Identification of common glycosyl groups of flavonoid O‐glycosides by serial mass spectrometry of sodiated species

Flavonoid O‐glycosides are a ubiquitous and important group of plant natural products in which a wide variety of sugars are O‐linked to an aglycone. Determining the identity of the sugars, and the manner in which they are linked, by mass spectrometry alone is challenging. To improve the identification of common O‐linked di‐ and trisaccharides when analysing mixtures of flavonoid O‐glycosides by liquid chromatography/mass spectrometry (LC/MS), the fragmentation of electrosprayed sodium adducts in an ion trap mass spectrometer was investigated. The sodium adducts [M + Na]⁺ of kaempferol 3‐O‐glycosides generated sodiated glycosyl groups by the neutral loss of kaempferol. The product ion spectra of these sodiated glycosyl groups differed between four isomeric kaempferol 3‐O‐rhamnosylhexosides and four isomeric kaempferol 3‐O‐glucosylhexosides in which the primary hexose was either glucose or galactose and bore the terminal glucose or rhamnose at either C‐2 or C‐6. Fragmentation of sodiated glycosyl groups from linear O‐triglucosides and branched O‐glucosyl‐(1 → 2)‐[rhamnosyl‐(1 → 6)]‐hexosides produced sodiated disaccharide residues, and the product ion spectra of these ions assisted the identification of the complete sugar. The product ion spectra of the sodiated glycosyl groups were consistent among flavonoid O‐glycosides differing in the position at which the sugar was O‐linked to the aglycone, and the nature of the aglycone. The abundance of sodiated species was enhanced by application of a pre‐trap collision voltage, without the need to dope with salt, allowing automated LC/MS methods to be used to identify the glycosyl groups of common flavonoid O‐glycosides, such as rutinosides, robinobiosides, neohesperidosides, gentiobiosides and sophorosides. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro formation of phase I and II metabolites of propranolol and determination of their structures using chemical derivatization and liquid chromatography–tandem mass spectrometry

Derivatization with 1,2‐dimethylimidazole‐4‐sulfonyl chloride (DMISC) has been successfully used as a tool to differentiate between aromatic and aliphatic O‐glucuronides of hydroxypropranolol. The analyses were performed with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) with both a triple quadrupole and an ion trap instrument. Hydroxylated forms of propranolol can be glucuronidated in aliphatic as well as aromatic positions. These isoforms are not distinguishable by tandem MS alone, as they both initially lose 176 Da, i.e. monodehydrated glucuronic acid, giving back the aglycone. Two in vitro systems were set up for the production of propranolol metabolites. The obtained isomers of 4′‐hydroxypropranolol glucuronide were determined to correspond to one aliphatic and one aromatic form, using chemical derivatization with DMISC and LC‐MSⁿ. DMISC was shown to react with the secondary amine in the case where the naphtol was occupied by the glucuronyl moiety, resulting in a different fragmentation pattern compared with that of the aliphatic glucuronide, where the naphtol group was accessible to derivatization. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification and quantification of atractylenolide I and atractylenolide III in Rhizoma Atractylodes Macrocephala by liquid chromatography–ion trap mass spectrometry

Rhizoma Atractylodes Macrocephala (RAM) is an important traditional Chinese medicinal herb that is used for treatment of dyspepsia and anorexia. The active ingredients, atractylenolide I (AO‐I) and atractylenolide III (AO‐III), were identified by direct‐injection ion trap‐mass spectrometry (IT‐MS) for collecting MSⁿ spectra. The major fragment ions of AO‐I and AO‐III were confirmed by MSⁿ both in negative ion mode and in positive ion mode. The possible main cleavage pathway of fragment ions was studied. The determinations of AO‐I and AO‐III were accomplished by liquid chromatography (LC) with UV and MS. The analytes provided good signals corresponding to the protonated molecular ions [M + H]⁺ and product ions. The precursor ions and product ions for quantification of AO‐III and AO‐I were m/z 249 → 231 and m/z 233 → 215, respectively, using selected ion monitoring by LC‐IT‐MS. Two methods were evaluated for a number of validation characteristics (repeatability, limit of detection, calibration range, and recovery). MS provides a high selectivity and sensitivity for determination of AO‐III and AO‐I in positive mode. After optimization of the methods, separation, identification and quantification of the two components in RAM were comprehensively tested by HPLC with UV and MS. Copyright © 2012 John Wiley & Sons, Ltd.     

