Characterization of steroidal saponins in crude extract from Dioscorea nipponica Makino by liquid chromatography tandem multi-stage mass spectrometry

The liquid chromatography-electrospray ionization-tandem multi-stage mass spectrometry (LC-ESI-MSⁿ) method was developed for the analyses and characterization of steroidal saponins in plant extract from the rhizome of Dioscorea nipponica Makino. The HPLC experiments were performed by means of a reversed-phase C₁₈ column and a binary mobile phase system consisting of water and acetonitrile under gradient elution conditions. Pseudoprotodioscin, methyl protodioscin and dioscin were identified by comparing the retention times, UV spectra and the fragmentation properties of [M − H]⁻ ions with the authentic standards. Four groups of steroidal saponin isomers possessed the [M − H]⁻ ions at m/z 1063, 1045, 901 and 1047, respectively, were observed during the LC-ESI(−)-MS analysis, and three groups of them except the pair of isomers with the [M − H]⁻ ions at m/z 1047 could be differentiated by LC-ESI(−)-MS³. Furthermore, the ESI-MSⁿ fragmentation behaviors of the [M + Li]⁺ ions of pseudoprotodioscin and methyl protodioscin have been investigated, and the observed information helped the structural elucidation of the more abundant isomer with the [M − H]⁻ ion at m/z 1047. As the result, a special sugar sequence of the saccharide chains was observed that not glucose but rhamnose might be connected with the hydroxyl group at C-3 position of the steroidal aglycone.     

Determination of ephedra alkaloids by liquid chromatography/tandem mass spectrometry

In conjunction with an AOAC Task Group on dietary supplements, a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was validated for measurement of 6 major alkaloids in raw ephedra sinica herb, ephedra extracts, ephedra tablets, complex dietary supplements containing ephedra, and a high-protein drink mix containing ephedra. The amount of ephedrine-type alkaloids present was determined by LC with mass selective detection. Six replicates of each matrix were analyzed on 3 separate occasions. The presence of 6 ephedrine-type alkaloids was detected at a level > 0.5 microg/g based on a 0.5 g sample. The standard curve range for this assay is from 0.02 to 1.0 microg/mL. Appropriate dilutions covered a wide range of specific alkaloid concentrations. The calibration curves for all 6 analytes had correlation coefficients > 0.995.     

Rapid structural characterization of triterpenoid saponins in crude extract from Symplocos chinensis using liquid chromatography combined with electrospray ionization tandem mass spectrometry

Triterpenoid saponins in bioactive crude extract from Symplocos chinensis were rapidly identified using electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with sequential mass spectrometry (LC-MSn). According to the characteristic fragmentation behavior of known glucuronide-type triterpenoid saponins isolated from this plant, a total of fourteen constituents in the crude extract were structurally characterized on the basis of their retention time and tandem mass spectrometric analysis, including five pairs of naturally occurring isomers. Except one known saponin formerly obtained, the other constituents were new compounds. The analytical method of LC-MSn combined with ESI-MSn in positive and negative ion modes has been developed for the direct structural elucidation of triterpenoid saponins of this kind in plant extracts.     

Rapid structural determination of alkaloids in a crude extract of Stemona saxorum by high‐performance liquid chromatography/electrospray ionization coupled with tandem mass spectrometry

The electrospray ionization (ESI) mass spectrometric behavior of five Stemona alkaloids, stemokerrin, oxystemokerrin, oxystemokerrilactone, oxystemokerrin N‐oxide and stemokerrin N‐oxide, was studied using an ESI tandem mass technique (MSⁿ). These compounds, isolated from Stemona saxorum endemic in Vietnam, represent a class of alkaloids containing a pyrido[1,2‐a]azepine A,B‐ring core with a 1‐hydroxypropyl side chain attached to C‐4. Their fragmentation pathways were elucidated by ESI‐MSⁿ results and the elemental composition of the major product ions was confirmed by accurate mass measurement. In order to rationalize some fragmentation pathways, the relative Gibbs free energies of some product ions were estimated using the B3LYP/6‐31+G(d) method. Based on the ESI‐MSⁿ results of five reference compounds, a reversed‐phase high‐performance liquid chromatography with tandem mass spectrometry (RP‐HPLC/MSⁿ) method was developed for the characterization of Stemona alkaloids with a pyrido[1,2‐a]azepine A,B‐ring core from the extract of S. saxorum. A total of 41 components were rapidly identified or tentatively characterized, of which 12 compounds were identified as Stemona alkaloids with a pyrido[1,2‐a]azepine A,B‐ring core, including four new compounds. This method is convenient and sensitive, especially for minor components in complex natural product extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Analyses of Stemona alkaloids in Stemona tuberosa by liquid chromatography/tandem mass spectrometry

