Positive Electrospray Ionization Mass Spectrometry Research Articles

Determination of pharmaceutical compounds in aqueous dimethyl sulfoxide by electrospray ionization mass spectrometry

Standard solutions of reserpine, dextromethorphan, imipramine and amitriptyline in dimethyl sulfoxide (DMSO), DMSO containing 0.1% formic acid, and DMSO/H(2)O (1:1, v/v) containing 0.1% formic acid were analyzed by positive electrospray ionization mass spectrometry (ESI-MS). A triple-quadrupole mass spectrometer equipped with a TurboIonspray (TIS) interface was used for the ESI-MS analyses. The samples dissolved in the respective DMSO solution were infused directly into the mass spectrometer at 10 microL/min using an infusion pump. The positive Q1 full-scan (m/z 50-650) mass spectrum of DMSO (MW = 78) showed three main peaks at m/z 79, 101 and 179, corresponding to the protonated molecule [DMSO+H](+), and the sodiated adducts [DMSO+Na](+) and [2DMSO+Na](+), respectively. The ESI of the compounds in DMSO and DMSO containing 0.1% formic acid was promoted by using the TIS gas (GS2) combined with the nebulizer gas (GS1), and TIS source temperature set to 250 degrees C. In contrast, samples dissolved in DMSO/H(2)O (1:1, v/v) containing 0.1% formic were sprayed at a lower temperature (100 degrees C) using only the nebulizer gas. The TIS voltage (V) was optimized in order to establish the lowest voltage necessary to achieve optimum ESI of each pharmaceutical compound with the voltage maintained below the onset potential required to produce a corona discharge at the TIS probe (sprayer). Detection limits of 10 ng/mL were achieved for reserpine, dextromethorphan, imipramine and amitriptyline in each solvent composition.

Simultaneous Determination of Ipriflavone and its Main Metabolites M1 and M5 in Human Plasma by Liquid Chromatography with Tandem Mass Spectrometry

A Liquid chromatography and tandem mass spectrometry (LC/MS/MS) method has been described for the simultaneous determination of ipriflavone and its main metabolites, 7‐hydroxy‐3‐phenyl‐chromen‐4‐one (M1) and 7‐(4‐oxo‐3‐phenyl‐4H‐chromen‐7‐yloxy)‐propionic acid (M5) in human plasma using protein precipitation. Ipriflavone, M1, M5, and the internal standard [2‐(3,4‐dimethoxy‐phenyl)‐5,7‐dihydroxy‐chromen‐4‐one] were analyzed on XTerra C18 column with the mixture of acetonitrile‐ammonium formate (10 mM, pH 3.0) (53:47, v/v) as mobile phase. The analytes were determined using electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode. The standard curves for ipriflavone, M1, and M5 were linear over the concentration range of 1.00–500 ng/mL. The lower limit of quantification was 1.00 ng/mL using 50 µL plasma sample. The relative error and the coefficients of variation of intra‐ and interday assays for ipriflavone, M1, and M5 at three quality control levels ranged from −5.6 to 1.6% and 1.0 to 4.6%, respectively. This work was supported by Korea Research Foundation Grant.

An immunomodulator used to protect young in the pouch of the Tammar wallaby, Macropus eugenii

Eugenin [pGluGlnAspTyr(SO(3))ValPheMetHisProPhe-NH(2)] has been isolated from the pouches of female Tammar wallabies (Macropus eugenii) carrying young in the early lactation period. The sequence of eugenin has been determined using a combination of positive and negative ion electrospray mass spectrometry. This compound bears some structural resemblance to the mammalian neuropeptide cholecystokinin 8 [AspTyr(SO(3))MetGlyTrpMetAspPhe-NH(2)] and to the amphibian caerulein peptides [caerulein: pGluGlnAspTyr(SO(3))ThrGlyTrpMetAspPhe-NH(2)]. Eugenin has been synthesized by a route which causes only minor hydrolysis of the sulfate group when the peptide is removed from the resin support. Biological activity tests with eugenin indicate that it contracts smooth muscle at a concentration of 10(-9) M, and enhances the proliferation of splenocytes at 10(-7) M, probably via activation of CCK(2) receptors. The activity of eugenin on splenocytes suggests that it is an immunomodulator peptide which plays a role in the protection of pouch young.

