Identification and Determination of the Major Constituents in the Traditional Uighur Medicinal Plant Saussurea involucrata by LC-DAD-MS

A liquid chromatography coupled with diode array detector and electrospray ionization mass spectrometry method was developed for the simultaneous analysis of the major constituents in Saussurea involucrata (SI). A comprehensive validation of the developed method was conducted, and the unique properties of the present method were confirmed by analyzing 11 SI samples. Seventeen compounds including phenolic acids, flavonoids, and lignanoids were identified by online ESI-MS and by comparison with known data in the literature and standard compounds; of these, eight were simultaneously quantified by LC-DAD. All linear regressions were acquired with R ² > 0.99, and the limits of detection ranged from 0.85 to 3.03 ng. Repeatability was evaluated by intra- and inter-day assays; the relative standard deviation (RSD) value was within 4.95%. Recovery studies for the quantified compounds were found to be within the range 97.31–101.17% with an RSD of less than 2.18%. Overall, the present hyphenation procedure is highly efficient and reliable, and hence suitable for qualitative and quantitative analysis of a large number of samples.     

Qualitative and quantitative analysis of the major constituents in Jin‐Mu‐Gan‐Mao tablet by high‐performance liquid chromatography with diode‐array detection and quadrupole time‐of‐flight tandem mass spectrometry

Jin‐Mu‐Gan‐Mao tablet is a well‐known traditional Chinese medicinal preparation, which has been used to treat the common cold in China. In this study, a systematic method was established for the qualitative and quantitative analysis of the major constituents in Jin‐Mu‐Gan‐Mao tablet. First, a method of high‐performance liquid chromatography with diode‐array detection and quadrupole time‐of‐flight mass spectrometry was developed for identification of the multi‐constituents. Thirty‐one compounds including ten phenolic acids, 18 flavonoids, and three iridoid glycosides were clearly identified by comparison with the reference standards, and 11 compounds were deduced by comparison with the literature data. Second, a new quantitative analysis method of Jin‐Mu‐Gan‐Mao tablet was established by high‐performance liquid chromatography with diode‐array detection. Twelve compounds, either with high contents or strong bioactivities, were chosen as marker components. This analytical method was validated through intra‐ and interday precision, repeatability, and stability, with respective relative standard deviations less than 1.74, 2.54, 2.44, and 2.48%. The limits of detection and quantification were less than 0.327 and 0.881 μg/mL, respectively. The overall recoveries ranged from 97.04–102.76% (relative standard deviation ≤ 2.91%). Then this validated method was applied to determine ten batches of Jin‐Mu‐Gan‐Mao tablet. The results indicated that these new approaches can be applicable for the qualitative and quantitative analysis of Jin‐Mu‐Gan‐Mao tablet.     

Qualitative and quantitative analysis of the major bioactive phenolic compounds of Glechoma longituba by LC-coupled with PAD and ESI-MS detection

A sensitive and selective method based on HPLC-PAD and Quadrupole TOF ESI-MS-MS is presented for quality control of Glechoma longituba, the major bioactive phenolic compounds of which could be quantified simultaneously. LC-ESI-MS was applied for nine fully identified compounds and another eight partially identified compounds in the plant by comparing their mass spectral data and retention times with those of selected standards and literature data. For quantitative analysis, the chromatographic conditions and extraction procedure were optimized in order to ensure the exhaustive extraction of the plant material. The calibration curves for determination of the nine compounds showed good linearity over the tested ranges (r2 > 0.999). Measurement of intra-day and inter-day variability was conducted to assess precision of the method, and their RSDs were between 2.22%-2.92% and 1.35%-2.95%. The RSDs of repeatability and stability were between 0.90%-2.53% and 1.73%-2.94%. The recoveries of the nine compounds were between 94.7%-106.5%, with RSD value ranging from 0.56%-2.88%. These validation results demonstrated the suitability of the method for the precise and accurate determination of the main constituents of G. longituba. The method was used for the quality evaluation of twenty batches of G. longituba obtained from different regions of China. The multi-index comprehensive evaluation method for the different kinds of compounds can ensure the efficacy and safety of the traditional Chinese medicine.     

