Ultra‐fast LC–ESI‐MS/MS method for the simultaneous determination of six highly toxic Aconitum alkaloids from Aconiti kusnezoffii radix in rat plasma and its application to a pharmacokinetic study

A fast, sensitive, and efficient ultra‐fast LC–ESI‐MS/MS method was developed for the simultaneous quantitation of six highly toxic Aconitum alkaloids, that is, aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, in rat plasma after oral administration of crude ethanol extracts from Aconiti kusnezoffii radix by ultrasonic extraction, reflux extraction for 1 h, and reflux extraction for 3 h, respectively. The separation of six Aconitum alkaloids and aminopyrine (internal standard) was performed on an InertSustain® C₁₈ column, and the quantification of the analytes was performed on a 4000Q ultra‐fast LC–MS/MS system with turbo ion spray source in the positive ion and multiple‐reaction monitoring mode. Absolute recoveries ranged within 65.06–85.1% for plasma samples. The intra‐ and interday precision and accuracy of analytes were satisfactory. The methods were validated with sensitivity reaching the lower LOQ for aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine, which were 0.025, 0.025, 0.050, 0.025, 0.025, and 0.100 ng/mL, respectively. The method was successfully applied to a pharmacokinetic study of six Aconitum alkaloids in rat plasma after oral administration of crude ethanol extracts from the raw root of Aconitum kusnezoffii Reichb. by three different extraction processes.     

Targeted profiling and relative quantification of benzoyl diterpene alkaloids in Aconitum roots by using LC–MS/MS with precursor ion scan

Benzoyl aconite alkaloids have myocardial protective effects at a low dose and produce toxic effects at high dose. Due to lack of enough reference compounds, most of the benzoyl alkaloids had few concerns, except the typical ones, i.e. aconitine, mesaconitine, and hypaconitine. To rapidly screen out and quantify benzoyl alkaloids, a high performance liquid chromatography combined with tandem mass spectrometry was proposed based on precursor ion scanning mode. First, a diagnostic ion at m/z 105 corresponding to benzoyl group was observed by using tandem mass spectrometry, which could be used for the rapid identification of benzoyl alkaloids. The targeted screening of these alkaloids was then conducted by using precursor ion scan of characteristic ion at m/z 105. Shengfuzi (the lateral root of A. carmichaelii) was taken as example, and 24 benzoyl‐containing alkaloids were identified. The six major alkaloids including aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine were determined in the precursor ion scan mode by the standard curve method. Reliable linearity, sensitivity, precision, accuracy, and repeatability were obtained and validated. Then the relative response factors between these six analytes were calculated, which were not more than two times using any alkaloid as reference. Thus, the other 18 alkaloids lacking reference compounds were relatively quantified. This approach provides a useful tool for rapid identification and quantitative analysis of toxic benzoyl alkaloids, and also an efficient method for the safety assessment of Aconitum roots.     

The comparative research on constituents of Radix Aconiti and its processing by HPLC quadrupole TOF-MS

Based upon the regulations stipulated by the State Food and Drug Administration of China, only the processed, detoxified tubers and roots of Aconitum are allowed to be administered orally, used in clinical decoctions and adopted as raw materials for pharmaceutical manufacturing, so the processing principle of preparation of Radix Aconiti is important for ensuring the Radix Aconiti praeparata quality. A simple approach was described for HPLC‐Q‐TOF‐MS screening and identification of many of the aconitine alkaloids present in unprocessed Radix Aconiti and Radix Aconiti praeparata. To compare their fingerprints, the processing principle of preparation of Radix Aconiti was developed. Twenty‐nine compounds and 26 compounds were assigned to aconitine alkaloids and tentatively identified by comparing accurate mass and fragments information with that of the authentic standards or by mass spectrometry analysis and retrieving the reference literature. The nonester alkaloids were almost the same. The diester diterpene alkaloids were decreased, the monoester‐diterpene alkaloids were increased and lipo‐alkaloids decreased obviously in the processing of the preparation. These transformed components could be regarded as potential chemical markers that can be used to distinguish between raw and processed herbs. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification and Determination of <em>Aconitum</em> Alkaloids in <em>Aconitum</em> Herbs and <em>Xiaohuoluo Pill</em> Using UPLC-ESI-MS