Liquid chromatography–mass spectrometry method for determination of tetramethylpyrazine and its metabolite in dog plasma

A liquid chromatography–mass spectrometry method is described for the determination of tetramethylpyrazine (TMP) and its active metabolite, 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP) in dog plasma. This method involves a plasma clean-up step using protein precipitation procedure followed by LC separation and positive electrospray ionization mass spectrometry detection (ESI-MS). Chromatographic separation of the analytes was achieved on a C18 column using a mobile phase of methanol, water and acetic acid (50:50:0.6, v/v/v) at a flow rate of 1.0 ml/min. Selected ion monitoring (SIM) mode was used for analyte quantitation at m/ z 137.2 for TMP, m/ z 153.2 for HTMP and m/ z 195.2 for caffeine. The linearity was obtained over the concentration ranges of 20–6000 ng/ml for TMP and 20–4000 ng/ml for HTMP and the lower limit of quantitation was 20 ng/ml for both analytes. For each level of QC samples, both inter- and intra-day precisions (R.S.D.) were ≤7.4% for TMP and ≤6.0% for HTMP, and accuracy (R.E.) was ±6.0% for TMP and ≤3.5% for HTMP. The proposed LC–MS method was successfully applied to the pharmacokinetic studies of a TMP formulation preparation after oral administration to beagle dogs.

Liquid chromatography–positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

Liquid chromatography–positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

Quantitation of 17α‐ethinylestradiol in aquatic samples using liquid‐liquid phase extraction, dansyl derivatization, and liquid chromatography/positive electrospray tandem mass spectrometry

A rapid and sensitive method for the analysis of 17alpha-ethinylestradiol (EE2) in environmental aqueous samples has been developed. Aquatic samples were extracted using liquid-liquid extraction, and organic phase extracts were concentrated and derivatized with dansyl chloride. Analysis was performed using high-performance liquid chromatography with positive electrospray ionization and tandem mass spectrometry (HPLC/ESI-MS/MS). Deuterated 17alpha-ethinylestradiol was used as internal standard and was added to samples before extraction. A limit of quantitation of 1 ng/L was obtained using a 25 mL aqueous sample. The average recovery of EE2 spiked into a 25 mL tapwater sample was 100%. This highly sensitive quantitation method is useful for measuring low levels of EE2 in aqueous environmental samples.

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich’s ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C∘G to T∘A basepairs. A melting pH (pH m) of 7.6 was observed for the destabilization of the (GAA) 2T 4(TTC) 2T 4(CTT) 2 triplex to the corresponding Watson–Crick duplex and the T 4(CTT) 2 overhang. The mass spectrometric analyses of (TTC) 6•(GAA) 6∘(TTC) 6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC) 6•(GAA) 6∘(TTC) 6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA–ligand complexes.

Liquid chromatography–positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

A rapid, sensitive and novel narrow-bore liquid chromatography–mass spectrometric method was developed and fully validated for the quantification of citalopram in human plasma. The analyte and internal standard (imipramine) were extracted by liquid–liquid extraction with a mixture of hexane–heptane–isopropanol (88:10:2, v/v/v). The use of a Hypersil BDS C 8 micro-bore column (250 mm × 2.1 mm i.d.; 3.5 μm particle size), results in substantial reduction in solvent consumption. The mobile phase consisted of 10 mM ammonium formate–formic acid (pH 4.5) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.15 ml min −1. The analytes were detected after positive electrospray ionization using the selected ion-monitoring mode of the species at m/ z 325 for citalopram and m/ z 281 for imipramine. The method had a chromatographic run time of 10.0 min and a linear calibration curve over the range 0.50–250 ng ml −1 ( r 2 > 0.996). The limit of quantitation was 0.50 ng ml −1. Accuracy and precision were below the acceptance limits of 15%.