Development and validation of an UHPLC-LTQ-Orbitrap MS method for non-anthocyanin flavonoids quantification in Euterpe oleracea juice

Euterpe oleracea fruits have gained much attention because of their phenolic constituents that have shown potential health benefits. The aim of this work was to quantify the major non-anthocyanin flavonoids (NAF) in the fruit juice by an accurate method coupling ultra-high pressure liquid chromatography with a linear ion trap-high resolution Orbitrap mass spectrometry system (UHPLC-LTQ-Orbitrap MS). Fruits were processed to juice, and then the juice was lyophilized and defatted. The residue was then extracted in the presence of methanol by sonication. The extraction time was optimized and recovery rates of the extraction were >90 %. The extracts were dried and solubilized again in 40 % MeOH, which showed the best compromise for MS detection. For the UHPLC quantification, a HSS C18 column (1.8 μm) was used with a gradient elution of methanol and water both with 0.1 % formic acid. Total error and accuracy profiles were used as validation criteria. Seven compounds and their isomers were successfully separated, including the major NAF. Calibration in the matrix was found to be more accurate than calibration without matrix. Trueness (<15 % relative bias), repeatability, and intermediate precision (<13 % RSD), selectivity, response function, linearity, LOD (ranged from 0.04 to 0.81 μg/mL) and LOQ (0.15–5.78 μg/mL) for 12 compounds were evaluated and the quantification method was validated. Its applicability was demonstrated on real samples from different suppliers. Their qualitative and quantitative profiles were similar and some compounds were for the first time quantified. In addition, eriodictyol was identified for the first time in this fruit along with five other flavonoids for which possible structures were proposed.     

Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q‐TOF‐MS/MS combined with UPLC/QQQ‐MS/MS

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4‐dihydroxybenzoic acid, salidroside, p‐ coumaric acid‐4‐O ‐β ‐d ‐glucopyranoside, bergeninum, 4‐hydroxybenzoic acid, 4‐hydroxyphenylacetic acid, syringate, 6′′‐O ‐galloylsalidroside, rhodiosin, rhodionin and kaempferol‐7‐O ‐α ‐l ‐rhamnoside, were simultaneously quantified by the developed ultra‐performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB‐C₁₈ column (3.0 × 100 mm, 1.8 μm) with linear gradient elution of methanol–0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R , 0.9979–0.9997), precision (RSD, 1.3–4.7%), repeatability (RSD, 1.7–4.9%), stability (RSD, 2.2–4.9%) and recovery (RSD, 0.6–4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.     

Comparative Analysis of the Major Constituents in the Traditional Tibetan Medicinal Plants Saussurea laniceps and S. medusa by LC–DAD–MS

Liquid chromatography coupled with diode array detector and electrospray ionization mass spectrometry was developed for the qualitative and quantitative comparison of the main constituents in Saussurea laniceps (SL) and S. medusa (SM), two species of plants used under the name “Xuelianhua” in traditional Tibetan medicine. A method validation including linearity, limit of detection, precision and recovery was performed. The results showed that a good linearity with R ² > 0.99 was achieved, and the limit of detection of the quantified constituents was reported to be between 0.8 and 3.3 ng. The relative standard deviation value was below 3.82% for repeatability, and recovery studies for the quantified compounds were found to be within the range 90.92–103.12%. The unique properties of the present method were evaluated by analyzing twelve related herbal samples including five S. laniceps samples and seven S. medusa samples. Twenty-two compounds including phenolic acids, cumarins, lignanoids and flavonoids were identified by online ESI–MS and by comparison with literature data and standard compounds, and seven of them were quantified by LC–DAD simultaneously. The results demonstrated that the common constituents in the two herbs were protocatechuic acid, syringoside, chlorogenic acid, isoquercitroside, 1,5-dicaffeoylquinic acid, apigenin 7-O-β-d-glucoside, chrysoeriol 7-O-β-d-glucoside, acacetin 7-O-β-d-glucoside, apigenin and chrysoeriol. In the present study, it was found that the characteristic constituents were umbelliferone, scopoletin and their glucosides in S. laniceps, as well as arctiin and arctigenin in S. medusa. It was feasible to choose these characteristic compounds for the quality evaluation as well as chemical authentication of the two related herbs. The results also support discrimination between the two species when using them in folk medicine.     

Fractionation of polyphenols in hawthorn into polymeric procyanidins, phenolic acids and flavonoids prior to high-performance liquid chromatographic analysis

Polymeric procyanidins, phenolic carboxylic acids and flavonoids of hawthorn (Crataegus laevigata) were fractionated prior to HPLC analysis using column chromatography and solid-phase extraction (SPE). The flavonoid fraction also contained (-)-epicatechin. The three groups of phenolics, each with clearly different UV spectra, were examined by means of high-performance liquid chromatography-diode array detection (HPLC-DAD) analysis. The average repeatability of the method (RSD) was in the range of 8-13% for chlorogenic acid, (-)-epicatechin and hyperoside. The polymeric procyanidins of hawthorn flowers consisted mainly of (-)-epicatechin subunits, and their mean degree of polymerization (DP) was 22.2. The HPLC methods developed can be used for the qualitative and quantitative analysis of different phenolic compounds in hawthorn plant material and their extracts.     

