detected by negative ion CIMS using carbon tetrachloride as reagent gas. While the total ion current in the latter technique is lower than that obtained with positive ion CIMS, the background noise in the mass spectra of samples obtained from natural sources is greatly reduced. CIMS has been used to show the presence of aromatic amino acids in acid extracts of samples of two different classes of tumour without sample derivatization.     

Subphthalocyanines as fluoro-chromogenic probes for anions and their application to the highly selective and sensitive cyanide detection

The use of a subphthalocyanine derivative as a selective chromo-fluorogenic reporter for the anion cyanide in mixed aqueous solutions is reported.     

Determination of hexamethylphosphoramide and other highly polar phosphoramides in water samples using reversed-phase liquid chromatography/electrospray ionization time-of-flight mass spectrometry

The widely used solvent hexamethylphosphoramide (HMPA) and its biological (metabolic) and chemical (abiotic) phosphoramide-based oxidation products may cause adverse health effects through occupational exposure and intake of contaminated groundwater. However, no current methods exist for the separation and the detection of the many polar HMPA oxidation products. Thus, we developed a new RPLC/ESI-TOF-MS method and further investigated the chromatographic performances of two columns (i.e., XTerra Phenyl and XBridge Phenyl). In addition, the impact of (forced) acid hydrolysis for optimized chromatographic performance of the XTerra Phenyl column is investigated. The XTerra Phenyl column showed the best separation of the less polar major metabolic oxidation products pentamethylphosphoramide and hydroxymethyl-pentamethylphosphoramide, however, only after treating the column with formic acid (acid-treated). The XTerra column separated most of the investigated HMPA oxidation products (11 of 16 compounds) in a single chromatographic run. In contrast, the XBridge Phenyl column requires one method for the less polar and another method for the more polar oxidation products. However, this results in an overall better separation performance of the XBridge Phenyl column, especially for the less polar major abiotic oxidation products hydroxymethyl-pentamethylphosphoramide and formyl-pentamethylphosphoramide, as well as for 11 highly polar oxidation products (R(S)>1.5). The RPLC/ESI-TOF-MS method presented and validated in this study is the first analytical method that can be used to separate and detect HMPA (LOD 0.10 μM without preconcentration) and all of its oxidation products.     

Simultaneous quantitation of sphingoid bases and their phosphates in biological samples by liquid chromatography/electrospray ionization tandem mass spectrometry

We developed a liquid chromatography/electrospray ionization tandem mass spectrometry method for the simultaneous quantitative determination of C18 sphingosine (Sph), C18 dihydrosphingosine (dhSph), C18 phytosphingosine (pSph), C18 sphingosine-1-phosphate (S1P), C18 dihydrosphingosine-1-phosphate (dhS1P), and C18 phytosphingosine-1-phosphate (pS1P). Samples were prepared by simple methanol deproteinization and analyzed in selected reaction monitoring modes. No peak tailing was observed on the chromatograms using a Capcell Pak ACR column (1.5 mm i.d. × 250 mm, 3 μm, Shiseido). The calibration curves of the sphingoids showed good linearity (r > 0.996) over the range of 0.050-5.00 pmol per injection. The accuracy and precision of this method were demonstrated using four representative biological samples (serum, brain, liver, and spleen) from mice that contained known amounts of the sphingoids. Samples of mice tissue such as plasma, brain, eye, testis, liver, kidney, lung, spleen, lymph node, and thymus were examined for their Sph, dhSph, pSph, S1P, dhS1P, and pS1P composition. The results confirmed the usefulness of this method for the physiological and pathological analysis of the composition of important sphingoids.     

High-performance liquid chromatography with paired ion electrospray ionization (PIESI) tandem mass spectrometry for the highly sensitive determination of acidic pesticides in water

A novel method based on the paired ion electrospray ionization (PIESI) mass spectrometry has been developed for determination of acidic pesticides at ultratrace levels in surface and ground waters. The proposed approach provides greatly enhanced sensitivity for acidic pesticides and overcomes the drawbacks of the less sensitive negative ion mode ESI-MS. The limits of detection (LODs) of 19 acidic pesticides were evaluated with four types of dicationic ion-pairing reagent (IPR) in both single ion monitoring (SIM) and selected reaction monitoring (SRM) mode. The LOD of 19 pesticides obtained with the use the optimal dicationic ion-pairing reagent ranged from 0.6 pg to 19 pg, indicating the superior sensitivity provided by this method. The transition pathways for different pesticide-IPR complexes during the collision induced dissociation (CID) were identified. To evaluate and eliminate any matrix effects and further decrease the detection limits, off-line solid-phase extraction (SPE) was performed for DI water and a river water matrix spiked with 2000 ng L⁻¹ and 20 ng L⁻¹ pesticides standards respectively, which showed an average percent recovery of 93%. The chromatographic separation of the acidic pesticides was conducted by high-performance liquid chromatography (HPLC) using a C18 column (250 mm × 2.1 mm) in the reversed phase mode using linear gradient elution. The optimized HPLC–PIESI-MS/MS method was utilized for determination of acidic pesticide at ng L⁻¹ level in stream/pond water samples. This experimental approach is 1–3 orders of magnitude more sensitive for these analytes than other reported methods performed in the negative ion mode.     