Regiospecific determination of short‐chain triacylglycerols in butterfat by normal‐phase HPLC with on‐line electrospray‐tandem mass spectrometry

This study uses normal-phase HPLC with on-line positive ion electrospray mass spectrometry (ESI-MS) to obtain quantitative compositional data on both synthetic and butterfat short-chain TAG. The product ion tandem MS of standards averaged 11.1 times lower in abundance of the ion formed by cleavage of FA from the sn-2-position for the pairs of regioisomers in the TAG classes: L/L/S-L/S/L and L/S/S-S/L/S, where L denotes long and S short acyl chain (C2-C6). The molar correction factors, determined for 42 regioisomeric pairs of short-chain TAG of 20 randomized mixture of standards, differed by 1.4-80% as the ratios varied between 0.217 and 5.847. Butterfat TAG were resolved into four fractions on short flash chromatography grade silica gel columns. Pairs of regioisomers in the TAG classes L/S/S-S/L/S with predominance of L/S/S isomers and the sole regioisomers in the TAG classes L/L(M)/S were identified by tandem MS, where M denotes either 8:0 or 10:0 acyl chain. The total proportion of L/L(M)/S isomers was estimated at 34.7 and that of L/S/S-S/L/S at 1.0 mol%, including a small proportion of S/S/S. In contrast to previous work, the present data indicate the presence of a small proportion of butyric and caproic acids in the sn-1-position. The overall distribution of the FA in the short-chain TAG of butterfat, calculated from direct MS measurements, was consistent with the results of indirect determinations based on stereospecific analyses of total butterfat.

Comparison of the binding stoichiometries of positively charged DNA-Binding drugs using positive and negative ion electrospray ionization mass spectrometry

Positive and negative ion electrospray ionization (ESI) mass spectra of complexes of positively charged small molecules (distamycin, Hoechst 33258, [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2) have been compared. [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2 bind to DNA by intercalation. Negative ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA showed ions from DNA-ligand complexes consistent with solution studies. In contrast, only ions from free DNA were present in positive ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA, highlighting the need for obtaining ESI mass spectra of non-covalent complexes under a range of experimental conditions. Negative ion spectra of mixtures of the minor groove binder Hoechst 33258 with DNA containing a known minor groove binding sequence were dominated by ions from a 1:1 complex. In contrast, in positive ion spectra there were also ions present from a 2:1 (Hoechst 33258: DNA) complex, suggesting an alternative binding mode was possible either in solution or in the gas phase. When Hoechst 33258 was mixed with a DNA sequence lacking a high affinity minor groove binding site, the negative ion ESI mass spectra showed that 1:1 and 2:1 complexes were formed, consistent with existence of binding modes other than minor groove binding. The data presented suggest that comparison of positive and negative ion ESI-MS spectra might provide an insight into various binding modes in both solution and the gas phase.

Elucidation of O‐phosphoryl and N‐phosphoryl amino acids by electrospray ionization tandem mass spectrometry

AbstractMass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI‐MS) in conjunction with tandem mass spectrometry (MS/MS). Besides N‐diisopropyloxyphosphoryl amino acids (N‐DIPP‐AA), O‐phospho‐ and O‐diisopropyloxyphosphoryl amino acids (O‐DIPP‐AA) were studied and compared to N‐DIPP‐AA. The fragmentation pathways of [M+H]+, [M+Na]+ and [M−H]− ions of phosphoryl amino acids were summarized. In addition to several similar patterns. each of them showed its characteristic fragmention.

Simultaneous determination of phenyl- and sulfonylurea herbicides in water by solid-phase extraction and liquid chromatography with UV diode array or mass spectrometric detection

A method for the simultaneous determination of 10 sulfonyl- and phenylurea herbicides (chlorsulfuron, triasulfuron, fenuron, monuron, fluometuron, chlorotoluron, isoproturon, diuron, metobromuron, linuron) and one of their most common degradation products (3-chloro-4-methyl-phenyl urea) has been developed. Liquid chromatography with diode array UV detection (LC–DAD UV) and positive ion electrospray mass spectrometry (LC–ESI–MS) were used for the separation, identification and quantification of these analytes. In the LC–DAD UV method, chromatographic separation was achieved in about 27 min; however, analysis time can be reduced to less than 13 min when LC–MS is applied, since no complete separation of the chromatographic peaks was required. A preconcentration step based on solid-phase extraction was applied for the simultaneous extraction of sulfonylureas (acidic in nature) and phenylureas (neutral compounds) from water samples. Different types of sorbents were evaluated: silica-based C 18 and two polymeric sorbents, Oasis HLB and LiChrolut EN. The best results were obtained with Oasis HLB, a co-polymer of poly(divinylbenzene-co- N-vinylpyrrolidone). Other parameters, such as the elution solvent and sample volume, were optimised in order to maximise extraction efficiency; the recoveries obtained varied between 70 and 95% for the acidic and neutral herbicides extracted jointly without acidification of the sample. Both procedures, SPE LC–DAD UV and SPE–LC–MS, were applied to spiked river waters and were compared in terms of selectivity and sensitivity. The detection limits obtained in river water samples were below 50 ng L −1 for both procedures.