A rapid, specific, and sensitive ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) method to examine the chemical differences between Aconitum herbs and processed products has been developed and validated. Combined with chemometrics analysis of principal component analysis (PCA) and orthogonal projection to latent structural discriminate analysis, diester-diterpenoid and monoester-type alkaloids, especially the five alkaloids which contributed to the chemical distinction between Aconitum herbs and processed products, namely mesaconitine (MA), aconitine (AC), hypaconitine (HA), benzoylmesaconitine (BMA), and benzoylhypaconitine (BHA), were picked out. Further, the five alkaloids and benzoylaconitine (BAC) have been simultaneously determined in the Xiaohuoluo pill. Chromatographic separations were achieved on a C₁₈ column and peaks were detected by mass spectrometry in positive ion mode and selected ion recording (SIR) mode. In quantitative analysis, the six alkaloids showed good regression, (r) > 0.9984, within the test ranges. The lower limit quantifications (LLOQs) for MA, AC, HA, BMA, BAC, and BHA were 1.41, 1.20, 1.92, 4.28, 1.99 and 2.02 ng·mL^-1, respectively. Recoveries ranged from 99.7% to 101.7%. The validated method was applied successfully in the analysis of the six alkaloids from different samples, in which significant variations were revealed. Results indicated that the developed assay can be used as an appropriate quality control assay for Xiaohuoluo pill and other herbal preparations containing Aconitum roots.     

Method for the determination of Aconitum alkaloids in dietary supplements and raw materials by reversed-phase liquid chromatography with ultraviolet detection and confirmation by tandem mass spectrometry: single-laboratory validation

A study of single-laboratory validation (SLV) of a reversed-phase liquid chromatography (RP-LC) method was conducted for the determination of diester-diterpene Aconitum alkaloids, viz., aconitine, mesaconitine, and hypaconitine, in a variety of dietary supplements, including single- and multiple-ingredient dry powder extracts, pills, capsules, and raw materials. The Aconitum alkaloids in the samples were extracted by diethyl ether in the presence of ammonia. After cleanup with solid-phase extraction to remove the matrix interferences, the alkaloids were determined by RP-LC with UV detection at 235 nm, and the results were confirmed by tandem mass spectrometry. The linear responses for aconitine, mesaconitine, and hypaconitine based on the present LC system ranged from 0.5 to 200 microg/mL. Relative standard deviations of 2.0 to 6.9% were obtained from duplicate analysis of 6 test materials of different matrixes for the 3 Aconitum alkaloids performed by 2 analysts on 5 different days. The recoveries determined for supplements and raw materials spiked with 3 Aconitum alkaloids at levels of 2.5-10 microg/g were in the range of 86-99%. In view of the attainment of satisfactory results for accuracy, precision, and recovery in the SLV study, it is recommended that the method validation process proceed to a collaborative study.     

利用液质联用技术分析干姜对乌头类生物碱在大鼠肠内吸收的影响

为从吸收的角度考察干姜对乌头类双酯型生物碱的解毒机理, 采用外翻肠囊法展开实验. 利用超高液相与三重四极杆质谱联用技术定量检测双酯型生物碱成分, 采用标准曲线法计算乌头碱、中乌头碱、次乌头碱在肠囊内吸收的绝对含量, 采用质谱峰面积直接分析其它双酯型生物碱的相对变化, 结果加入干姜提取液后, 乌头碱、中乌头碱、次乌头碱的单位肠管面积累计吸收量均降低, 10-羟基中乌头碱的的累积峰面积降低; 加入维拉帕米后, 双酯型生物碱的单位肠管面积累计吸收量及累积峰面积均增加; 向含有地高辛的肠营养液中加入干姜提取液后, 地高辛在各实验时间点的单位肠管面积累计吸收量均降低, 根据以上结果推测干姜抑制乌头类双酯型生物碱在大鼠肠囊内吸收的可能机制是通过诱导肠内P-葡糖蛋白, 从而抑制作为P-葡糖蛋白底物的双酯型生物碱的吸收, 最终起到减毒作用.     

Analysis of the aconitine alkaloids in traditional Chinese medicines by nonaqueous capillary electrophoresis using a new recording mode

The determination of three aconitine alkaloids (hypaconitine, aconitine, mesaconitine) in five traditional Chinese medicines including two Tibetan medicines, Chuanwu, Caowu, Fuzi, Aconitum Tanguticum Maxim and Aconitum Gymnandrum Maxim by non-aqueous capillary electrophoresis using a new recording mode is described. The dissociation constants of aconitine, mesaconitine and hypaconitine have also been determined by CZE and were 7.71, 6.60 and 6.25, respectively. The separation was achieved by optimizing the applied voltage, the pH and the concentration of the buffer. The electrophoretic medium was 20 mM borax-70% (v/v) methanol (pH 8.5) and an uncoated capillary (50 cm x 75 microm i.d.) was used. Detection was carried out with a UV monitor at 214 nm. The total time for separation and determination was under 13 min.     

