Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS

The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD‐HPLC‐ESI‐TOF‐MS and IT‐MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF‐MS in order to obtain molecular formula and exact mass. Posterior analyses with IT‐MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode.     

Simultaneous Determination Baicalin and Forsythin in Shuanghuanglian Oral Liquid by HPLC/DAD and LC‐ESI‐MS

Rapid, simple and reliable HPLC/DAD and LC‐ESI‐MS methods for the simultaneous determination of baicalin and forsythin in the traditional Chinese medicinal preparation Shuanghuanglian oral liquid were described and validated. The separation condition for HPLC/DAD was optimized using a BDS hypersil C18 column (Thermo, 2.1 × 150 mm, particle size 5 μm) by gradient elution using methanol‐0.2 % ammonium acetate as the mobile phase. The suitable detection wavelength was set at 277 nm for the quantitative analysis of baicalin and forsythin in this method. Some operational parameters of the ESI interface were optimized, negative m/z 445[M?H]^? for baicalin and negative m/z 593[M+CH₃COO]^? for forsythin, positive m/z 447[M+H]⁺ for baicalin and positive m/z 552[M+NH 4]⁺ for forsythin, respectively. These HPLC/DAD and LC‐ESI‐MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). These methods can be used as a complementary method for the commercial quality control of Shuanghuanglian oral liquid and its pharmaceutical preparations.     

Identification and analysis of alkaloids in cortex Phellodendron amurense by high‐performance liquid chromatography with electrospray ionization mass spectrometry coupled with photodiode array detection

Alkaloids from Cortex Phellodendron amurense Rupr. were identified to determine the material basis for the bioactivity of this herb. HPLC–ESI‐MS with photodiode array detection coupled to XCharge C₁₈ column was applied to analyze the alkaloids qualitatively and quantitatively. A total of 37 alkaloids were identified and tentatively characterized from the ethanol extract by online ESI‐MSⁿ fragmentation and UV spectral analysis. A total of ten alkaloids, including four novel natural products, were tentatively identified for the first time in P. amurense. The fragmentation pathways for certain compounds were analyzed. The contents of a pair of isomers (columbamine and jatrorrhizine) and four main alkaloids (phellodendrine, magnoflorine, berberine, and palmatine) were simultaneously quantified using the aforementioned method. Results showed that the newly discovered and known components of P. amurense were helpful in determining the material basis for the bioactivity of the herb. The application of the XCharge C₁₈ column is a suitable and practical method for the isolation of alkaloids in plants.     

Preparative separation of flavonoids in plant extract of Smilacis Glabrae Roxb. by high performance counter‐current chromatography

Four flavonoids, isoastilbin, astilbin, isoengelitin, and engelitin were isolated and purified simultaneously from Smilacis Glabrae Roxb. for the first time by high performance counter‐current chromatography using a system consisting of n‐hexane–n‐butanol–water (1:2:3, v/v/v). A total of 392.6 mg of astilbin, 71.4 mg of isoastilbin, 47.4 mg of engelitin, and 10.3 mg of isoengelitin were purified from 1.89 g of the ethyl acetate extract of Smilacis Glabrae Roxb. in six runs, each at over 94.51% purity as determined by HPLC. The structures of the four compounds were identified by their retention time, the LC‐ESI‐MSⁿ in the negative ion mode, and confirmed by ¹H‐NMR experiments. The characteristic LC‐ESI‐MS fragmentation patterns of the four compounds were discussed.     

Characterization of protoberberine alkaloids in Coptidis Rhizoma (Huanglian) by HPLC with ESI‐MS/MS

This study aims to qualitatively analyze protoberberine alkaloids in crude extract of Coptidis Rhizoma using HPLC with ESI‐MS/MS. Possible specific molecular weights of protoberberine alkaloids were firstly deduced according to literatures and were adopted to screen the alkaloids in the HPLC with ESI‐MS of crude extract of Coptidis Rhizoma. As a result, 21 protoberberine alkaloids were found, including compounds of very low concentration and compounds coeluted in one peak. Among these, two compounds were positively identified and verified by comparison with standards. Ten of these compounds were first reported in this study for Coptidis Rhizoma. In addition, chromatographic retention parameters a and c of all compounds were obtained using their retention times under five gradient conditions and were applied to confirm the deduction about the structures of protoberberine alkaloids by tandem mass data.     