Tandem mass spectrometric analysis of distinct fragmentation patterns to [M+Na]/z and [M+H]/z of dendritic Al(III) and Zn(II) quinolates.

The synthesis of two novel dendritic aluminum and zinc quinolates, which are soluble in common solvents, was monitored effectively by positive ion electrospray ionization mass spectrometry (ESI-MS). Through tandem mass spectrometric analysis of the complexes, distinct fragmentation pathways for sodium adduct molecular ion [M+Na]+ and protonated molecular ion [M+H]+ were observed.

Fragmentation patterns of novel dispirocyclopiperazinium dibromides with strong analgesic activity under electrospray ionization tandem mass spectrometry conditions

The fragmentation patterns of a series of dispirocyclopiperazinium dibromides with strong analgesic activity were analyzed by positive ion electrospray ionization mass spectrometry in conjunction with tandem mass spectrometry (ESI-MS n ). The [C 2+Br −] + ions showed the characteristic isotopic peaks with high intensity. In each of their MS 2 spectra, only the [C 2+Br −HBr] + ion peak was observable. Further analysis indicated that a selective rearrangement occurred in the unsaturated spirocyclopiperazine ring to achieve dihydropyrrole moiety. Meanwhile, the [C] 2+ ions were unique and always the base peaks. The ions [C 2+Br −] + and [C] 2+ were formed from the equilibrium of precursor molecules 1 in solution, and the latter ions could not be observed in the MS 2 spectra of ions [C 2+Br −] +. The related fragmentation mechanisms were proposed.

Syntheses and Characterization of Ruthenium(II) Tetrakis(Pyridine) Complexes. An Advanced Coordination Chemistry Experiment or Mini-Project

This experiment involves the syntheses of several coordination complexes of ruthenium(II) and their characterization by using various spectroscopic and spectrometric techniques. Because ruthenium is a precious metal all of the reactions are carried out on a small scale, giving students experience in careful laboratory manipulation. This experiment includes the option of extension to allow students to carry out simple experiments of their own devising, acting as an introduction to research work. The known complex trans-RuIICl2(py)4 (2, py = pyridine) is prepared from cis-RuIICl2(DMSO)4 (1, DMSO = dimethylsulfoxide) and then used as a precursor to the chloride-substituted derivative trans-RuII(NCS)2(py)4, 4. Further reactions of 2 with chosen salts can also be carried out, if desired. The new complex trans-RuIICl2(acpy)4 (3, acpy = 4-acetylpyridine) is also prepared from 1. Complexes 2–4 and any further product(s) are characterized by using 1H NMR, infrared, and UV-visible spectroscopies and positive ion electrospray or FAB mass spectrometry. The results of these measurements are related to the molecular structures. In particular, the 1H NMR spectra confirm the trans coordination geometries, while the UV–visible spectra show intense dπ(RuII) → π*(py or acpy) metal-to-ligand charge-transfer (MLCT) absorptions. The relative energies of these MLCT bands can be qualitatively correlated with the electronic properties of the ligands.

Identification of betalains from petioles of differently colored Swiss chard (Beta vulgaris L. ssp. cicla [L.] Alef. Cv. Bright Lights) by high-performance liquid chromatography-electrospray ionization mass spectrometry.

