Characterization of G-rich and T-rich oligonucleotides using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry

Characteristics of G-rich and T-rich oligonucleotides were investigated to compare their retention time, total ion current (TIC) intensity, charge-state distribution and product ion using ion-pair reversed-phase high- performance liquid chromatography/tandem electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) at room temperature. Three commonly used mobile phases for the analysis of oligonucleotides, triethylammonium acetate (TEAA), triethylammonium bicarbonate (TEAB) and triethylammonium hexafluoroisopropanol (HFIP) have been utilized. Retention time of G-rich and T-rich oligonucleotides was significantly different in TEAA and TEAB buffer systems, while in the HFIP buffer system it was affected more by the length of oligonucleotides. On the other hand, the ESI-MS ion abundance in the HFIP buffer system was higher than that in both TEAA and TEAB buffers. The TIC intensity of T-rich oligonucleotides was much higher than that of G-rich oligonucleotides in all mobile phases. In addition, much higher charge-state fragments were observed in HFIP buffer system than that in the case of TEAA and TEAB buffer systems. Product ions of both G-rich and T-rich oligonucleotides were affected by charge state of parent ions and collision energy.     

Systematic fragmentation patterns of archaeal intact polar lipids by high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry

Archaea are ubiquitous and abundant microorganisms on Earth that mediate key global biogeochemical cycles. The headgroup attached to the sn-1 position of the glycerol backbone and the ether-linked isoprenoid lipids are among the diagnostic traits that distinguish Archaea from Bacteria and Eukarya. Over the last 30 years, numerous archaeal lipids have been purified and described in pure cultures. Coupled high-performance liquid chromatography (HPLC) ion-trap mass spectrometry (ITMS) now enables the detection and rapid identification of intact polar lipids in relatively small and complex samples, revealing a wide range of archaeal lipids in natural environments. Although major structural groups have been identified, the lack of a systematic evaluation of MS/MS fragmentation patterns has hindered the characterization of several atypical components that are therefore considered as unknowns. Here, we examined mass spectra resulting from lipid analysis of natural microbial communities using HPLC/electrospray ionization (ESI)-ITMS(n), and depicted the systematics in MS(2) fragmentation of intact archaeal lipids. This report will be particularly useful for environmental scientists interested in a rapid and straightforward characterization of intact archaeal membrane lipids.     

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.     

Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry

Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was utilized in tandem with negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of native and chemically modified oligonucleotides. Separation was performed on a 1.0 x 50 mm column packed with porous C(18) sorbent with a particle size of 2.5 microm and an average pore diameter of 140 A. A method was developed which maximizes both chromatographic separation and mass spectrometric sensitivity using an optimized buffer system containing triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol with a methanol gradient. The ESI-TOFMS tuning parameters were also optimized in order to minimize in-source fragmentation and achieve the best sensitivity. Analyses of native, phosphorothioate, and guanine-rich oligonucleotides were performed by LC/MS. Detection limits were at sub-picomole levels with an average mass accuracy of 125 ppm. The described method allowed for the LC/MS analysis of oligonucleotides up to 110mer in length with little alkali cation adduction. Since sensitive detection of oligonucleotides was achieved with ultraviolet (UV) detection, we utilized a combination of UV-MS for quantitation (UV) and characterization (MS) of oligonucleotides and their failure sequence fragments/metabolites.     

Determination of Tripamide in human urine by high-performance liquid chromatography and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Tripamide is a drug widely used in clinical practice for the treatment of hypertension and edema. This work evaluated a screening method for Tripamide and its urinary metabolites in human urine, using high-performance liquid chromatography diode-array detection (HPLC/DAD). Identification of these metabolites was investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) after dosing with 15 mg Tripamide. Acid hydrolysis showed that Tripamide is conjugated in the body. Two suspected metabolites were detected by HPLC/DAD. HPLC/ESI-MS/MS analysis suggested that these metabolites were probably hydroxylated together with loss of the -NH(2) group and dehydrogenation. These results will be useful in confirmation methods for Tripamide in doping control.     

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.     