Effect of pH on the Metabolism of Aconitine under Rat Intestinal Bacteria and Analysis of Metabolites Using HPLC/MS-MSn Technique

A semi‐quantitative method of mass spectrometry (MS) has been described for the analysis of metabolites of aconitine by rat intestinal bacteria at different pH. At pH 7.0, the rat intestinal bacteria exhibit optimal activity for the metabolism of aconitine. A high‐performance liquid chromatography‐electrospray ionization multiple‐stage mass spectrometry (HPLC/ESI‐MSⁿ) method has been applied to investigate the characteristic product ions of metabolites. Then, the logical fragmentation pathways of metabolites have been proposed. By comparing the retention time (t_R) of HPLC and the ESI‐MSⁿ data with the data of standard compounds and reports from literature, ten metabolites have been identified and a distinctive metabolite (15‐deoxyaconitine) has been deduced first time. The experimental results demonstrate that HPLC/ESI‐MSⁿ is a specific and useful method for the identification of metabolites of aconitine. Also, in the present paper, the HPLC‐MS method was introduced to determine the synthetical metabolite prior to the study of the toxicity by the method of Bliss.     

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.     

Bioflavonoid profile of citrus juices from Greece

High‐performance liquid chromatography with confirmation by UV–visible photodiode array detector–positive electrospray ionization–mass spectrometry [HPLC‐UV–vis‐DAD‐(+ESI)‐MS] with enhanced fragmentation by appropriate adjustment of the cone voltage was used to determine bioflavonoid content of five citrus species (tangerine, sanguine, sour orange, lemon and grapefruit) cultivated in Greece which come from citrus varieties analyzed for the first time. The main groups of bioflavonoids found in the juice of the citrus species according to HPLC retention times, spectral data and literature references were O‐glycosylated flavanones and flavones, C‐glucosylated flavones, O‐glucosylated flavones, O‐C‐glucosylated flavones like saponarin and a phenolic derivative. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Structural investigation and elucidation of new communesins from a marine‐derived Penicillium expansum Link by liquid chromatography/electrospray ionization mass spectrometry

Penicillium expansum is a ubiquitous species for which there are only few reports for chemical investigation in marine environments. Among the numerous secondary metabolites produced by this species, communesins represent a new class of cytotoxic and insecticidal indole alkaloids. In this study, we investigated a marine P. expansum strain exhibiting neuroactivity on a Diptera larvae bioassay. Bio‐guided purification led to the isolation and the identification of communesin B as the main active compound by HRMS and ¹H and ¹³C NMR. Liquid chromatography analyses with detection by electrospray ionization coupled with tandem mass spectrometry (LC/ESI‐MS/MS) and high‐resolution tandem mass spectrometry (LC/HRMS/MS) allowed the identification and characterization of four other known communesins (A, D, E and F) in the crude extract. A fragmentation model for dimethyl epoxide communesins was proposed after detailed interpretation of their MS/MS spectra. Further analyses of the extract using the modelled fragmentations led to the detection of seven new communesins found as minor compounds. Chemical structural elucidation of these new derivatives is discussed based on their fragmentation characteristics. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation study of iridoid glycosides including epimers by liquid chromatography‐diode array detection/electrospray ionization mass spectrometry and its application in metabolic fingerprint analysis of Gardenia jasminoides Ellis

