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.     

Fragmentation of 2‐aroylbenzofuran derivatives by electrospray ionization tandem mass spectrometry

We investigated the gas‐phase fragmentation reactions of a series of 2‐aroylbenzofuran derivatives by electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The most intense fragment ions were the acylium ions m/z 105 and [M+H–C₆H₆]⁺, which originated directly from the precursor ion as a result of 2 competitive hydrogen rearrangements. Eliminations of CO and CO₂ from [M+H–C₆H₆]⁺ were also common fragmentation processes to all the analyzed compounds. In addition, eliminations of the radicals •Br and •Cl were diagnostic for halogen atoms at aromatic ring A, whereas eliminations of •CH₃ and CH₂O were useful to identify the methoxyl group attached to this same ring. We used thermochemical data, obtained at the B3LYP/6‐31+G(d) level of theory, to rationalize the fragmentation pathways and to elucidate the formation of E , which involved simultaneous elimination of 2 CO molecules from B .     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Studies of iridoid glycosides using liquid chromatography/electrospray ionization tandem mass spectrometry

Fragmentation pathways of five iridoid glycosides have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)). The first-stage MS data of the five iridoid glycosides were compared. The MS spectra showed that the adduct ions of iridoid glycosides and the formate anion were diagnostic ions to distinguish iridoid glycosides with a carboxyl group at the C-4 position or an ester group at the C-4 position. The MS fragmentation pathways of the five iridoid glycosides were also studied. Analyzing the product ion spectra of iridoid glycosides, some neutral losses were observed, such as H(2)O, CO(2) and glucose residues, which were very useful for the identification of the functional groups in the structures of iridoid glycosides. Furthermore, specific loss of one molecule of methyl 3-oxopropanoate or 3-oxopropanic acid was firstly discussed, which corresponded to the isomerization of the hemiacetal group in the structure of iridoid aglycone. According to the fragmentation mechanisms and HPLC/MS(n) data, the structures of five iridoid glycosides in a crude extract of Gardenia jasminoisdes fruit have been identified. Three compounds were compared with standards and the other two were identified as shanzhiside and genipin gentibioside by their MS(n) data without standard compounds. In order to further validate the veracity of the deduction, genipin gentiobioside was isolated from the extract of Gardenia jasminoisdes fruit using Purification Factory and was further identified by C- and H-NMR.     

High-resolution liquid chromatography/electrospray ionization time-of-flight mass spectrometry combined with liquid chromatography/electrospray ionization tandem mass spectrometry to identify polyphenols from grape antioxidant dietary fiber

Grape antioxidant dietary fiber (GADF) is a dietary supplement that combines the benefits of both fiber and antioxidants that help prevent cancer and cardiovascular diseases. The antioxidant polyphenolic components in GADF probably help prevent cancer in the digestive tract, where they are bioavailable. Mass spectrometry coupled to liquid chromatography is a powerful tool for the analysis of complex plant derivatives such as GADF. We use a combination of MS techniques, namely liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) on a triple quadrupole, for the identification of the polyphenolic constituents of the soluble fraction of GADF. First, we separated the mixture into four fractions which were tested for phenolic constituents using the TOF system in the full scan mode. The high sensitivity and resolution of the TOF detector over the triple quadrupole facilitate the preliminary characterization of the fractions. Then we used LC/ESI-MS/MS to identify the individual phenols through MS/MS experiments (product ion scan, neutral loss scan, precursor ion scan). Finally, most of the identities were unequivocally confirmed by accurate mass measurements on the TOF spectrometer. LC/ESI-TOF-MS combined with MS/MS correctly identifies the bioactive polyphenolic components from the soluble fraction of GADF. High-resolution TOF-MS is particularly useful for identifying the structure of compounds with the same LC/ESI-MS/MS fragmentation patterns.     

Quantitation of glutathione by liquid chromatography/positive electrospray ionization tandem mass spectrometry

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. It is present in practically all cells and has several important roles, such as preventing the oxidation of the sulfhydryl groups of proteins within a cell. Evidence for GSH deficiency or depletion has been found in a variety of diseases and toxicity-related studies, including diabetes and induction of oxidative stress to form reactive oxygen species which cause DNA, lipid, and protein oxidations. A simple, selective, and sensitive analytical method for measuring low levels of GSH in biological fluids would therefore be desirable to conduct GSH deficiency or depletion-related mechanistic toxicity studies. Here a method for both low- and high-level quantitation of GSH from cultured cells and rat liver tissues via liquid chromatography/positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed. The lower limit of quantitation (LOQ) of the method was 5 ng/mL. The method is linear over a wide dynamic concentration range of 5.0 to 5000.0 ng/mL, with a correlation coefficient R2 > 0.99. The intra-day assay precision relative standard deviation (RSD) values for all quality control (QC) samples were < or =16.31%, with accuracy values ranging from 94.13 to 97.80%. The inter-day assay precision RSD values for all QC samples were < or =15.94%, with accuracy values ranging from 94.51 to 100.29%. With this method, low levels of GSH from diethyl maleate (DEM)-treated mouse lymphoma cells, and GSH in rat liver tissues, were quantified.     