Assigning glucose or galactose as the primary glycosidic sugar in 3‐O‐mono‐, di‐ and triglycosides of kaempferol using negative ion electrospray and serial mass spectrometry

Kaempferol 3‐O‐β‐glucopyranoside, kaempferol 3‐O‐β‐galactopyranoside and higher glycosides of these two flavonoids with α‐rhamnose at C‐2 and/or C‐6 of the primary sugar were studied by negative ion electrospray ionisation and serial mass spectrometry in a three‐dimensional (3D) ion trap mass spectrometer. Kaempferol 3‐O‐β‐glucopyranoside and kaempferol 3‐O‐α‐rhamnopyranosyl(1→6)‐β‐glucopyranoside could be distinguished from their respective galactose analogues by differences in the ratio of the radical aglycone ion [Y₀ – H]^?? to the rearrangement aglycone ion Y following MS/MS of the deprotonated molecules. Kaempferol 3‐O‐rhamnopyranosyl(1→2)‐β‐glucopyranoside and kaempferol 3‐O‐α‐rhamnopyranosyl(1→2)[α‐rhamnopyranosyl(1→6)]‐β‐glucopyranoside could be distinguished from their respective galactose analogues by differences in the product ion spectra of the [(M – H) – rhamnose]^? ion following serial mass spectrometry. In the triglycoside, it was deduced that this ion resulted from the loss of the rhamnose substituted at 2‐OH of the primary sugar by observing that MS/MS of deprotonated kaempferol 3‐O‐β‐glucopyranosyl(1→2)[α‐rhamnopyranosyl(1→6)]‐β‐glucopyranoside showed the loss of glucose and not rhamnose. Thus the class of sugar (hexose, deoxyhexose, pentose) at C‐2 and C‐6 of the primary sugar can be determined. These observations aid the assignment of kaempferol 3‐O‐glycosides, having glucose or galactose as the primary glycosidic sugar, in LC/MS analyses of plant extracts, and this can be done with reference to only a few standards. Copyright © 2009 John Wiley & Sons, Ltd.     

Fast analysis of isobaric grape anthocyanins by Chip‐liquid chromatography/mass spectrometry

In a previous work, direct‐infusion electrospray ionization ion trap tandem mass spectrometry (ESI‐IT‐MS/MS) was applied to the study of anthocyanins in extracts from the skins of Clinton grapes, a non‐Vitis vinifera red grape variety qualitatively and quantitatively rich in anthocyanins. A good characterization of anthocyaninins was obtained, but it was impossible to differentiate some compounds with the same nominal mass but with different elemental composition. In this work, the capabilities of quadrupole time‐of‐flight mass spectrometry (QTOF‐MS) coupled with Chip‐liquid chromatography (LC‐Chip) were applied to the study of Clinton anthocyanins and this method provided the complete sample anthocyanin fingerprint in less than 5 min. Multi‐stage mass spectrometry (MSⁿ; n >2) was not necessary to identify isobaric compounds, nor were deuterium‐exchange experiments necessary to distinguish between compounds containing the same aglycone. The fast separation bypasses the problem of petunidin‐3‐O‐(6‐O‐acetyl)monoglucoside and delphinidin‐3,5‐O‐diglucoside quantification, present in the direct‐infusion ESI‐ITMS approach, due to overlapping with matrix interferences. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a CE‐MS2 method for the enantiomeric separation of L/D‐carnitine: Application to the analysis of infant formulas