离心超滤质谱筛选川乌中与人血清白蛋白相互作用的乌头类生物碱

采用离心超滤质谱技术从川乌总生物碱提取物中筛选与人血清白蛋白相互作用的乌头类生物碱成分,并用LC-MSn技术对筛选出的活性成分进行了分离鉴定.结果表明,从川乌总生物碱中筛选并鉴定出9种与人血清白蛋白存在相互作用的乌头类生物碱:苯甲酰新乌头原碱、苯甲酰乌头原碱、10-OH-中乌头碱、中乌头碱、10-OH-乌头碱、乌头碱、次乌头碱、脱氧乌头碱和3,13-脱氧乌头碱.     

HPLC测定植物性农药——0.25%乌头总碱乳油中的乌头类生物碱

 选择十八烷基键合相柱 ,以甲醇 水 氯仿 三乙胺 (体积比为 6 8∶32∶2∶0 1)混合溶液为流动相 ,用高效液相法测定了一种植物性农药 0 2 5 %乌头总碱乳油中的乌头生物碱。实验结果表明中乌头碱、乌头碱及次乌头碱与其他杂质能够得到很好的分离。以安宫黄体酮作内标物 ,用峰面积比测定各生物碱含量 ,在其线性范围内分析结果准确 ,回收率高 (>92 % ) ,重现性好 (RSD <3 2 % ) 。     

Microcalorimetric investigation of six alkaloids from Radix Aconite Lateralis Preparata (Fuzi) on the metabolic activity of mitochondria isolated from rat liver

Using the TAM air isothermal microcalorimeter, the thermogenic power–time (HP–time) curves of isolated mitochondrial energy metabolism were measured, and the effects of six alkaloids from Radix Aconite Lateralis Preparata (Fuzi) including mesaconitine, aconitine, hypaconitine, benzoylmesaconine, benzoylaconine, and benzoylhypaconine on mitochondrial energy metabolism were investigated. Some quantitative information, such as k, P _max, T _max, Q, P _av, and T _lag, was obtained from the HP–time curves. The results revealed that mesaconitine, aconitine, and hypaconitine had inhibitory effect on rat-isolated mitochondrial energy metabolism, and their IC₅₀ value was 1.82, 2.22, and 2.48 μg mL⁻¹, respectively, while benzoylmesaconine, benzoylaconine, and benzoylhypaconine promoted mitochondrial energy metabolism, and their EC₅₀ value was 30.95, 14.36, and more than 30 μg mL⁻¹, respectively. Further structure–activity relationship analysis showed that the ester bond in C(8) position, hydroxyl in C(3) position, and ethyl in N atom could significantly influence the energy metabolism. These results provide scientific basis for the treatment of mitochondrial abnormalities and energy dysfunction diseases using Fuzi, and it will be of great interest to doctors and pharmacists.

     

Qualitative and quantitative analysis of aconitine-type and lipo-alkaloids of Aconitum carmichaelii roots

By optimizing the extraction and analytical conditions, a reliable and precise HPLC method coupled with photodiode array detection (HPLC–DAD) has been developed for the identification and quantification of three major aconitine-type alkaloids (aconitine, mesaconitine, hypaconitine) in the roots of Aconitum carmichaelii Debeaux. The qualitative analysis of the plant material was carried out by LC-APCI-MSⁿ. By means of this method, 26 lipo-alkaloids were also identified from the roots of A. carmichaelii. The effect of processing on aconitine-type alkaloids, lipo-alkaloids and pure aconitine was studied. As part of our investigation, two lipo-alkaloids, 14-benzoylaconine-8-palmitate and 14-benzoylaconine-8-linoleate were produced semisynthetically. The COX-1, COX-2 and LTB₄ formation inhibitory activity of aconite root extracts and different types of diterpene alkaloids and the toxicity of lipo-alkaloids were also investigated.     