Alkaloid profiles in Stemona tuberosa were found to be highly variable. Six Stemona alkaloids isolated from the plant were subjected to on-line high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and tandem mass spectrometry (MS/MS) analyses. Their fragmentation patterns and products were useful for their characterization. The LC/MS fingerprints of these alkaloids, though variable among samples, could provide an overall characterization of the authenticity and quality of this species and help to differentiate it from S. japonica and S. sessilifolia, as all three species are recognized as genuine sources of the herb Radix Stemonae in the Pharmacopoeia of the People's Republic of China.     

Characterization of phenolic compounds in the crude extract of Hedysarum multijugum by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

The fragmentation behavior of four types of phenolic compounds: coumestans, pterocarpenes, benzofurans and isoflavones, were studied using electrospray ionization multistage mass spectrometry (ESI-MS(n)) (n = 2-5) in negative ion mode. Losses of methyl (15 Da), CO (28 Da), CO(2) (44 Da), isopropyl (43 Da) and isobutenyl (55 Da) were dominating fragmentation patterns. Different positions and numbers of the substituents also led to the different fragmentation behavior. These fragmentation rules were applied for the identification of constituents in methanolic extracts of Hedysarum multijugum, in which 29 compounds were characterized, including nine new compounds. The method established in this study could be applied to the comprehensive quality control of the herb and its formulations. It could also be applied to the biological and pharmacological research of traditional Chinese medicines.     

On-line identification of tropane alkaloids from Erythroxylum vacciniifolium by liquid chromatography-UV detection-multiple mass spectrometry and liquid chromatography-nuclear magnetic resonance spectrometry

The bark of catuaba (Erythroxylum vacciniifolium Martius, Erythroxylaceae), a tree native to the northern part of Brazil, was investigated for its alkaloid content. With the aim of obtaining preliminary structure information on-line, the alkaloid extract was analysed by high-performance liquid chromatography coupled to diode array UV detection, to mass spectrometry and to nuclear magnetic resonance. Interpretation of on-line spectroscopic data obtained from this extract led to structural elucidation of six new alkaloids and partial identification of 18 potentially original alkaloids bearing the same tropane skeleton esterified in positions 3 and 6 by 1-methyl-1H-pyrrol-2-carboxylic acid and/or 4-hydroxy-3,5-dimethoxybenzoic acid.     

Direct characterization of isoquinoline alkaloids in a crude plant extract by ion-pair liquid chromatography-electrospray ionization tandem mass spectrometry: example of Eschscholtzia californica

An ion-pair HPLC-ESI-MS-MS method has been developed for the direct and rapid characterization of isoquinoline alkaloids in a crudely purified extract of the aerial parts of Eschscholtzia californica (Papaveraceae). This plant was chosen because of its increasing use in pharmaceutical industries and because its well known alkaloid composition allows the optimization of the experimental procedure through an on-line analytical sequence. Thus, 14 isoquinoline alkaloids of different types were detected and characterized. The identities of these compounds were confirmed unambigously by their fragmentation and UV spectra obtained by LC-diode-array detection. Various experiments including tandem mass spectrometry and in-orifice collision induced dissociation were performed and prove that MS-MS is a very efficient technique to identify these compounds. An explanation for each isoquinoline alkaloid type MS-MS fragmentation pattern is proposed and indicates similar neutral and/or radical losses. The order of the fragmentation depended on the type of compound but the lost fragments were similar.     