Structural elucidation of V‐type nerve agents by liquid chromatography/electrospray ionization mass spectrometry

V‐nerve agents present information‐poor spectra, both in GC‐EI‐MS and LC‐ESI‐MS/MS, with dominant fragments/product ions corresponding to the amine‐containing residue. Hence, derivatives/isomers with the same amine residue exhibit similar mass spectral patterns, leading to ambiguity in the phosphonate structure. We present a simple approach for their structural elucidation based on two complementary experiments: ESI‐MS/MS of the original compound, which provides information about the amine moiety, and ESI‐MS/MS of the phosphonic acid hydrolysis products generated by N‐iodosuccinimide, which provides ions' characteristic of the phosphonate structure. This approach enables the structural elucidation of the original V‐agents with a higher degree of certainty.     

Evaluation of tramadol and its main metabolites in horse plasma by high‐performance liquid chromatography/fluorescence and liquid chromatography/electrospray ionization tandem mass spectrometry techniques

Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat pain in humans. The clinical response of tramadol is strictly correlated to its metabolism, because of the different analgesic activity of its metabolites. O‐Desmethyltramadol (M1), its major active metabolite, is 200 times more potent at the µ‐receptor than the parent drug. In recent years tramadol has been widely introduced in veterinary medicine but its use has been questioned in some species. The aim of the present study was to develop a new sensible method to detect the whole metabolic profile of the drug in horses, through plasma analyses by high‐performance liquid chromatography (HPLC) coupled with fluorimetric (FL) and photodiode array electrospray ionization mass spectrometric (PDA‐ESI‐MS) detection, after its sustained release by oral administration (5 mg/kg). In HPLC/FL experiments the comparison of the horse plasma chromatogram profile with that of a standard mixture suggested the identification of the major peaks as tramadol and its metabolites M1 and N,O‐desmethyltramadol (M5). LC/PDA‐ESI‐MS/MS analysis confirmed the results obtained by HPLC/FL and also provided the identification of two more metabolites, N‐desmethyltramadol (M2), and N,N‐didesmethyltramadol (M3). Another metabolite, M6, was also detected and identified. The present findings demonstrate the usefulness and the advantage of LC/ESI‐MS/MS techniques in a search for tramadol metabolites in horse plasma samples. Copyright © 2008 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.     

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MS(n) data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MS(n), non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools for rapid screening and structural assignment of saponins in plant extracts.     

Synthesis and reactions of 11H‐benzo[b]pyrano[3,2‐f]indolizines an pyrrolo[3,2,1‐ij]pyrano[3,2‐c]quinolines

2‐Methyl‐3H‐indoles 1 cyclize with two equivalents of ethyl malonate 2 to form 4‐hydroxy‐11H‐benzo[b]pyrano[3,2‐f]indolizin‐2,5‐diones 3, whereas 2‐mefhyl‐2,3‐dihydro‐1H‐indoles 9 give under similar conditions regioisomer 8‐hydroxy‐5‐methyl‐4,5‐dihydro‐pyrrolo[3,2,1‐ij]pyrano[3,2‐c]quinolin‐7,10‐diones 10 . The pyrone rings of 3 and 9 can be cleaved either by alkaline hydrolysis to give 7‐acetyl‐8‐hydroxy‐10H‐pyrido[1,2‐a]indol‐6‐ones 4 or 5‐acetyl‐6‐hydroxy‐2‐methyl‐1,2‐dihydro‐4H‐pyrrolo‐[3,2,1‐ij]quinolin‐4‐ones 11 , respectively. Chlorination of 3 and 9 with sulfurylchloride gives under subsequent ring opening 7‐dichloroacetyl‐8‐hydroxy‐10H‐pyrido[1,2‐a]indol‐6‐ones 5 or 5‐dichloracetyl‐6‐hydroxy‐2‐methyl‐1,2‐dihydro‐4H‐pyrrolo[3,2,1‐ij]quinolin‐4‐ones 12 . The dichloroacetyl group of 5 can be reduced with zinc to 7‐acetyl‐8‐hydroxy‐10H‐pyrido[1,2‐a]indol‐6‐ones 7. Treatment of the acetyl compounds 4, 7 and 11 with 90% sulfuric acid cleaves the acetyl group and yields 8‐hydroxy‐10H‐pyrido[1,2‐a]‐indol‐6‐ones 6 and 8 , and 6‐hydroxy‐2‐methyl‐1,2‐dihydro‐4H‐pyrrolo[3,2,1‐ij]quinolin‐4‐ones 13 . Reaction of dichloroacetyl compounds 12 with sodium azide yields 6‐hydroxy‐2‐methyl‐5‐(1H‐tetrazol‐5‐ylcarbonyl)‐1,2‐dihydro‐4H‐pyrrolo[3,2,1‐ij]quinolin‐4‐ones 14 via intermediate geminal diazides.     