A new chiral analytical method based on CE‐MS is proposed for the identification and simultaneous quantification of D /L ‐carnitine in infant formulas. Previous derivatization of carnitine with FMOC enabled the optimization of the chiral separation using CE with UV detection. An optimization of electrospray‐MS parameters using a partial filling of the non‐volatile chiral selector (succinyl‐γ‐CD) was performed. A selective fragmentation using MS² experiments with an ion trap analyser was carried out to confirm the identity of D /L ‐carnitine according to the current legislation. Satisfactory results were obtained in terms of linearity, precision, and accuracy. Interestingly, the CE‐MS² method developed allowed a sensitivity enhancement with respect to UV detection of 100‐fold, obtaining an LOD of 100 ng/g for D ‐carnitine. The determination of L ‐carnitine and its enantiomeric purity in 14 infant formulas supplemented with carnitine was successfully achieved, sample preparation only requiring an ultrafiltration with centrifugal filter devices to retain the components with the highest molecular weights.     

Simultaneous determination and pharmacokinetic study of three flavonoid glycosides in rat plasma by LC–MS/MS after oral administration of Rubus chingii Hu extract

A simple and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of isoquercitrin, kaempferol‐3‐O‐rutinoside and tiliroside in rat plasma. Plasma samples were deproteinized with methanol and separated on a Hypersil Gold C₁₈ column (2.1 × 50 mm, i.d., 3.0 μm) using gradient elution with the mobile phase of water and methanol at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed with negative ion electrospray ionization in selected reaction monitoring mode. All analytes showed good linearity over their investigated concentration ranges (r² > 0.99). The lower limit of quantification was 1.0 ng/mL for isoquercitrin and 2.0 ng/mL for kaempferol‐3‐O‐rutinoside and tiliroside, respectively. Intra‐ and inter‐day precisions were <8.2% and accuracy ranged from −11.5 to 9.7%. The mean extraction recoveries of analytes and IS from rat plasma were >80.4%. The assay was successfully applied to investigate the pharmacokinetic study of the three ingredients after oral administration of Rubus chingii Hu to rats.     

First report of non‐coloured flavonoids in Echium plantagineum bee pollen: differentiation of isomers by liquid chromatography/ion trap mass spectrometry

Apicultural products have been widely used in diet complements as well as in phytotherapy. Bee pollen from Echium plantagineum was analysed by high‐performance liquid chromatography/photodiode‐array detection coupled to ion trap mass spectrometry (HPLC‐PAD‐MSⁿ) with an electrospray ionisation interface. The structures have been determined by the study of the ion mass fragmentation, which characterises the interglycosidic linkage in glycosylated flavonoids and differentiates positional isomers. Twelve non‐coloured flavonoids were characterised, being kaempferol‐3‐O‐neohesperidoside the major compound, besides others in trace amounts. These include quercetin, kaempferol and isorhamnetin glycosides, with several of them being isomers. Acetylated derivatives are also described. This is the first time that non‐coloured flavonoids are reported from this pollen, with MS fragmentation proving to be most useful in the elucidation of isomeric structures. Copyright © 2010 John Wiley & Sons, Ltd.     

High‐sensitivity simultaneous quantification of tacrolimus and 13‐O‐demethyl tacrolimus in human whole blood using ultra‐performance liquid chromatography coupled to tandem mass spectrometry

Blood concentrations of tacrolimus show large variability among patients and the narrow therapeutic range is related to adverse effects. Therefore, therapeutic drug monitoring is needed for strict management. 13‐O‐Demethyl tacrolimus (13‐O‐DMT) was reported as the major metabolite formed by cytochrome P450 (CYP)3A such as CYP3A5. In previous studies, the best lower limit of quantification (LLOQ) was 0.1 ng/mL for both substances. However, this LLOQ may not be low enough now because the dosage of tacrolimus has decreased in recent years. The purpose of this study was to develop and validate a high‐sensitivity and high‐throughput assay for simultaneous quantification of tacrolimus and 13‐O‐DMT in human whole blood using ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). Thirty‐five stable kidney transplant recipients receiving tacrolimus were recruited in this study. The calibration curve range was 0.04–40 ng/mL. All calibration samples and quality control samples fulfilled the requirements of the US Food and Drug Administration and the European Medicines Agency guidelines for assay validation. Trough concentrations of tacrolimus and 13‐O‐DMT in 35 stable kidney transplant recipients receiving tacrolimus were within the range of the respective calibration curve. Our novel UPLC–MS/MS method is more sensitive than previous methods for quantification of tacrolimus and 13‐O‐DMT.     