A high‐performance liquid chromatography‐diode array detection/electrospray ionization mass spectrometry (HPLC‐DAD/ESI‐MS) method was applied to the characterization of ten iridoid glycosides in Gardenia jasminoides Ellis, a traditional Chinese medicine. During the process of structural elucidation, two groups of isomers including two epimers were structurally characterized and differentiated according to their distinctive fragmentation patterns which were closely related to their isomeric differentiations. Subsequently, the major compounds were purified by multi‐dimensional chromatography and semi‐preparative HPLC and the structure identification was confirmed with NMR techniques. The major fragmentation pathways of iridoid glycosides in Gardenia jasminoides Ellis obtained through the MS data were schemed systematically, which provided the best sensitivity and specificity for characterization of the iridoid glycosides especially the isomers so far. Based on the fragmentation patterns of iridoid glycosides concluded, seven major iridoid glycosides were characterized in rat plasma after intravenous administration of Gardenia jasminoides Ellis. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid screening and identification of lycodine‐type alkaloids in Lycopodiaceae and Huperziaceae plants by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Lycodine‐type alkaloids have gained significant interest owing to their unique skeletal characteristics and acetylcholinesterase activity. This study established a rapid and reliable method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐ESI‐Q/TOF‐MS/MS) for comprehensive characterization of lycodine‐type alkaloids for the first time. The lycodine‐type alkaloids were detected successfully from Lycopodiastrum casuarinoides, Huperzia serrata and Phlegmarirus carinatus in seven plants of the Lycopodiaceae and Huperziaceae families, based on the established characteristic MS fragmentation of five known alkaloids. Furthermore, a total of 13 lycodine‐type alkaloids were identified, of which three pairs of isomers were structurally characterized and differentiated. This study further improves mass analysis of lycodine‐type alkaloids and demonstrates the superiority of UPLC with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of other trace active compounds. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization and quantification of 10‐hydroxycamptothecine in Camptotheca acuminate and its medicinal preparation by liquid chromatography–ion trap mass spectrometry

A rapid and sensitive method for the identification and quantification of 10‐hydroxycamptothecine (HCPT) in Camptotheca acuminata Decne is described. The HCPT standard solution was directly infused into the ion trap mass spectrometers (IT/MS) for collecting the MSⁿ spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of HCPT was proposed and the ESI‐MSⁿ fragmentation behavior of HCPT was deduced in detail. The major fragment ions of HCPT were confirmed by MSⁿ in both negative ion and positive ion mode. The possible main cleavage pathway of fragment ions was studied. Quantification of HCPT was assigned in negative‐ion mode at a product ion at m/z 363 → 319 by LC‐MS. The LC‐MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the HCPT. Lastly, the LC‐MS method was successfully applied to determine HCPT in real samples of Camptotheca acuminate Decne and its medicinal preparation in the first time. Copyright © 2013 John Wiley & Sons, Ltd.     

Challenges in the identification process of phenidate analogues in LC‐ESI‐MS/MS analysis: Information enhancement by derivatization with isobutyl chloroformate

A new analytical technique for the structural elucidation of four representative phenidate analogues possessing a secondary amine residue, which leads to a major/single amine‐representative fragment/product ion at m/z 84 both in their GC‐EI‐MS and LC‐ESI‐MS/MS spectra, making their identification ambiguous, was developed. The method is based on “in vial” chemical derivatization with isobutyl chloroformate in both aqueous and organic solutions, followed by liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS/MS). The resulting carbamate derivatives promote rich fragmentation patterns with full coverage of all substructures of the molecule, enabling detailed structural elucidation and unambiguous identification of the original compounds at low ng/mL levels.     

Identification and characterization of impurities in an insecticide,bifenthrin technical

The HPLC‐DAD and GC/MS methods were successfully used for the identification and characterization of the impurities in an agrochemical insecticide, bifenthrin technical. Three impurities ranging from 0.175%–0.541% were detected by the HPLC‐DAD method. The LC/MS technique with ESI or APCI source failed to detect the impurities detected by HPLC‐DAD, due to lack of ionization in ESI or APCI. The three impurities were enriched by prep‐HPLC, and then their structures were elucidated based on the GC/EIMS and CIMS data. The EI mass spectra of bifenthrin and its impurities displayed molecular ion and provided structure indicative fragment ions; the CIMS data further confirmed their molecular weight. The identity of the impurity 1 was further confirmed by the synthesis of the authentic sample followed by NMR and GC/MS data.     

