Regioisomer characterization of triacylglycerols by non‐aqueous reversed‐phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag⁺ after their separation by non‐aqueous reversed‐phase liquid chromatography (NARP‐LC) before MS to improve MS sensitivity. Electrospray ionization–MS (ESI‐MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO₃]⁺ ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS⁵ spectra. The observation of ions produced by LC‐MS⁵ of on‐line Ag⁺‐cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS⁵ experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils. Copyright © 2010 John Wiley & Sons, Ltd.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Characterisation of interferon α‐2b by liquid chromatography and mass spectrometry techniques

Interferon α‐2b produced by Escherichia coli consists of 165 amino acids and contains two disulphide bonds; its purity was confirmed by LC‐UV (DAD)‐FLD and LC‐MS techniques. A C₄ column was used with UV detection at 214 nm; diode array detector (DAD) spectra were recorded from 200–400 nm and fluorescence detection was performed at specific wavelengths of trypthophan emission and excitation. Peptide mapping was performed with trypsin. Peptides produced by trypsin digestion were analysed by LC‐UV (DAD)‐FLD, LC‐MS, and LC‐MS/MS using a C₁₈ column. Amino acid sequence coverage was about 95%. UV spectra in the range from 200 nm to 400 nm, emission (Em) and excitation (Ex) spectra of each separated peptide were additionally compared with spectra of the same peptide produced by digestion of European Pharmacopaeia interferon α‐2b standard (spectral matching). The chromatogram of any interferon α‐2b (drug substance or certificated standard) sample produced in the same manner with the same amino acid composition should be similar to the chromatogram obtained by the method described in this paper. Molecular masses of peptides were obtained from MS experiments and MS/MS experiments gave additional structural information. The molecular mass of interferon α‐2b was obtained by MALDI‐TOF MS analysis in linear mode, with an accuracy comparable to the theoretical average mass ± 5 atomic mass units. The molecular mass was obtained from the deconvoluted ESI mass spectrum.     

Separation and determination of homogenous fatty alcohol ethoxylates by liquid chromatography with mulitstage mass spectrometry

Alcohol ethoxylates (AEs) are a significant component of a stream of surfactants directed to the aquatic environment. The aim of this work was the investigation of the dependence of the analytical signals of homogeneous AE homologues on liquid chromatography with tandem mass spectrometry conditions, as well as the separation of AEs from the water matrix and, on this basis, the development of an analytical procedure suitable for the determination of AEs in environmental samples. Homogeneous homologues containing dodecyl moiety and 2–9 oxyethylene subunits were investigated. The analytical signals of the investigated homologues were optimized in terms of concentration of ammonium acetate in the mobile phase (optimum 5 mM) and a column temperature (optimum 35°C) of the liquid chromatography with tandem mass spectrometry system. A separation of AEs from the water matrix by liquid–liquid extraction (ethyl acetate, chloroform) or solid‐phase extraction (C₁₈, styrene divinylbenzene, H‐RX) was investigated. In a model investigation, the best recoveries (>90%) were obtained with a styrene divinylbenzene cartridge eluted with a 1:1 mixture of chloroform and methanol. However, much worse recoveries were obtained from the river water sample. Better results were obtained for liquid–liquid extraction with ethyl acetate. Recoveries of 62–80% were obtained for homologues having 4–9 oxyethylene subunits, at the lowest spike.     

Determination of nonylphenol ethoxylate oligomers by liquid chromatography-electrospray mass spectrometry in river water and non-ionic surfactants

Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for the quantitative determination of five nonylphenol ethoxylate (NPE) oligomers in river water was described. These NPE oligomers were separated on a poly(vinyl alcohol) gel column using acetonitrile-30 mM ammonium acetate as the mobile phase followed by ESI-MS detection without any sample concentration steps. The sample was only filtered using the disposable filter and the aliquot (100 micro1) of this sample was injected into the LC-ESI-MS system. All NPE oligomers were detected using the [M+NH4]-ion. Detection limits ranged from 160 pg/ml (NPE4) to 240 pg/ml (NPE2), repeatability and reproducibility ranged from 4.2% (NPE2) to 6.2% (NPE6) and from 7.4% (NPE5) to 9.8% (NPE6).     