Comparison of A+T-rich oligonucleotides with and without self-complementary sequence using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry

Both A+T-rich oligonucleotides with and without self-complementary sequences were analyzed using ion- pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) by tryethylammonium acetate (TEAA) and hexafluoroisopropanol (HFIP) buffer systems to study the characteristics of their retention behavior and electrospray ionization tandem mass spectrometry (ESI-MS/MS) response. The results indicated that the chain length had the same effect on the retention of A+T-rich oligonucleotides in both of TEAA and HFIP buffer systems but the sequence had a different impact on the retention in the two buffer systems. A+T- rich oligonucleotides with a self-complementary sequence were much shorter than those without in the TEAA buffer system whereas a slight difference was observed in the HFIP buffer system. Similar total ion current (TIC) intensity was observed both in oligonucleotides with or without self-complementary sequence. The opposite trend of a change in the TIC intensities with increasing chain length were observed in both the TEAA and HFIP buffer systems. A lower charge state was predominant in the TEAA buffer system whereas a higher charge state was mainly distributed in the HFIP buffer system. The oligonucleotides without self-complementary sequences had a higher charge state than those with self-complementary sequences. A- and T- are more esily formed at a higher charge state whereas the sequence fragments will be formed more easily at a lower charge state in both A+T-rich oligonucleotides with and without self-complementary sequences.     

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.     

Analysis of metal complex azo dyes by high-performance liquid chromatography/electrospray ionization mass spectrometry and multistage mass spectrometry

Five metal complex azo compounds were analyzed using negative-ion electrospray ionization mass spectrometry (ESI-MS). Mass spectra of all compounds yield intense peaks corresponding to [M - H](-) ions without any fragmentation, where M denotes the neutral compound with a proton as the counterion. Under collision induced dissociation (CID) conditions, structurally important fragment ions were studied using the ion trap analyzer with a multistage mass spectrometry (MS(n) facility. Synthesized compounds with (15)N atoms in the azo group facilitated the fragmentation pattern recognition. A reversed-phase high-performance liquid chromatography (HPLC) method using 5 mM ammonium acetate in 70% aqueous acetonitrile as mobile phase was developed making possible the separation of all complex compounds tested. The lower detection limits of the ESI-MS method are in the range 10-20 ng of each compound. The HPLC/ESI-MS method makes possible the monitoring of ligand exchange in aqueous solutions of metal complex azo dyes, and also investigation of the stabilities of the complexes in solution. Copyright 2000 John Wiley & Sons, Ltd.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

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.     

Quantification of cow milk adulteration in goat milk using high-performance liquid chromatography with electrospray ionization mass spectrometry

A method was developed for the quantification of cow milk adulteration in goat milk, based on solvent separation of whey proteins followed by high-performance liquid chromatography with electrospray ionization mass spectrometry (HPLC/ESI-MS). The presence of cow milk was determined using beta-lactoglobulin whey protein as the molecular marker. The adulterants were identified using both retention time and molecular mass derived from multiply charged molecular ions. Standard solutions containing cow and goat milk in different volume ratios were prepared and analyzed. Good linearity covering cow milk content from 5% and above was obtained. The proposed method identifies the adulterants using accurate molecular masses for protein identification and detects the addition of cow milk to goat milk at levels as low as 5%.     

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.     

Quantitative determination of DNA adducts using liquid chromatography/electrospray ionization mass spectrometry and liquid chromatography/high-resolution inductively coupled plasma mass spectrometry

The quantitative determination of nucleotides from DNA modified by styrene oxide is described using a combination of inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) and electrospray ionization mass spectrometry (ESI-MS), both interfaced to reversed-phase high-performance liquid chromatography (HPLC). LC/ICP-MS (resolution > 1500 to discriminate against 15N16O+ and 14N16OH+) was employed to determine quantitatively the content of modified nucleotides in standard solutions based on the signal of phosphorus; phosphoric acid served as an internal standard. By means of the standard addition technique the sensitivity of the LC/ESI-MS approach was subsequently determined. Since a comparison of UV, ICP and ESI-MS data suggested that in ESI-MS the ionization efficiency of the adducts is identical within the error limits, quantitative determination of all adducts is possible. For LC/ESI-MS with single ion monitoring, the detection limit for styrene oxide adducts of nucleotides was determined to be 20 pg absolute or 14 modified in 10(8) unmodified nucleotides in a 5 micrograms DNA sample, which comes close to the best methods available for the detection of chemical modifications in DNA.