Direct analysis of herbal powders by pipette-tip electrospray ionization mass spectrometry

Conventional electrospray ionization mass spectrometry (ESI-MS) is widely used for analysis of solution samples. The development of solid-substrate ESI-MS allows direct ionization analysis of bulky solid samples. In this study, we developed pipette-tip ESI-MS, a technique that combines pipette tips with syringe and syringe pump, for direct analysis of herbal powders, another common form of samples. We demonstrated that various herbal powder samples, including herbal medicines and food samples, could be readily online extracted and analyzed using this technique. Various powder samples, such as Rhizoma coptidis, lotus plumule, great burdock achene, black pepper, Panax ginseng, roasted coffee beans, Fructus Schisandrae Chinensis and Fructus Schisandrae Sphenantherae, were analyzed using pipette-tip ESI-MS and quality mass spectra with stable and durable signals could be obtained. Both positive and negative ion modes were attempted and various compounds including amino acids, oligosaccharides, glycosides, alkaloids, organic acids, ginosensides, flavonoids and lignans could be detected. Principal component analysis (PCA) based on the acquired mass spectra allowed rapid differentiation of closely related herbal species.     

Structural analysis of saponins from medicinal herbs using electrospray ionization tandem mass spectrometry

The underivatized saponins from Tribulus terrestris and Panax ginseng have been investigated by electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)). In ESI-MS spectra, a predominant [M + Na](+) ion in positive mode and [M - H](-) ion in negative mode were observed for molecular mass information. Multi-stage tandem mass spectrometry of the molecular ions was used for detailed structural analysis. Fragment ions from glycoside cleavage can provide information on the mass of aglycone and the primary sequence and branching of oligosaccharide chains in terms of classes of monosaccharides. Fragment ions from cross-ring cleavages of sugar residues can give some information about the linkages between sugar residues. It was found that different alkali metal-cationized adducts with saponins have different degrees of fragmentation, which may originate from the different affinity of a saponin with each alkali metal in the gas phase. ESI-MS(n) has been proven to be an effective tool for rapid determination of native saponins in extract mixtures, thus avoiding tedious derivatization and separation steps.     

Analysis of oxidizer salt mixtures by electrospray ionization mass spectrometry

Binary aqueous mixtures of NaNO3, KNO3 and NaClO4 oxidizers were analyzed using electrospray ionization mass spectrometry. Sodium nitrate solutions were observed to form doubly charged clusters of the type [(NaNO3)n2Na]2+ and [(NaNO3)n2NO3]2-, where n = 11, 13, 15, etc., in addition to singly charged cluster ions that have been reported previously. The identity of the doubly charged clusters was determined by tandem mass spectrometry. Two-component NaNO3-KNO3 salt solutions were observed to form cluster ions of the type [(NaNO3)i(KNO3)jNO3]- in the negative ion mode and [(NaNO3)i(KNO3)jNa]+ and [(NaNO3)i(KNO3)jK]+ in the positive ion mode, where i + j = 1, 2, 3 ... 10. Two-component solutions of KNO3-NaClO4 formed ions of the type [(KNO3)i(NaClO4)j(KClO4)k(NaNO3)lK](+) and [(KNO3)i(NaClO4)j(KClO4)k(NaNO3)lNa]+ in the positive ion mode, where i + j + k + l = 1, 2, 3 ... 10. Similar clusters containing excess nitrate and perchlorate to provide the charge are formed in the negative ion mode. In each case, the maximum number of spectral lines for a cluster of size n can be calculated as the number of combinations of n(th) order (where n = i + j) of N different cation-anion pairs taken with replication and without regard for the ordering of the N cation-anion pairs. The actual number of lines observed may be reduced due to degeneracy of nominal m/z values for some ions.     

The mass spectral analysis of isolated hops A-type proanthocyanidins by electrospray ionization tandem mass spectrometry

Comprehensive mass spectral fragmentation patterns have been established for sequencing chromatographically isolated A-type proanthocyanidins (PAs) using electrospray ionization tandem mass spectrometry (ESI-MS(n)) in the positive ion mode similar to those used for sequencing previously reported B-type PAs. Sequence-identifying fragmentations for A-type PAs include heterocyclic ring fission (HRF), retro-Diels-Alder (RDA) fission, benzofuran-forming (BFF) fission, and quinone methide (QM) fission. There is commonality in fragmentation patterns between A-type and B-type PAs, but distinguishing features in the mass spectral patterns between the two classes include 2-Da mass differences in the pseudo molecular ions, the propensity for the A-type PAs to undergo QM fissions and yield bis-quinoid ions as opposed to mono-quinoid ions in the upper unit of the sequence, and the reluctance of A-type linkages to undergo RDA, BFF, and BFF/H(2)O fissions from the upper unit. The positions of one or more A-type (C2-->O-->C7') ether linkages have been located in sequences of PAs ranging in chain lengths of two to five monomer units using ESI-MS(n) data. Using the fragmentation information from ESI-MS(n) experiments, a total of 17 PAs were structurally sequenced by systematic real time ESI-MS(n). Among them ten A-type and six B-type hop PAs are reported here for the first time.     

Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry

Diiodothyronines 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,3'-diiodothyronine (3,3'-T2) are important metabolites of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3). In this paper, a novel and rapid method for identifying and quantifying 3,5-T2, 3',5'-T2 and 3,3'-T2 has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed on the basis of our data obtained by ESI-MS/MS. MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate 3,5-T2, 3',5'-T2 and 3,3'-T2. On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation.     

Differentiation of monoiodothyronines using electrospray ionization tandem mass spectrometry

In this work two monoiodothyronines, 3-T1 and 3'-T1, have been analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS. MS2 spectra in either negative or positive ion mode can be used to differentiate 3-T1 and 3'-T1. Based on the relative abundance of fragment ions in MS2 spectra in the negative ion mode, quantification of the 3-T1 and 3'-T1 isomers in mixtures is achieved without prior separation. Solid-phase extraction in combination with ESI-MS/MS provides a practicable procedure that can be used to determine the molar ratio of 3-T1 and 3'-T1 in human serum with an error less than 3%. The detection limits for 3-T1 and 3'-T1 were 0.5 and 0.7 pg/microL, respectively.     

Discrimination of cyclic peptide diastereomers by electrospray ionization tandem mass spectrometry

In this research, the characteristic ions' abundance ratio between two isomers A and B in MS/MS mass spectra was defined as a parameter for discriminating diastereomers. Through this ratio, the discrimination of four pairs of cyclic peptide (CP) diastereomers was successfully achieved. Furthermore, in the analysis of diastereomers' mixtures, both calibration curve and calculational methods were substantiated to have high precision and accuracy. The average absolute errors of the two methods were 2.0 and 2.5% in the 48 measurements of 16 samples, respectively. This research provided a promising approach for the analysis of the CP diastereomers in the fields of asymmetrical synthesis, chiral natural products and structural biology by ESI‐MS/MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural determination of sphingomyelin by tandem mass spectrometry with electrospray ionization

Alkaline metal adduct ions of sphingomyelin were formed by electrospray ionization in positive ion mode. Under low energy collisionally activated dissociation (CAD), the product ion spectra yield abundant fragment ions representative of both long chain base and fatty acid which permit unequivocal determination of the structure. Tandem spectra obtained by constant neutral loss scanning permit identification of sphingomyelin class and specific long chain base subclass in the mixture. The fragmentation pathways under CAD were proposed, and were further confirmed by source CAD tandem mass spectrometry. The total analysis of sphingomyelin mixtures from bovine brain, bovine erythrocytes, and chicken egg yolk is also presented.     

Sediment extraction and clean-up for organic phosphorus analysis by electrospray ionization tandem mass spectrometry

A method to prepare NaOH sediment extracts for organic P compound analysis with electrospray ionization tandem mass spectrometry (ESI-MS-MS) was developed on natural samples. Ion exchange, rotary evaporation and mass cut-off filtering proved to be suitable for sample preparation. Samples were analyzed with ESI-MS-MS, and reproducibility and repeatability of the method was calculated. In addition, ³¹P-nuclear magnetic resonance spectroscopy (³¹P NMR) was used to measure recovery of different P compound groups such as orthophosphate (Ortho-P), orthophosphate monoesters (Monoester-P), orthophosphate diesters (Diester-P) and pyrophosphates (Pyro-P).

The developed sample preparation method resulted in an easy-to-spray liquid for the ESI-MS-MS instrumentation. The overall P recovery was 65% and ³¹P NMR showed that Diester-P, possibly in the form of DNA, was apparently lost through the filtering step most likely due to their size. Variances in the total intensities of the MS scans (relative standard deviation (R.S.D.) 35–54%) were for about 50% due to repeated MS runs. Covariances of the peaks in the MS spectra were calculated to be for about 30% due to the sample preparation procedure. Finally, with the ESI-MS-MS approach, 11 peaks in the mass spectra were found likely to represent phosphate containing compounds.     

Fragmentation of plumeran indole alkaloids from Aspidosperma spruceanum by electrospray ionization tandem mass spectrometry

The fragmentation of six plumeran indole alkaloids (PIAs) previously isolated from Aspidosperma spruceanum has been investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) in the positive ion mode. The fragmentation pathways have been established on the basis of MS/MS experiments using fragment ions generated in‐source and deuterium‐labeled alkaloids as precursor ions and on the basis of accurate mass measurements. Our results demonstrated that the fragmentation routes observed for the protonated PIAs are essentially derived from a pericyclic reaction and from the opening of rings D and E, followed by 1,4‐hydrogen rearrangements. Product ions resulting from radical eliminations were also observed, contrary to the ‘even‐electron rule’. Our data reveals that some product ions from protonated PIAs provide crucial information for the characterization of the acyl substituent at N‐1, the methoxyl and hydroxyl groups at the aromatic moiety, and give evidence of an ether bridge between C‐18 and C‐21. The data reported here were used for the dereplication of these compounds in a stem bark methanolic extract of Aspidosperma spruceanum. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of a novel microperoxidase from Marinobacter hydrocarbonoclasticus by electrospray ionization tandem mass spectrometry