Development of an HPLC‐DAD method for the determination of five alkaloids in Stephania yunnanensis Lo and in rat plasma after oral dose of Stephania yunnanensis Lo extracts

For the rational utilization and the quantitative quality control of the Stephania yunnanensis Lo, an HPLC‐DAD method was developed for the quantitative and simultaneous determination of five alkaloids in rat plasma (stepharine, sinomenine, palmatine, isocorydine and tetrahydropalmatine), which were the main active chemical constituents of this plant and belong to four kinds of isoquinoline‐type alkaloids (protoberberine, morphine, aporphine and protaporphine alkaloids). The contents of five alkaloids ranged from 0.09 to 2.32% (w/w). The method validation was tested for the linearity (r² > 0.9975), precision (intra‐day RSD < 4.8% and inter‐day RSD < 4.9%), extraction recovery (85.49 ± 2.29% to 99.21 ± 1.48%) and stability (98.5 ± 5.3% to 101.2 ± 3.4%). We developed an HPLC‐DAD method to simultaneously measure these alkaloids in rat plasma after oral administration of the extract of this plant to rats. The results supported the hypothesis that isoquinoline alkaloids were the compounds responsible for the main pharmacological activities for anti‐inflammatory and analgesic.     

Characterization of antioxidant phenolics in Syringa vulgaris L. flowers and fruits by HPLC‐DAD‐ESI‐MS

In this study the polyphenolic composition of lilac flowers and fruits was determined for the first time. For the identification of compounds, accurate molecular masses and formulas, acquired by LC and ESI‐TOF‐MS and fragmentation pattern given by LC‐ESI/MS/MS analyses, were used. Our chromatographic system in conjunction with tandem MS was found to be valuable in the rapid separation and determination of the multiple constituents in methanolic extracts of lilac flowers and fruits. Altogether 34 phenolics, comprising 18 secoiridoids, seven phenylpropanoids, four flavonoids and five low‐molecular‐weight phenols, were identified. As marker compounds two secoiridoids (oleuropein and nuzhenide), two phenylpropanoids (acteoside and echinacoside) and rutin were quantified by validated methods. As a result of quantitative analysis, it was confirmed that flowers contain significant amounts of phenylpropanoids (acteoside, 2.48%; echinacoside, 0.75%) and oleuropein (0.95%), while in fruits secoiridoid oleuropein (1.09%) and nuzhenide (0.42%) are the major secondary metabolites. The radical scavenging activities of the extracts and the constituents were investigated by DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) and ABTS [2,2′‐azino‐bis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid)] assays. Both extracts show remarkable antioxidant activities. Our results clearly show that lilac flowers and fruits are inexpensive, readily available natural sources of phenolic compounds with pharmacological and cosmetic applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Oxidation‐assisted structural elucidation of compounds containing a tertiary amine side chain using liquid chromatography mass spectrometry

A novel analytical technique for the structural elucidation of compounds bearing a tertiary amine side chain via “in vial” instantaneous oxidation and liquid chromatography mass spectrometry (LC‐MS) was developed. A series of lidocaine homologs and benzimidazole derivatives with a major/single amine representative base peak in both their EI‐MS and ESI‐MS/MS spectra were subjected to oxidation by a 0.1% solution of hydrogen peroxide (including several ¹⁶O/¹⁸O exchange experiments), followed by LC‐ESI‐MS/MS analysis. The N‐oxide counterparts promoted extensive fragmentation with complete coverage of all parts of the molecule, enabling detailed structural elucidation and unambiguous identification of the unoxidized analytes at low nanogram per milliliter levels.