On-line identification of the antifungal constituents of Erythrina vogelii by liquid chromatography with tandem mass spectrometry,ultraviolet absorbance detection and nuclear magnetic resonance spectrometry combined with liquid chromatographic micro-fractionation

In our continuing search for new antifungal agents of plant origin, the investigation of Erythrina vogelii Hook. f. (Leguminosae), a plant used in the traditional medicine of Ivory Coast to treat various infectious ailments, was undertaken. In order to rapidly identify the active principles, the crude extract was analysed by low-flow LC-1H nuclear magnetic resonance spectrometry (NMR) which gave a sensitive detection of all the main peaks. LC microfractionation was performed just after LC-NMR detection and all peaks collected were submitted to antifungal bioautography assays against Cladosporium cucumerinum. By this means, the antifungal activity could be efficiently linked to three of the LC peaks. In order to obtain complementary on-line structural information for all peaks of interest, high-resolution LC-MS-MS together with LC-UV with post-column addition of UV shifts reagents was undertaken on the crude extract. This chemical screening strategy with integrated antifungal bioassays has permitted the on-line identification of numerous constituents and has given useful information for an efficient peak-guided isolation procedure.     

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.     

In vitro metabolite identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry.

Metabolite identification can provide critical information for further synthesis during the lead optimization stage of drug discovery. Fast chromatographic gradient methodology was explored to reduce the analysis time for metabolite identification by liquid chromatography/tandem mass spectrometry while maintaining chromatographic separation of all components of interest. Short HPLC columns (2 cm) containing 3 microm particles were used and the chromatographic conditions involved elevated flow rates (1.0-2.5 mL/min) and fast, full range linear gradients (<2 min). Detection was accomplished by fast scanning triple quadrupole mass spectrometers or a quadrupole time-of-flight mass spectrometer. Analysis of liver microsomal incubations of several compounds indicates that separation of isomeric metabolites as well as the parent compound is feasible with analysis times not exceeding 2 minutes.     

Characterization and identification of triterpenoid saponins in crude extracts from Clematis spp. by high-performance liquid chromatography/electrospray ionization with multi-stage tandem mass spectrometry

High-performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC/ESI-MS(n)) was applied to characterize and identify triterpenoid saponins in crude extracts from nine species of Clematis L. After separation on a Zorbax SB-C(18) column, negative ESI-MS experiments were performed. The quasi-molecular ions [M-H]-, [M+Cl]- and [M+HCOO]- were observed in the full-scan MS spectra of all compounds. The MS(n) (n = 2-4) data of the [M-H]- ions provided structural information on the sugar sequence of the oligosaccharide chains and on the aglycone of the saponins. In addition, the fragmentation mechanisms could be deduced from the fragment ions. As a result, eight saponins were unambiguously identified in C. ganpiniana by comparison with reference compounds. In addition, a new compound was tentatively identified as 3-O-ribopyranosyl --> rhamnopyranosyl --> (glucopyranosyl) --> arabinopyranosylhederagenin 28-O-rhamnopyranosyl --> glucopyranosyl --> glucopyranosyl ester (peak 1), and another one was tentatively deduced to be 3-O-glucopyranosyl --> ribopyranosyl --> rhamnopyranosyl --> arabinopyranosylhederagenin 28-O-rhamnopyranosyl --> glucopyranosyl --> glucopyranosyl ester (peak 5) from the genus Clematis L. for the first time. By ESI-MS(n), non-isomeric saponins could be discriminated rapidly. It is of interest that cleavage preferentially occurrs at the ester bond at C-28 and the charge is easy to transfer onto the oligosaccharide chain when the ester bond of a monodesmosidic saponin like HNH cleaves.     