Biomarkers of alcohol abuse in racehorses by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method was developed for the screening, quantification and confirmation of ethyl glucuronide (EG) and ethyl sulfate (ES) as biomarkers for alcohol administration to racehorses using liquid chromatography coupled on-line with triple quadrupole tandem mass spectrometry. Urine sample aliquots (0.1 mL) were pre-treated by protein precipitation. Separation of EG and ES was achieved on an Ultra PFP column. Isocratic elution with a flush step was performed using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Analysis was performed by negative electrospray ionization in multiple reaction monitoring mode. The retention times for EG and ES were 1.7 +/- 0.30 and 3.4 +/- 0.30 min, respectively. The internal standard used was d(5)-ethyl glucuronide with a retention time of 1.7 +/- 0.30 min. The entire separation was completed in <5 min. The limit of detection (LOD) and of quantification (LOQ) for both analytes were 100 ng/mL (S/N > or =3) and 500 ng/mL, respectively. The limit of confirmations (LOC) for EG and ES were 500 ng/mL and 1.0 microg/mL, respectively. The assay was linear over a concentration range of 0.5-100 microg/mL (r(2) > 0.995). Intra- and inter-day accuracy and precision were less than 15%. The analytes were stable in urine for 24 h at room temperature, 10 days at 4 degrees C and 21 days at -20 degrees C and -70 degrees C. Ion suppression or enhancement due to matrix effect was negligible. The measurement uncertainty was <14% for EG and <25% for ES. This method was successfully used for the quantification of EG and ES in urine samples following alcohol administration to research horses and for screening and confirmation of EG and ES in urine samples obtained from racehorses post-competition. The method is simple, rapid, inexpensive, and reliably reproducible.     

Poly(amic acid) and polyimide characterization using gas chromatography/mass spectrometry and particle beam liquid chromatography/mass spectrometry

A number of polymers were hydrolyzed in NH₄OH and studied using gas chromatography/ mass spectrometry (GC/MS) and particle beam liquid chromatography/mass spectrometry (particle beam LC/MS) techniques. The polymers studied in this report were as follows: BPDA-PDA, BPDA-PDA-ODA, BPDA-PDA-GFDA, PMDA-ODA, and BTDA-APB. Some of the polymer samples were hydrolyzed in both their acid and imide forms to see if any mass spectrometric differences could be detected. ln all cases, the acid and imide spectra looked the same. GC/MS was unable to determine either the amine or acid portion of these polymers via a direct injection of the sample, but when the samples were first extracted with diethyl ether and this ether extract was injected into the chromatograph, the amine portion of the polymers was readily detected. The acid portion was, again, not detected in either the sample or the ether extract. The particle beam was able to detect both the amine and acid monomeric units in the nonextracted sample.     

Comprehensive analysis of fatty alcohol ethoxylates by ultra high pressure hydrophilic interaction chromatography coupled with ion mobility spectrometry mass spectrometry using a custom‐designed sub‐2 μm column

Comprehensive analysis of fatty alcohol ethoxylates has been conducted by coupling ultra high pressure hydrophilic interaction chromatography and ion mobility spectrometry mass spectrometry. A custom‐designed sub‐2 μm column was used for the chromatographic separation of fatty alcohol ethoxylates by hydrophilic interaction chromatography. Ion mobility spectrometry provided a post‐ionization resolution during a very short period of 6.4 ms. Distinguishable families of singly, doubly, and triply charged fatty alcohol ethoxylates were clearly observed. By virtue of the combination of hydrophilic interaction chromatography and ion mobility spectrometry, comprehensive resolution based on both hydrophobicity difference and mobility disparity has been achieved for fatty alcohol ethoxylates. The orthogonality of the developed separation and analysis system was evaluated with the correlation coefficient and peak spreading angle of 0.0224 and 88.72°, respectively. The actual peak capacity obtained was individually 40 and 193 times than those when hydrophilic interaction chromatography and ion mobility spectrometry were used alone. The collision cross‐sections of fatty alcohol ethoxylates were calculated by calibrating the traveling wave ion mobility device with polyalanine.     

Simultaneous determination of 2‐arachidonoylglycerol, 1‐arachidonoylglycerol and arachidonic acid in mouse brain tissue using liquid chromatography/tandem mass spectrometry