Rapid and highly sensitive liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantitation of buspirone in human plasma: application to a pharmacokinetic study

A rapid and sensitive method for the quantitation of buspirone in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was developed. Plasma samples were treated by liquid-liquid extraction with methyl tert-butyl ether (MTBE). The chromatographic separation was performed isocratically on a reversed-phase Shiseido C18 column (50 mm x 2.0 mm, 3 microm) with a mobile phase of acetonitrile/0.1% acetic acid (1:1, v/v). The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 386 --> 122 for buspirone and m/z 409 --> 238 for amlodipine (the internal standard). The method was validated to determine its specificity, recovery, limit of quantitation, accuracy and precision. The lower limit of quantitation was 0.02 ng/mL with a relative standard deviation of less than 10%. The present method provides an accurate, precise and sensitive tool for buspirone and was successfully applied to a pharmacokinetic study in eight subjects.     

Rapid separation and identification of phenolic and diterpenoid constituents from Radix Salvia miltiorrhizae by high-performance liquid chromatography diode-array detection, electrospray ionization time-of-flight mass spectrometry and electrospray ionization quadrupole ion trap mass spectrometry

High-performance liquid chromatography-diode array detection (HPLC-DAD), electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) and electrospray ionization quadrupole ion trap mass spectrometry (HPLC-ESI-MSn) were used for the isolation, identification and structural analysis of water-soluble phenolic and nonpolar diterpenoid constituents in Dan-shen (Radix Salvia miltiorrhizae) which was prepared by sonication in 70% methanol. Mass spectra were obtained by ESI-TOF-MS and electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS). A formula database of known constituents in Dan-shen was established and most constituents were rapidly identified by HPLC-DAD/ESI-TOF-MS by matching their accurate molecular masses with the formulae of the compounds in the database. Compounds with the same molecular formula could not be differentiated by TOF-MS; however, QIT-MS could differentiate those compounds and elucidate their structures based on their characteristic fragmentation. HPLC-DAD, HPLC/ESI-TOF-MS and HPLC/ESI-MSn provided complementary information for the identification of the constituents in Dan-shen. Forty constituents were identified in 30 min based on their positive and negative ion ESI mass spectra and liquid chromatographic information. Thus the method described is useful for the rapid analysis of multiple constituents in Dan-shen.     

Synthesis of benzofurazan derivatization reagents for short chain carboxylic acids in liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS)

Benzofurazan derivatization reagents, 4‐[2‐(N,N‐dimethylamino)ethylaminosulfonyl]‐7‐(2‐aminopentylamino)‐2,1,3‐benzoxadiazole (DAABD‐AP) and 4‐[2‐(N,N‐dimethylamino) ethylaminosulfonyl]‐7‐(2‐aminobutylamino)‐2,1,3‐benzoxadiazole (DAABD‐AB), for short‐chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60°C for 60 min. The generated derivatives were separated on the reversed‐phase column and detected by ESI‐MS/MS with the detection limits of 0.1–0.12 pmol on column. Upon collision‐induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD‐AP and DAABD‐AB are suitable as the derivatization reagents in LC/ESI‐MS/MS analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Unexpected synthesis of conformationally restricted analogues of gamma-amino butyric acid (GABA): mechanism elucidation by electrospray ionization mass spectrometry

From previous results with lower homologues, dehydroiodination of the three alkenyl-beta-enamino esters 3a-c was expected to provide six-membered N-heterocyclic products. The reactions of 3a-c with triethylamine are found to lead, however, to the unexpected stereoselective synthesis of the trisubstituted cyclopentane derivatives 4a-c, as confirmed by IR and NMR spectroscopy. Cyclopentanes 4a-c bear two chiral centers and a gamma-amino ester moiety, and are therefore conformationally restricted analogues of gamma-amino butyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system. Use of electrospray ionization mass (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) allowed the key iminium ion intermediates 5a-c(+), as well as the protonated molecules of both the reactant and final products, [3a-c + H](+) and [4a-c + H](+), to be intercepted and structurally characterized. From these findings a mechanism for this unexpected but synthetically attractive and efficient stereoselective reaction is proposed.     