Comparative study on effects of single and multiple oral administration of mungbean (Phaseolus radiatus L.) seed extract on the pharmacokinetics of aconitine by UHPLC‐MS

The study was aimed to investigate the effects of single and multiple oral administration of mungbean (Phaseolus radiatus L.) seed extract (ME) on the pharmacokinetics of aconitine in rats. The Sprague–Dawley rats were randomly divided into three groups (six rats each group). In group 1, rats were orally administered 500 µg/kg aconitine after receiving a single oral dose of 1 g/kg ME. In group 2, rats were orally administered with 500 µg/kg aconitine at day 7 of treatment with 1 g/kg/day ME. In group 3, rats were orally administered with 500 µg/kg aconitine. Blood samples were collected at different time points (0.083, 0.25, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0 and 10.0 h). The concentration of aconitine in rats plasma was determined by a fully validated ultra‐high‐performance liquid chromatography coupled with mass spectrometry method. The results showed that single and multiple oral co‐administration of ME significantly altered the pharmacokinetic parameters of aconitine. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Matrix Solid-phase Dispersion Extraction of Alkaloids from the Roots of Aconitum kusnezoffii Reichb

Matrix solid-phase dispersion(MSPD) was developed for the extraction of four alkaloids, including aconitine, mesaconitine, hypaconitine and deoxyaconitine, from the roots of Aconitum kusnezoffii Reichb. The determination of the analyte was carried out by high performance liquid chromatography with UV detection. The alkaline alumina was used as sorbent. The mixture of acetonitrile and water was used as elution solvent. Several extraction parameters, such as type of sorbent, the ratio of sample to solid suppo...     

Enhancement by Glycyrrhizae Radix of hepatic metabolism of hypaconitine,a major bioactive and toxic component of Aconiti Laterlis Radix,evaluated by HPLC‐TQ‐MS/MS analysis

Glycyrrhizae Radix (GR) is often prescribed together with Aconiti Laterlis Radix (ALR) (a so‐called compatible drug pair) in traditional Chinese medicinal practice to reduce toxicity of ALR. However, the mechanisms involved remain to be addressed. In this study, the metabolic interactions between GR–ALR drug pair were investigated for the first time. First, an HPLC‐TQ‐MS/MS method was developed to analyze hypaconitine, a major bioactive and toxic component of ALR, in rat liver S9. Then the in vitro metabolic rates of hypaconitine by different rat liver S9 were compared using the established method. The experiments were designed in four groups: pure hypaconitine (group I) and ALR extract (group II) incubated with liver S9 of normal rats, and pure hypaconitine (group III) and ALR extract (group IV) incubated with liver S9 of GR‐pretreated rats. When incubated for more than 4 h, the metabolic rates of hypaconitine in group III were significantly higher than those in group I, and when incubated for more than 2 h, the metabolic rates of hypaconitine in group IV were significantly higher than those in group II, suggesting that GR can enhance metabolic rate of hypaconitine, the mechanism of which might be related to hepatic metabolizing enzyme induction by GR. Copyright © 2012 John Wiley & Sons, Ltd.     

液相色谱/质谱/质谱联用法测定乌头属药材及中成药中乌头类生物硷含量

探讨了用LC／MS／MS联用法测定乌头属药材及有关中成药中乌头硷、次乌头硷和中乌头硷含量的方法。应用优化后的技术条件及采用多反应监测扫描方式,结果表明分析样品中各乌头硷浓度在0．5－40mg/L范围内,色谱峰面积与浓度有良好的线性关系。此法亦应用于14个不同的乌头属药材及10个不同的中成药样品加的乌头硷含量检测。     

电喷雾串联质谱分析附子炮制中的化学成分变化

利用电喷雾质谱方法(ESI-MS)分析了附子加辅料(甘草)炮制前后水煎液中二萜类生物碱在种类和含量方面的变化,通过加入内标化合物,建立了电喷雾质谱的半定量分析方法。此方法具有快速、准确、灵敏的特点,能够更加全面地反映中药配伍炮制过程中多种化学成分的含量变化,并能根据电喷雾串联质谱的分析结果鉴定配伍后产生的新的化学成分,在共煎液中的次乌头碱、中乌头碱和乌头碱的相对含量分别是单煎液中的5.67%、4.05%和4.88%。通过研究附子与甘草的单煎液、共煎液以及药渣中化学成分的变化,揭示了甘草作为辅料,在炮制过程中对附子减毒作用机理。     

Alkaloid profiling of the Chinese herbal medicine Fuzi by combination of matrix-assisted laser desorption ionization mass spectrometry with liquid chromatography–mass spectrometry

A matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) method was developed for the high throughput and robust qualitative profiling of alkaloids in Fuzi—the processed lateral roots of the Chinese herbal medicine Aconitum carmichaeli Debx (A. carmichaeli). After optimization, powdered roots – without any further sample preparation – could be used to screen for the presence of Aconitum alkaloids. Furthermore, the semi-quantitative potential of MALDI-MS was confirmed using liquid chromatography–mass spectrometry (LC–MS) as reference. In total over sixty alkaloids were detected by LC–MS and fifteen of them were tentatively identified. Both MALDI-MS and LC–MS analysis revealed significant variation in alkaloid content in different (commercial) samples. LC–MS analysis of three toxic alkaloids in 14 batches of Fuzi resulted in a variation of their concentrations expressed as RSDs of 138%, 99% and 221% for aconitine, hypaconitine and mesaconitine, respectively. The variation in concentrations (expressed as RSD) of about the ninety constituents detected were classified as follows: 13 constituents showed an RSD of 77–100%, 46 with an RSD of 100–150%, 21 with an RSD of 150–200% and 9 constituents with an RSD in concentration of 200–235%. These results demonstrate a strong difference in chemical composition of the various Fuzi and illustrate the necessity of adequate QA/QC procedures for both safety and efficiency of herbal medicine. The described analytical procedures for alkaloid profiling could play a role in these procedures.     

Separation and determination of aconitine alkaloids in traditional Chinese herbs by nonaqueous capillary electrophoresis

An easy, rapid, and simple nonaqueous capillary electrophoresis (NACE) method was developed for the identification and determination of three aconitine alkaloids, hypaconitine (HN), aconitine (AN), and mesaconitine (MN) within 6 min. The most suitable running buffer was composed of 60 mM ammonium acetate, 0.5% acetic acid, and 15% acetonitrile (ACN) in methanol with a fused-silica capillary column (50 cm x 75 microm ID). In the concentration range 12.5-1000 mg/L the calibration curves reveal linear relationships between the peak area for each analyte and its concentration (correlation coefficients: 0.9997 for HN, 0.9999 for AN, and 0.9995 for MN). The relative standard deviations of the migration time and peak area of the three alkaloids were 0.13, 0.57, 0.33 and 2.87, 1.06, 3.49%, respectively. The method was successfully applied to determine the three alkaloids in two commonly used traditional Chinese herbal medicines, the recoveries of the three constituents ranging between 94.7-101.9% for HN, 98.3-102.3% for AN, and 98.1-104.6% for MN.     

Metabolomics study on Fuzi and its processed products using ultra-performance liquid-chromatography/electrospray-ionization synapt high-definition mass spectrometry coupled with pattern recognition analysis

The lateral root of Aconitum carmichaelii Debx is named "Fuzi" which is widely distributed across Asia and North America and has been used to relieve joint pain and treat rheumatic diseases for over two thousand years. However, it has very narrow therapeutic ranges and despite the toxicological risk, its usage remains very high. A traditional Chinese processing approach (Paozhi, detoxifying measure) is necessary to remove the poisonous Aconitum alkaloids mainly deriving from the diester diterpene alkaloids (DDAs) including aconitine, mesaconitine and hypaconitine. They can be decomposed into less or non-toxic derivatives through Paozhi that plays an essential role in detoxification. Processed Fuzi is mainly focused on the three main forms of Yanfuzi (YFZ), Heishunpian (HSP) and Baifupian (BFP) which are highly desirable in order to guarantee the clinical safety and their low toxicity in decoctions. The difference in metabolomic characters between Fuzi and its processed preparations is still completely unclear. Therefore, this paper was designed to investigate a comprehensive metabolome of Fuzi and its processed products by ultra-performance liquid-chromatography/electrospray-ionization synapt high-definition mass spectrometry (UPLC-Q-TOF-HDMS) combined with pattern recognition methods. The difference in metabolic profiles between Fuzi and its processed preparations was well observed by the principal component analysis (PCA) of the MS spectra. Significant changes of 19 metabolite biomarkers were detected in the Fuzi samples and three preparations. The underlying regulations of Paozhi-perturbed metabolic pathways were also discussed according to the identified metabolites. The present study proves that UPLC-Q-TOF-HDMS based metabolomic analysis greatly contributes to the investigation of Fuzi metabolism through Paozhi techniques, and provides useful information to further comprehensively understand the pharmacological activity and potential toxicity of processed Fuzi in a clinical environment.