Microperoxidases are small heme-peptides obtained by proteolytic digestion of cytochrome c, exhibiting peroxidase activity. They consist of a short- or medium-length polypeptide chain, covalently linked to an iron protoporphyrin IX moiety via two thioether bonds involving Cys residues at the c-porphyrin A and B pyrrole rings. These small molecules are interesting for a wide range of possible applications. We have structurally characterized, by means of electrospray ionization (ESI) mass and tandem mass spectrometric experiments, a novel microperoxidase called MMP-5 (Marinobacter MicroPeroxidase-5), obtained by proteolytic digestion of cytochrome c552, a monoheminic electron-transfer protein isolated from Marinobacter hydrocarbonoclasticus. This microperoxidase, which still maintains the functional peptide moieties for peroxidase activity, is devoid of the two amino acids intercalating the Cys residues linked to the c-porphyrin, thus increasing its water solubility. Once submitted to the ESI source potential, MMP-5 showed an interesting tendency for the reduction of the iron protoporphyrin substructure. This behaviour was clearly evidenced by the mass shift exhibited by the reduced form.     

Structural characterization of isoprenylated flavonoids from Kushen by electrospray ionization multistage tandem mass spectrometry

Eighteen isoprenylated flavonoids (8 flavanones, 3 flavanols, and 7 chalcones) isolated from Kushen or synthesized were studied by positive and negative ion electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). Plausible fragmentation patterns were obtained by comparing their MS(n) spectra with each other, which were further supported by high-resolution MS data and two model compounds. It was shown that the 2'-OH group would make the C-ring of flavonoids studied more labile through a six-membered mechanism, resulting in base peaks of (1,3)A+ (positive mode) and (1,4)A(-) (negative mode). In addition, the 2'-OH is also responsible for the neutral loss of water in (+)ESI/MS(2) of flavanones. The neutral loss of water (or methanol) in (-)ESI/MS(2) of flavanols was elucidated by a E2 elimination mechanism. Different relative abundances (RA) of (1,3)A(+) and S(+) in (+)ESI/MS(2) spectra were used to discriminate flavanones with their open-ring products, chalcones, since the equilibrium for flavanone<-->chalcone isomerization in ESI ion source could not be obtained in positive mode.     

Direct analysis of camptothecin from Nothapodytes nimmoniana by desorption electrospray ionization mass spectrometry (DESI-MS)

Desorption electrospray ionization was employed for fast and direct ambient detection of the anti-tumor drug, camptothecin, and its derivative, 9-methoxycamptothecin in Nothapodytes nimmoniana. Different parts of the plant such as leaves, stems and bark were examined. The ion intensities suggest that the concentration in bark is higher than that in the leaves and stems. The method does not require any sample preparation or preseparation. The identity of the alkaloids was further confirmed by tandem mass spectrometry.     

Characterization of isomeric 1,2,4-oxadiazolyl-N-methylpyridinium salts by electrospray ionization tandem mass spectrometry

The mass spectrometry behavior of 1,2,4-oxadiazolyl-N-methylpyridinium salts has been investigated. These substances are of current interest as perspective ionic liquids, compounds used as green solvents for synthesis, and for their catalytic properties. The studies have been developed through ESI-MS/MS experiments. The obtained results demonstrate that a readily distinction between the two isomeric classes, 3- N-methylpyridinium- and 5-N-methylpyridinium-1,2,4-oxadiazoles, is possible through ESI-MS/MS experiments. A deeper investigation on the principal fragmentation pathways of characteristic ions has been also developed.     

Direct quantification of creatinine in human urine by using isotope dilution extractive electrospray ionization tandem mass spectrometry

Urinary creatinine (CRE) is an important biomarker of renal function. Fast and accurate quantification of CRE in human urine is required by clinical research. By using isotope dilution extractive electrospray ionization tandem mass spectrometry (EESI–MS/MS) a high throughput method for direct and accurate quantification of urinary CRE was developed in this study. Under optimized conditions, the method detection limit was lower than 50 μg L⁻¹. Over the concentration range investigated (0.05–10 mg L⁻¹), the calibration curve was obtained with satisfactory linearity (R² = 0.9861), and the relative standard deviation (RSD) values for CRE and isotope-labeled CRE (CRE-d3) were 7.1–11.8% (n = 6) and 4.1–11.3% (n = 6), respectively. The isotope dilution EESI–MS/MS method was validated by analyzing six human urine samples, and the results were comparable with the conventional spectrophotometric method (based on the Jaffe reaction). Recoveries for individual urine samples were 85–111% and less than 0.3 min was taken for each measurement, indicating that the present isotope dilution EESI–MS/MS method is a promising strategy for the fast and accurate quantification of urinary CRE in clinical laboratories.