Endocannabinoids (ECs), such as anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG), modulate a number of physiological processes, including pain, appetite and emotional state. Levels of ECs are tightly controlled by enzymatic biosynthesis and degradation in vivo. However, there is limited knowledge about the enzymes that terminate signaling of the major brain EC, 2‐AG. Identification and quantification of 2‐AG, 1‐AG and arachidonic acid (AA) is important for studying the enzymatic hydrolysis of 2‐AG. We have developed a sensitive and specific quantification method for simultaneous determination of 2‐AG, 1‐AG and AA from mouse brain and adipose tissues by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a simple brain sample preparation method. The separations were carried out based on reversed phase chromatography. Optimization of electrospray ionization conditions established the limits of detection (S/N = 3) at 50, 25 and 65 fmol for 2‐AG, 1‐AG and AA, respectively. The methods were selective, precise (%R.S.D. < 10%) and sensitive over a range of 0.02–20, 0.01–10 and 0.05–50 ng/mg tissue for 2‐AG, 1‐AG and AA, respectively. The quantification method was validated with consideration of the matrix effects and the mass spectrometry (MS) responses of the analytes and the deuterium labeled internal standard (IS). The developed methods were applied to study the hydrolysis of 2‐AG from mouse brain extracts containing membrane bound monoacylglycerol lipase (MAGL), and to measure the basal levels of 2‐AG, 1‐AG and AA in mouse brain and adipose tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of explosives by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry using electrospray ionization and parent-ion scanning techniques

Analytical techniques for the detection of small amounts of explosives (in the picogram range) are now involved in various application. Some of them concern soil, water and air monitoring in order to face environmental problems related to improper handling procedures either in stocking or in wasting of the explosive products. Other areas are strictly related to forensic analysis of samples coming either from explosion areas where the matrix is various (metal, glass, wood, scraps), or from explosives transportation related to international terrorism. Generally speaking, for these applications the bulk of the matrix seriously interferes in the detection of the explosive analyte, which is usually present at trace levels. Unfortunately, despite some improvements, analytical techniques developed up today in this domain are still faced to two main constraints: the introduction of new products with unanticipated chemico-physical properties and the requirement of a routine and fast analytical method which can handle any matrix with a minimal clean-up and performing a sensitivity compatible either with the ever-decreasing demanded detection limit and with the ever-decreasing available specimen amount. These requirements can be fulfilled now by the new LC-MS and LC-MSMS techniques: mass spectrometry (MS) is likely an universal detector but even specific, especially when implemented in tandem MS (MSMS); LC is by far the most suitable technique to handle such a kind of compounds. Moreover, of a particular concern are some explosives which are reported to be thermally stable but difficult to dissolve. Some of the experiments on characterization of explosives [Octagen (HMX), Ethyleneglycol dinitrate (EGDN), Exogen (RDX), Propanetriol trinitrate (NG), Trinitrotoluene (TNT), N-Methyl-N-tetranitrobenzenamine (TETRYL), Dintrotoluene (DNT), Bis-(nitrooxy-methyl) propanediol dinitrate (PETN), Hexanitrostilbene (HNS), Triazido-trinitrobenzene (TNTAB), Tetranitro-acridone (TENAC), Hexa-nitrodiphenylamine (HEXYL), Nitroguanidine (NQ)] by LC-MS and LC-MSMS with the API-IonSpray source and using the Parent-Scan technique are presented.     

Fast liquid chromatography/multiple‐stage mass spectrometry of coccidiostats

Drugs that are used as medicines and also as growth promoters in veterinary care are considered as emerging environmental contaminants and in recent years concern about their potential risk to ecosystems and human health has risen. In this paper we used a method based on liquid chromatography/electrospray tandem mass spectrometry to analyze eight coccidiostatic compounds: diclazuril, dinitrocarbanilide (the main metabolite of nicarbazin), robenidine, lasalocid, monensin, salinomycin, maduramicin and nasarin. Multiple‐stage mass spectrometry (MSⁿ) based on the precursor ions [M+Na]⁺ (polyether ionophores), [M+H]⁺ (robenidine) and [M–H]^? (diclazuril and dinitrocarbanilide) was used to study the fragmentation of these compounds. MSⁿ data and genealogical relationships were used to propose a tentative assignment of the different fragment ions. Loss of water, decarboxylations, ketone β‐cleavages and rearrangement of cyclic ethers and amide groups were some of the fragmentations observed for these compounds. Liquid chromatography with a sub‐2 µm particle size column was coupled to tandem mass spectrometry (LC/MS/MS) allowing the separation of these compounds in less than 7 min. Method detection limits ranging from 11 to 71 ng L^?1 and run‐to‐run values in terms of relative standard deviation (RSD) (up to 12%) were obtained. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of Xipamide metabolite in human urine by high‐performance liquid chromatography/diode‐array detection,high‐performance liquid chromatography/electrospray ionization mass spectrometry and gas chromatography/mass spectrometry

The original article to which this Erratum refers was published in Rapid Commun. Mass Spectrom. 2004; 18 : 2505–2512