Analysis of Amaryllidaceae alkaloids from Crinum by high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A high‐performance liquid chromatography/electrospray ionization multi‐stage tandem mass spectrometry (HPLC/ESI‐MSⁿ) method was developed to analyze two structurally related groups of Amaryllidaceae alkaloids (AmAs), crinane‐ and tazettine‐type alkaloids, in the species Crinum latifolium and C. asiaticum, as well as different organs of C. latifolium. In ESI‐MSⁿ spectra of the two types of alkaloids, characteristic fragmentation reactions were observed that allowed us to determine and differentiate them. Based on the fragmentation rules of reference standards, crinane‐type alkaloids displayed concurrent neutral loss of C₂H₅N (43 u) and C₂H₆N (44 u) as well as characteristic ions of m/z 213 and 211, whereas tazettine‐type alkaloids exhibited neutral loss of C₃H₇N (57 u) [or C₂H₅N (43 u), C₃H₇NO (73 u)] from the [M+H]⁺ and [M+H–H₂O]⁺ ions. These were supported by quadrupole time‐of‐flight (Q‐Tof)‐MS/MS analysis. The chemical complexity of the mixture was resolved by profiling. The compositions of the main crinane‐ and tazettine‐type alkaloids in the above‐mentioned species and organs were also compared. Overall, 28 AmAs comprising 14 crinane‐type and 14 tazettine‐type alkaloids were identified and studied by MS. Among them, 14 AmAs were tentatively characterized from the two species for the first time. This method allowed a rapid analysis of alkaloid distribution and composition of Crinum species, and may also be used for quality control and screening of extracts designated for pharmaceutical application. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization and identification of steroidal alkaloids in Fritillaria species using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC/ESI-QTOF-MS/MS) was performed to study the fragmentation behaviors of steroidal alkaloids from Fritillaria species, the antitussive and expectorant herbs widely used in traditional Chinese medicine. We propose, herein, a strategy that combining key diagnostic fragment ions and the relative abundances and amounts of major fragment ions (the ions exceeding 10% in abundance) to distinguish different sub-classes of Fritillaria alkaloids (FAs). It was found that hydrogen rearrangement and induction effects result in ring cleavage of the basic skeletons occurred in the MS/MS process and produced characteristic fragment ions, which are useful for structural elucidation. This method was finally used to investigate the primary steroidal alkaloids in the extracts of eight major Fritillaria species. As a result, 41 steroidal alkaloids (29 cevanine type, 1 jervine type, 6 veratramine type and 5 secosolanidine type alkaloids) were selectively identified in these Fritillaria species. Twenty-six compounds were unambiguously identified by comparing with the reference compounds and 15 compounds were tentatively identified or deduced according to their MS/MS data. Logical fragmentation pathways for different types of FAs have been proposed and are useful for the identification of these types of steroidal alkaloids in natural products especially when there are no reference compounds available.     

Simultaneous quantitation of seven alkaloids in processed Fuzi decoction by rapid resolution liquid chromatography coupled with tandem mass spectrometry

A rapid analytical method based on rapid resolution LC coupled with MS/MS was first established to quantify seven alkaloids in processed Fuzi decoction. The chromatographic method was optimized to allow simultaneous analysis of all analytes in 5 min and demonstrated good linearity (r > 0.9995), repeatability (RSD < 4.36%), intra‐ and interday precisions (RSD < 5.07%) with good accuracies (97.76–105.08%) and good recovery (95.0–107.5%) of seven alkaloids, namely higenamine, benzoylhypaconine, benzoylmesaconine, benzoylaconine, aconitine, hypaconitine, and mesaconitine. The LODs for these markers were in the range of 2.30–17.00 pg/mL. Quantitative analysis of the seven alkaloids in Baifupian decoction and Heishunpian decoction showed that the content of the seven marker chemicals varied significantly and concluded that the quality of Fuzi was greatly affected by different processed methods. The developed method could be used as a rapid, sensitive, and reliable approach for assessment of the quality of processed Fuzi and related decoction.     

Broad-spectrum drug screening of meconium by liquid chromatography with tandem mass spectrometry and time-of-flight mass spectrometry