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.     

Validated LC‐MS/MS method for the simultaneous determination of hyperoside and 2′′–O‐galloylhyperin in rat plasma: application to a pharmacokinetic study in rats

An LC‐MS/MS method was developed for the first time to simultaneously determine hyperoside and 2′′–O‐galloylhyperin, two major components in Pyrola calliantha extract, in rat plasma. Following extraction by one‐step protein precipitation with methanol, the analytes were separated on a Venusil MP‐C₁₈ column within 2 min, using methanol–water–formic acid (50:50:0.1, v/v/v) as the mobile phase at a flow rate of 0.4 mL/min. Detection was performed on electrospray negative ionization mass spectrometry by multiple‐reaction monitoring of the transitions of 2′′–O‐galloylhyperin at m/z 615.1 → 301.0, of hyperoside at m/z 463.1 → 300.1, and of internal standard at m/z 415.1 → 295.1. The limits of quantification were 2 ng/mL for both hyperoside and 2′′–O‐galloylhyperin. The precisions were <13.1%, and the accuracies were between ?9.1 and 5.5% for both compounds. The method was successfully applied in pharmacokinetic studies following intravenous administration of the total flavonoids of P. calliantha extract in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of flavonoid profile of Scutellaria bacalensis Georgi by high performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry

Scutellaria baicalensis Georgi is a well-known medicinal plant widely used in China and other East Asian countries. High performance liquid chromatography combined with diode array detection and electrospray ion trap mass spectrometry was used to determine the flavonoid profile of S. baicalensis. Under the optimized experiment conditions, 32 flavonoids were clearly detected. Eighteen main ones were doubtless identified by comparing their retention time, UV and MS (MSⁿ) data with isolated or commercial standards. The UV characteristics of these 18 known standards were studied in detail. The rules summarized provided valuable indications for the subsequent on-line identification processes. By interpreting both the MS and the UV data in detail, other 13 minor flavonoids in S. baicalensis were on-line identified successfully.     

Structural characterization and identification of iridoid glycosides,saponins, phenolic acids and flavonoids in Flos Lonicerae Japonicae by a fast liquid chromatography method with diode‐array detection and time‐of‐flight mass spectrometry

A fast liquid chromatography method with diode‐array detection (DAD) and time‐of‐flight mass spectrometry (TOF‐MS) has been developed for analysis of constituents in Flos Lonicerae Japonicae (FLJ), a traditional Chinese medicine derived from the flower bud of Lonicera japonica. The chromatographic analytical time decreased to 25 min without sacrificing resolution using a column packed with 1.8‐µm porous particles (4.6 × 50 mm), three times faster than the performance of conventional 5.0‐µm columns (4.6 × 150 mm). Four major groups of compounds previously isolated from FLJ were structurally characterized by DAD‐TOF‐MS: iridoid glycosides showed maximum UV absorption at 240 nm; phenolic acids at 217, 242, and 326 nm; flavonoids at 255 and 355 nm; while saponins had no absorption. In electrospray ionization (ESI)‐TOF‐MS experiments, elimination of a glucose unit (162 Da), and successive losses of H₂O, CH₃OH and CO, were generally observed in iridoid glycosides; saponins were characterized by a series of identical aglycone ions; phenolic acids typically generated a base peak at [M–H–caffeoyl]^? by loss of a caffeic acid unit (162 Da) and several marked quinic acid moiety ions; cleavage of the glycosidic bond (loss of 162 or 308 Da), subsequent losses of H₂O, CO, RDA and C‐ring fragmentation were the most possible fragmentation pathways for flavonoids. By accurate mass measurements within 4 ppm error for each molecular ion and subsequent fragment ions, as well as the ‘full mass spectral’ information of TOF‐MS, a total of 41 compounds including 13 iridoid glycosides, 11 phenolic acids, 7 saponins, and 10 flavonoids were identified in a methanolic extract of FLJ. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination and comparison of flavonoids and anthocyanins in Chinese sugarcane tips,stems, roots and leaves