High-performance liquid chromatography of selenium compounds utilizing perfluorinated carboxylic acid ion-pairing agents and inductively coupled plasma and electrospray ionization mass spectrometric detection

Increasing speciation demands in clinical chemistry, toxicology and nutrition have made the determination of the total elements in a sample inadequate; the amount of an element and the chemical forms in which it is present need to be known. Inductively coupled plasma mass spectrometry (ICP-MS) was used after high-performance liquid chromatographic (HPLC) separation, as was electrospray ionization mass spectrometry (ESI-MS). The effect of variation of the number of carbon atoms in perfluorinated carboxylic acids used as ion-pairing agents for the separation of selenium compounds was examined. Trifluoroacetic acid (0.1%), pentafluoropropanoic acid (0.1%) or heptafluorobutanoic acid (0.1%; HFBA) were alternatively used as additives to methanol-water (1:99, v/v) solutions as mobile phases. Reversed-phase HPLC-ICP-MS with 0.1% HFBA in the mobile phase allowed more than 20 selenium compounds to be separated in 70 min in an isocratic elution mode; the separation of natural selenium-enriched sample extracts was examined and explained. The pH of the 0.1% HFBA solution was modified with hydrochloric acid or ammonia and the pH of the sample extracts before injection was modified in order to overcome unwanted double peak formation in the chromatograms of sample extracts. Oxidations of standard gamma-glutamyl-Se-methylselenocysteine and Se-methylselenocysteine were carried out using 30% H2O2 solution and identifications of selenium-containing oxidation products were made using HPLC-ICP-MS and HPLC-ESI-MS. The principal organic oxidation product in both cases was methaneseleninic acid (MeSeO2H).     

Fast analysis of flavonoids in plant extracts by liquid chromatography-ultraviolet absorbance detection on poly(carboxylic acid)-coated silica and electrospray ionization tandem mass spectrometric detection

A highly hydrophilic poly(7-oxonorbornene-5,6-dicarboxylic acid-block-norbornene) [=poly-(ONDCA-b-NBE)]-coated silica was investigated for the liquid chromatographic (LC) determination of flavonoids in plant extracts of complex biological origin using UV absorbance and mass spectrometric (MS) detection. Compared to the most commonly used octadecyl derivatized silica this sorbent allowed fast separations even at extreme pH values. Furthermore, UV absorbance and MS detection were evaluated. As we found, UV detection at 254 nm allows the determination of flavonoids down to the ng range with a linearity of R2>0.9906. For the more selective characterization the validated LC system was coupled to a quadrupole ion trap mass spectrometer via an electrospray ionization (ESI) interface. MS detection showed high linearity (R2>0.9904) for all investigated flavonoids. Due to the relatively high flow-rate of 1 ml/min the limits of detection were found in the lower-microg range. Collision induced dissociation was applied to obtain characteristic fragmentation fingerprints. Finally, the validated LC-ESI-MS-MS method demonstrated that this poly-(ONDCA-b-NBE) stationary phase allows fast characterization and quantitation in onion, elderflower blossom, lime blossom, St. John's Wort and red wine.     

Rapid,simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05 − 5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.     

Quantification and remote detection of nitro explosives by helium plasma ionization mass spectrometry (HePI‐MS) on a modified atmospheric pressure source designed for electrospray ionization

Helium Plasma Ionization (HePI) generates gaseous negative ions upon exposure of vapors emanating from organic nitro compounds. A simple adaptation converts any electrospray ionization source to a HePI source by passing helium through the sample delivery metal capillary held at a negative potential. Compared with the demands of other He‐requiring ambient pressure ionization sources, the consumption of helium by the HePI source is minimal (20–30 ml/min). Quantification experiments conducted by exposing solid deposits to a HePI source revealed that 1 ng of 2,4,6‐trinitrotoluene (TNT) on a filter paper (about 0.01 ng/mm²) could be detected by this method. When vapor emanating from a 1,3,5‐trinitroperhydro‐1,3,5‐triazine (RDX) sample was subjected to helium plasma ionization mass spectrometry (HePI‐MS), a peak was observed at m/z 268 for (RDX●NO₂)^?. This facile formation of NO₂^? adducts was noted without the need of any extra additives as dopants. Quantitative evaluations showed RDX detection by HePI‐MS to be linear over at least three orders of magnitude. TNT samples placed even 5 m away from the source were detected when the sample headspace vapor was swept by a stream of argon or nitrogen and delivered to the helium plasma ion source via a metal tube. Among the tubing materials investigated, stainless steel showed the best performance for sample delivery. A system with a copper tube, and air as the carrier gas, for example, failed to deliver any detectable amount of TNT to the source. In fact, passing over hot copper appears to be a practical way of removing TNT or other nitroaromatics from ambient air. Copyright © 2012 John Wiley & Sons, Ltd.