Analysis of the major drugs of abuse in meconium has been established in clinical practice for detecting fetal exposure to illicit drugs, particularly for the ready availability of the sample and ease of collection from diapers, compared with neonatal hair and urine. Very little is known about the occurrence and detection possibilities of therapeutic and licit drugs in meconium. Meconium specimens (n = 209) were collected in delivery hospitals, from infants of mothers who were suspected to be drug abusers. A targeted analysis method by liquid chromatography–triple quadrupole mass spectrometry (LC-MS/MS) was developed for abused drugs: amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, morphine, codeine, 6-monoacetylmorphine, oxycodone, methadone, tramadol, buprenorphine, and norbuprenorphine. A separate LC-MS/MS method was developed for 11-nor-∆⁹-tetrahydrocannabinol-9-carboxylic acid. A screening method based on LC coupled to time-of-flight MS was applied to a broad spectrum of drugs. As a result, a total of 77 different compounds were found. The main drug findings in meconium were as follows: local anesthetics 82.5% (n = 172), nicotine or its metabolites 61.5% (n = 129), opioids 48.5% (n = 101), stimulants 21.0% (n = 44), hypnotics and sedatives 19.0% (n = 40), antidepressants 18.0% (n = 38), antipsychotics 5.5% (n = 11), and cannabis 3.0% (n = 5). By revealing drugs and metabolites beyond the ordinary scope, the present procedure helps the pediatrician in cases where maternal denial is strong but the infant seems to suffer from typical drug-withdrawal symptoms. Intrapartum drug administration cannot be differentiated from gestational drug use by meconium analysis, which affects the interpretation of oxycodone, tramadol, fentanyl, pethidine, and ephedrine findings.     

Pharmacokinetics and metabolism of ulixertinib in rat by liquid chromatography combined with electrospray ionization tandem mass spectrometry

The purpose of this study was to develop and validate a simple and sensitive liquid chromatography tandem mass spectrometry method for the determination of ulixertinib in rat plasma. The plasma samples were precipitated with acetonitrile and then separated on a C₁₈ column with water containing 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.3 mL/min. Analytes were monitored on a TSQ Vantage triple quadrupole tandem mass spectrometer operated in positive electrospray ionization mode. Selected reaction monitoring transitions were m/z 433.1→262.1 for ulixertinib and m/z 450.1→260.1 for internal standard. The assay achieved good linearity over the concentration range of 0.1‐1000 ng/mL with correlation coefficient > 0.9991. The validated assay has been successfully applied to pharmacokinetic study of ulixertinib in rat after oral and intravenous administration. The results revealed that ulixertinib showed high exposure in rat plasma, low clearance, moderate oral bioavailability (45.13%), and dose‐independent pharmacokinetic profiles over the oral dose range of 1‐15 mg/kg. In addition, six metabolites from rat plasma and hepatocytes were detected and structurally identified by ultra‐high performance liquid chromatography combined with high‐resolution mass spectrometry. The metabolic pathways of ulixertinib referred to hydroxylation and dealkylation and glucuronidation.     

Rapid screening for bisbibenzyls in bryophyte crude extracts using liquid chromatography/tandem mass spectrometry

A simple and rapid qualitative liquid chromatography-diode-array detection/tandem mass spectrometry (LC-DAD/MS/MS) method was developed and validated for screening bisbibenzyl compounds in bryophyte crude extracts at sub-ppm levels. After simple extraction with ethanol and analyte concentration with diethyl ether, the extracts were subjected to LC-DAD/MS/MS analysis. The overall instrument turnaround time was 50 min to obtain baseline separation of bisbibenzyl isomers in bryophytes. MS full scan, MS/MS precursor ion scan and MS/MS product ion scan modes were used for the screening. The bisbibenzyl standards studied gave limits of detection (LODs) at or below 10 ng/mL. The results also indicated that the method had acceptable precision to be used on a day-to-day basis for qualitative identification. The bisbibenzyl types, i.e. one biphenyl ether bond (A-type), two biphenyl ether bonds (B-type), one biphenyl ether and one biphenyl bond (C-type), or other biphenyl types can be differentiated by their ESI-MS/MS product profiles, and the number of alkoxyl substituents can also be identified. The linkage sites of biphenyl and biphenyl ether bonds cannot be identified for an unknown bisbibenzyl solely from its mass spectra. This system was used to support three screening assays of bryophytes including Marchantia polymorpha L., Ptagiochasm intermedium L. and Asterella angusta, which were collected from different places in China. From them, 7/12, 8/5 and 8/9 confirmed/unconfirmed bisbibenzyls were identified, respectively, based on their MS/MS data, UV spectra and the retention behavior. The screening method considerably reduced the time and the cost for the qualitative analyses, and the structure-fragmentation-UV relationships will facilitate the high-throughput screening (HTS) of bisbibenzyl compounds in bryophytes. It is also intended as a simple and convenient way for the determination of other structural families of natural products.