The flavonoids and anthocyanins in Chinese sugarcane (Saccharum sinensis Roxb.) tips, stems, roots and leaves were separately analyzed by HPLC‐UV diode array detector, respectively. The results indicated that the content of flavonoids in sugarcane leaves was considerably high in comparison with previous reports in Brazil sugarcane (S. officinarum L.) leaves. Moreover, the content of flavonoids in sugarcane tips and roots was also high in comparison with sugarcane stems. For another, the content of anthocyanins in sugarcane roots was higher than that in other parts of the sugarcane, such as leaves, tips and stems. In addition, two anthocyanins, named petunidin 3‐O‐(6″‐succinyl)‐rhamnoside and cyanidin‐3‐O‐glucoside, were first identified from S. sinensis by HPLC‐UV diode array detector and HPLC‐MS/MS.     

Identification and quantification of oleanolic acid and ursolic acid in Chinese herbs by liquid chromatography-ion trap mass spectrometry

A rapid and sensitive method for the identification and quantification of ursolic acid (UA) and oleanolic acid (OA) in Chinese herbs is described. The method combines liquid chromatography (LC) with ion trap‐mass spectrometry (IT‐MS) detection. The UA and OA standard solution were directly infused into IT‐MS for collecting MSⁿ spectra. The major fragment ions of UA and OA were confirmed by MSⁿ at m/z 455, 407, 391, 377 and 363 in negative ion mode, and m/z 457, 439, 411 and 393 in positive mode, respectively. The possible main cleavage pathway of fragment ions was studied. UA and OA provided good signals corresponding to the deprotonated molecular ion [M − H]⁻. The method is reliable and reproducible, and the detection limit is 5 ng/mL. The method was validated in the concentration range of 0.04–40 μg/mL; intra‐ and inter‐day precisions ranged from 0.78 to 2.15%, and the accuracy was 96.5–108.2% for UA and OA. The mean recovery of UA and OA was 97.1–106.2% with RSD less than 1.86%. An LC‐IT‐MS method was successfully applied to determine the UA and OA in nine Chinese herbs. Copyright © 2011 John Wiley & Sons, Ltd.     

A quantitative LC–MS/MS method for determination of a small molecule agonist of EphA2 in mouse plasma and brain tissue

Compound 27 {1, 12‐bis[4‐(4‐amino‐6,7‐dimethoxyquinazolin‐2‐yl)piperazin‐1‐yl]dodecane‐1,12‐dione} is a novel small molecule agonist of EphA2 receptor tyrosine kinase. It showed much improved activity for the activation of EphA2 receptor compared with the parental compound doxazosin. To support further pharmacological and toxicological studies of the compound, a method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC–MS/MS) has been developed for the quantification of this compound. Liquid–liquid extraction was used to extract the compound from mouse plasma and brain tissue homogenate. Reverse‐phase chromatography with gradient elution was performed to separate compound 27 from the endogenous molecules in the matrix, followed by MS detection using positive ion multiple reaction monitoring mode. Multiple reaction monitoring transitions m/z 387.3 → 290.1 and m/z 384.1 → 247.1 were selected for monitoring compound 27 and internal standard prazosin, respectively. The linear calibration range was 2–200 ng/mL with the intra‐ and inter‐day precision and accuracy within the acceptable range. This method was successfully applied to the quantitative analysis of compound 27 in mouse plasma and brain tissue with different drug administration routes.     

A novel UPLC‐MS/MS method for sensitive quantitation of boldine in plasma,a potential anti‐inflammatory agent: application to a pharmacokinetic study in rats