Determination of chlorpyrifos and its metabolites in rat brain tissue using coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry

A method has been developed to quantify chlorpyrifos (O,O-diethyl-O-[3,5,6,-trichloro-2-pyridyl] phosphorothionate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O-[3,5,6,trichloro-2-pyridinyl] phosphate) and TCP (3,5,6,-trichloro-2-pyridinol) in rat brain tissue by coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry (LC/LC/ESI-MS/MS). Rat brains were homogenized and treated by protein precipitation using ice-cold acetonitrile. The supernatant was directly injected onto the coupled-column system. Sample clean-up was achieved on a Zorbax Extend-C(18) column (2.1 x 50 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (40:60, v/v). The compounds were separated isocratically on a Zorbax Eclipse XDB C(8) column (2.0 x 150 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (75:25, v/v). Chlorpyrifos and chlorpyrifos-oxon were detected in positive ion mode using multiple reaction monitoring (MRM). TCP was detected in negative ion mode using precursor-to-precursor transition monitoring. The method was validated and the specificity, linearity, limit of quantitation (LOQ), precision, accuracy, stability, and recoveries were determined. Calibration curves for all three analytes yielded correlation coefficients of 0.993 or greater. The LOQs were 25.3 ng/g for chlorpyrifos and 6.3 ng/g for chlorpyrifos-oxon and TCP. All precision relative standard deviations (RSDs) were less than 16% for the LOQ and less than 11% for the other QC samples. This method was successfully applied to six rats that were injected subcutaneously with chlorpyrifos.     

Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

The metabolism of limonin 17-beta-D-glucopyranoside (LG) by non-cancerous (RWPE-1) and cancerous (PC-3) human prostate epithelial cells was investigated using high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation and tandem mass spectrometry (MS/MS). During positive ion LC/ESI-MS, LG formed an abundant sodiated species ([M+Na]+) while the protonated molecule was barely observable. [M+Na]+ further fragmented into the less abundant [LARL+H]+ and a predominantly protonated aglycone molecule (limonin) due to in-source fragmentation. The major metabolite, limonin A-ring lactone (LARL), formed an abundant protonated molecule that was fragmented into a protonated molecule of limonin by loss of one molecule of water. In MS/MS by collisionally activated dissociation (CAD), LG produced the sodiated aglycone, [aglycone+Na]+, while LARL fragmented into [M+H]+ of limonin and fragment ions resulted by further loss of water, carbon monoxide and carbon dioxide, indicating the presence of oxygenated-ring structures. The limits of detection of LG were 0.4 and 20 fmol in selected-ion monitoring (SIM) and selected-reaction monitoring (SRM) detection, respectively.     

Determination of flomoxef in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A specific, sensitive, rapid and reproducible method for the determination of flomoxef in human plasma using high‐performance liquid chromatography–tandem mass spectrometry was developed and validated. Flomoxef was detected using an electrospay ionization method operated in negative‐ion mode. Chromatographic separation was performed in gradient elution mode on a Luna® C₁₈(2) column (3 μm , 20 × 4.0 mm) at a flow rate of 1 mL/min and runtime 3.5 min. The mobile phase consisted of acetonitrile and water containing 0.1% formic acid as additive. Extraction of flomoxef from plasma and precipitation of plasma proteins was performed with acetonitrile with an absolute recovery of 86.4 ± 1.6%. The calibration curve was linear with a correlation coefficient of 0.999 over the concentration range 10–5000 ng/mL and the lower limit of quantification was 10 ng/mL. The intra‐ and inter‐day precisions were <11.8%, while the accuracy ranged from 99.6 to 109.0%. A stability study of flomoxef revealed that it could be successfully analyzed at 4ºС over 24 h, but it was unstable in solutions at room temperature during short‐term storage (4 h) and several freeze–thaw cycles. Copyright © 2013 John Wiley & Sons, Ltd.     

Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry

The histidine-containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic alpha,beta-unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO(4) (1 : 1, v/v) containing H-Tyr-His-OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow-rate of 0.2 ml min(-1), with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor --> product ion combinations: H-Tyr-His-OH (internal standard) m/z 319 --> 301; CAR m/z 227 --> 210 + 209; ANS m/z 241 --> 224 + 197 + 170; HCAR m/z 241 --> 156. The method was validated over the concentration range 15-1000 nmol g(-1) and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra- and inter-day precisions were <10% (< or =17.47% at the LOQ) and the intra- and inter-assay accuracies were within +/-10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine-containing dipeptides were detectable in plasma, liver, kidney and lung.     

High-precision isotopic analysis of palmitoylcarnitine by liquid chromatography/electrospray ionization ion-trap tandem mass spectrometry

Single quadrupole gas chromatography/mass spectrometry (GC/MS) has been widely used for isotopic analysis in metabolic investigations using stable isotopes as tracers. However, its inherent shortcomings prohibit it from broader use, including low isotopic precision and the need for chemical derivatization of the analyte. In order to improve isotopic detection power, liquid chromatography/electrospray ionization ion-trap tandem mass spectrometry (LC/ESI-itMS2) has been evaluated for its isotopic precision and chemical sensitivity for the analysis of [13C]palmitoylcarnitine. Over the enrichment range of 0.4-10 MPE (molar % excess), the isotopic response of LC/ESI-itMS2 to [13C]palmitoylcarnitine was linear (r = 1.00) and the average isotopic precision (standard deviation, SD) was 0.11 MPE with an average coefficient of variation (CV) of 5.6%. At the lower end of isotopic enrichments (0.4-0.9 MPE), the isotopic precision was 0.05 MPE (CV = 8%). Routine analysis of rat skeletal muscle [13C4]palmitoylcarnitine demonstrated an isotopic precision of 0.03 MPE for gastrocnemius (n = 16) and of 0.02 MPE for tibialis anterior (n = 16). The high precision enabled the detection of a small (0.08 MPE) but significant (P = 0.01) difference in [13C4]palmitoylcarnitine enrichments between the two muscles, 0.51 MPE (CV = 5.8%) and 0.43 MPE (CV = 4.6%), respectively. Therefore, the system demonstrated an isotopic lower detection limit (LDL) of < or =0.1 MPE (2 x SD) that has been impossible previously with other organic mass spectrometry instruments. LC/ESI-itMS2 systems have the potential to advance metabolic investigations using stable isotopes to a new level by significantly increasing the isotopic solving power.     

Simultaneous quantification of oleuropein and its metabolites in rat plasma by liquid chromatography electrospray ionization tandem mass spectrometry

Oleuropein (OE) is the cardinal bioactive compound derived from Olea europaea and possesses numerous beneficial properties for human health. However, despite the plethora of analytical methods that have studied the biological fate of olive oil‐derived bioactive compounds, no validated methodology has been published to date for the simultaneous determination of OE, along with all its major metabolites. In this study, a liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI MS/MS) method has been developed and validated for the quantification of OE, simultaneously with its main metabolites hydroxytyrosol, 2‐(3,4‐dihydroxyphenyl)acetic acid, 4‐(2‐hydroxyethyl)‐2‐methoxy‐phenol or homovanillyl alcohol, 2‐(4‐hydroxy‐3‐methoxyphenyl)acetic acid or homovanillic acid, and elenolic acid in rat plasma matrix. Samples were analyzed by LC‐ESI MS/MS prior to and after enzymatic treatment. A solid‐phase extraction step with high mean recovery for all compounds was performed as sample pretreatment. Calibration curves were linear for all bioactive compounds over the range studied, while the method exhibited good accuracy, intra‐ and inter‐day precision. The limit of detection was in the picogram range (per milliliterof plasma) for HT and OE and in the nanogram range (per milliliter of plasma) for the other analytes, and the method was simple and rapid. The developed methodology was successfully applied for the simultaneous quantification of OE and its aforementioned metabolites in rat plasma samples, thus demonstrating its suitability for pharmacokinetics, as well as bioavailability and metabolism studies. Copyright © 2009 John Wiley & Sons, Ltd.