The betalain pattern of differently colored Swiss chard (Beta vulgaris L. ssp. cicla [L.] Alef. cv. Bright Lights) was investigated for the first time. Nineteen betaxanthins and nine betacyanins were identified by RP-HPLC and positive ion electrospray mass spectrometry, co-injection experiments with semisynthetic reference compounds, and standards derived from authentic plant material, respectively. Histamine-betaxanthin and alanine-betaxanthin were found to be novel betaxanthins, which to the best of our knowledge have not been reported as natural compounds until now. Furthermore, tyramine-betaxanthin (miraxanthin III) and 3-methoxytyramine-betaxanthin, which to date were known only from families other than the Chenopodiaceae, were detected for the first time in colored Swiss chard. The betacyanin pattern of purple petioles was composed of betanin, isobetanin, betanidin, and isobetanidin. Although phyllocactin was present in only trace amounts, further acylated structures such as betanidin-monoferuloyl-5-O-beta-diglucoside and lampranthin II, accompanied by their corresponding C(15)-epimers, were identified. In addition, quantification of betalains and CIE LCh degrees measurements were performed with the colored extracts to correlate the visual appearance with the respective pigment patterns. Besides the novel phytochemical findings, the present study is useful for the evaluation of betalainic Swiss chard as a potential coloring foodstuff.

Determination of strophanthidin in ingesta and plant material by LC-MS/MS.

An LC-MS/MS method was developed for the semiquantitative determination of strophanthidin glycosides in ingesta from animals. Strophanthidin glycosides were simultaneously extracted and hydrolyzed to the strophanthidin aglycone using aqueous methanolic hydrochloric acid and the extracts cleaned up using solid-phase extraction. Extracts were analyzed using reverse-phase HPLC coupled with positive ion electrospray mass spectrometry. Characteristic product ion spectra were produced by fragmentation of the [M + H](+) precursor ion for each analyte. Quantitation was performed using the internal standard method with digitoxigenin serving as the internal standard. The method detection limit was calculated to be 0.075 microg/g, and the limit of quantitation was calculated to be 0.24 microg/g for strophanthidin in control rumen samples. This method was used in diagnostic investigations to confirm fatal strophanthidin glycoside poisonings in horses.

Determination of fatty acid positions in native lipid A by positive and negative electrospray ionization mass spectrometry.

Lipid A is the endotoxic principle of the lipopolysaccharide fraction from Gram-negative bacteria. It is involved in the elicitation of cytokine production that leads to massive inflammation and to septic shock as a lethal consequence. For this reason, the structural elucidation of lipopolysaccharides from toxic Gram-negative bacteria is an important and complicated task, mainly owing to its natural heterogeneity. Here, a new methodology to infer the distribution of the primary and secondary acyl residues is described, based on electrospray ionization mass spectrometry (ESI-MS) of intact lipid A under high cone voltage in order to achieve in-source fragmentation. Under these conditions, acyl fragmentation is induced and a different regioselective cleavage of secondary fatty acids is observed in positive and negative ESI-MS, allowing the rapid identification of the lipid A structure.

Mass spectrometry of 1,3- and 1,5-dicaffeoylquinic acids.

Samples of 1,3- (1) and 1,5-dicaffeoylquinic acid (2) and their hexaacetate derivatives were examined using positive and negative electrospray ionization mass spectrometry and tandem mass spectrometry. Differences in the various spectra allow the discrimination of each of the isomers. Specific losses in the spectra of 2 also permit the identification of the site of substitution of one of the caffeic acid moieties as being at the 5-position. The spectra of 3,5- (3) and 4,5-dicaffeoylquinic (4) acids and their hexaacetate derivatives were compared with those of 1 and 2 and their derivatives, and differences in ion abundances or the presence/absence of specific ions can be used to identify uniquely each of the compounds.

Fragmentation patterns of novel dispirocyclopiperazinium dibromides with strong analgesic activity under electrospray ionization tandem mass spectrometry conditions.

The fragmentation patterns of a series of dispirocyclopiperazinium dibromides with strong analgesic activity are analyzed by positive ion electrospray ionization mass spectrometry in conjunction with tandem mass spectrometry (ESI-MS(n)). Instead of the parent molecular ions, the fragment ions [M-Br](+) are detected as characteristic double peaks and usually the base peaks. Meanwhile, the fragment ions [M-2Br](2+) are unique for this series of diquaternary ammonium dibromides and show high intensity. Besides the common fragmentation patterns around the carbonyl group, elimination of hydrogen bromide from [M--Br](+) ions is another important pathway. Following the elimination, an interesting rearrangement takes place in the unsaturated spirocyclopiperazine and transforms it into a dihydropyrrole structure, whose fragmentations are similar to its precursor ion in the succeeding steps.