Boldine is a potential anti‐inflammatory agent found in several different plants. Published bioanalytical methods using HPLC with ultraviolet and fluorescent detection lacked enough sensitivity and required tedious sample preparation procedures. Herein, we describe the development of a novel ultra‐high performance LC with MS/MS for determination of boldine in plasma. Boldine in plasma was recovered by liquid–liquid extraction using 1 mL of methyl tert‐butyl ether. Chromatographic separation was performed on a C₁₈ column at 45°C, with a gradient elution consisting of acetonitrile and water containing 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min. The detection was performed on an electrospray triple‐quadrupole MS/MS by positive ion multiple reaction monitoring mode. Good linearity (r² > 0.9926) was achieved in a concentration range of 2.555–2555 ng/mL with a lower limit of quantification of 2.555 ng/mL for boldine. The intra‐ and inter‐day precisions of the assay were 1.2–6.0 and 1.8–7.4% relative standard deviation with an accuracy of ?6.0–8.0% relative error. This newly developed method was successfully applied to a single low‐dose pharmacokinetic study in rats and was demonstrated to be simpler and more sensitive than the published methods, allowing boldine quantification in reduced plasma volume. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of wogonin,oroxylin a,schisandrin, paeoniflorin and emodin in rat serum by HPLC–MS/MS and application to pharmacokinetic studies

Wogonin and oroxylin A in Scutellariae Radix, schisandrin in Chinensis Fructus, paeoniflorin in Moutan Cortex and emodin in Polygoni Cuspidate Rhizome et Radix are anti‐inflammatory active compounds. A method for simultaneous determination of the five compounds in rat was developed and validated using high‐performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS). The separation was performed on a Symmetry C₁₈ column (4.6 × 50 mm, 3.5 μm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The detection was performed using multiple‐reaction monitoring with electrospray ionization source in positive–negative ion mode. The calibration curves showed good linearity (r ≥ 0.9955). The lower limit of quantification (LLOQ) was 5 ng/mL for wogonin and schisandrin, 10 ng/mL for oroxylin A and emodin, and 15 ng/mL for paeoniflorin, respectively. The relative standard deviations of intraday and interday precisions were <11.49 and 14.28%, respectively. The extraction recoveries and matrix effects were acceptable. The analytes were stable under the experiment conditions. The validated method has been successfully applied to pharmacokinetic studies of the five compounds in rats after oral administration of Hu‐gan‐kan‐kang‐yuan capsule. This paper would be a valuable reference for pharmacokinetic studies of Chinese medicine preparations containing the five compounds.     

Comparison of LC‐UV and LC–MS methods for simultaneous determination of teriflunomide,dimethyl fumarate and fampridine in human plasma: application to rat pharmacokinetic study

This study describes a comparison between LC‐UV and LC–MS method for the simultaneous analyses of a few disease‐modifying agents of multiple sclerosis. Quantitative determination of fampridine (FAM), teriflunomide (TFM) and dimethyl fumarate (DMF) was performed in human plasma with the recovery values in the range of 85–115%. A reversed‐phase high‐performance liquid chromatography (HPLC) with UV as well as MS detection is used. The method utilizes an XBridge C₁₈ silica column and a gradient elution with mobile phase consisting of ammonium formate and acetonitrile at a flow rate of 0.5 mL min^?1. The method adequately resolves FAM, TFM and DMF within a run time of 15 min. Owing to low molecular weights, the estimation of DMF and FAM is more versatile in UV than MS detection. With LC‐UV, the detection limits of FAM, TFM and DMF were 0.1, 0.05, 0.05 μg and the quantification limit for all the analytes was 1 μg. With LC–MS, the detection and quantification limits for all of the analytes were 1 and 5 ng, respectively. The two techniques were completely validated and shown to be reproducible and sensitive. They were applied to a pharmacokinetic study in rats by a single oral dose. Copyright © 2016 John Wiley & Sons, Ltd.     

Five New C19‐Diterpenoid Alkaloids from Aconitum hemsleyanum

Five new C₁₉‐diterpenoid alkaloids, named hemsleyaconitines A–E ( 1 – 5 , resp.), were isolated from Aconitum hemsleyanum Pritz. By UV, IR, MS, 1D‐ and 2D‐NMR analyses, their structures were elucidated as 18‐dehydroxygeniculatine D ( 1 ), 6‐hydroxy‐14‐O‐veratroylneoline ( 2 ), 14‐O‐acetyl‐8‐ethoxysachaconitine ( 3 ), 18‐veratroylkaracoline ( 4 ) and 8‐O‐ethylaustroconitine B ( 5 ).