Qualitative and quantitative analysis of the chemical constituents in Mahuang‐Fuzi‐Xixin decoction based on high performance liquid chromatography combined with time‐of‐flight mass spectrometry and triple quadrupole mass spectrometers

High‐performance liquid chromatography coupled with time‐of‐flight mass spectrometry (HPLC‐TOF/MS) and high‐performance liquid chromatography–triple quadrupole mass spectrometry (HPLC‐QQQ/MS/MS) were utilized to clarify the chemical constituents of Mahuang‐Fuzi‐Xixin Decoction. There are 52 compounds, including alkaloids, amino acids and organic acids were identified or tentatively characterized by their characteristic high resolution mass data by HPLC‐QQQ/MS/MS. In the subsequent quantitative analysis, 10 constituents, including methyl ephedrine, aconine, songrine, fuziline, neoline, talatisamine, chasmanine, benzoylmesaconine, benzoylaconine and benzoylhypaconine were simultaneously determined by HPLC‐QQQ/MS/MS with multiple reaction monitoring mode. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r > 0.9992). The relative standard deviations (RSD) of inter‐ and intra‐day precisions were <3%. This method was also validated by repeatability, stability and recovery with RSD <3% respectively. A highly sensitive and efficient method was established for chemical constituents studying, including identification and quantification of Mahuang‐Fuzi‐Xixin decoction.     

Comparison of GC–MS and LC–MS methods for the analysis of antioxidant phenolic acids in herbs

Two methods were developed for the quantitative analysis of phenolic acids in herb extracts. The methods were based on liquid chromatography–time-of-flight mass spectrometry (LC–TOFMS) and gas chromatography–mass spectrometry (GC–MS). The methods were compared in terms of their linearity, repeatability, selectivity, sensitivity and the speed of the analysis. The sensitivity was good for both methods, with limits of detection of <80 ng/ml for most of the compounds. The relative standard deviations (RSD) of the peak areas were on average 7.2% for the LC–TOFMS method and 1.4% for the GC–MS method. Both methods were found to be suitable for the determination of the target analytes, although GC–MS was better suited to the quantitative determination of compounds present at low concentrations.     

Simultaneous quantification of five bioactive components of Acanthopanax senticosus and its extract by ultra performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

A simple and reliable ultra performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry method (UPLC-TOF-MS) was developed and validated for the simultaneous determination of the major bioactive constituents in Acanthopanax senticosus and its extract. The separation of five compounds was performed on a UPLC? HSS T3 column (100 mm × 2.1 mm, 1.7 μm) with gradient elution using a mobile phase consisting of 0.1% aqueous formic acid and acetonitrile containing 0.1% formic acid. All targeted compounds (syringin, chlorogenid acid, caffeic acid, eleutheroside E and isofraxidin) were baseline separated within 5.3 min in samples, which represented an approximate six-fold reduction in the analysis time in comparison to published HPLC method. Quantitation was carried out working in the V mode using the narrow widow extracted ion chromatograms (nwXICs) of each compound (extracted using a 20 mDa window). Furthermore, all calibration curves showed good linearity (r > 0.999) within the test ranges. The precision was evaluated by intra- and inter-day tests, which revealed relative standard deviation (RSD) values of less than 3.88%. The recoveries for the quantified compounds were between 96.3% and 103.7%, with RSD values below 2.89%. According to the literature, this study represents the first investigation of the simultaneous analysis of multiple components and the method can be applied to determine the amounts of the major compounds in Acanthopanax senticosus and its extract by UPLC-TOF-MS.     

Optimisation of ultra-performance LC conditions using response surface methodology for rapid separation and quantitative determination of phenolic compounds in Artemisia minor

A method that couples rapid, sensitive, reproducible and accurate ultra-performance LC (UPLC) with quadrupole-TOF-MS was established for the first simultaneous qualitative and quantitative analysis of phenolic compounds in Artemisia minor. Box-Behnken designs (BBDs) were applied as an effective tool to optimise major parameters that influence the resolution of UPLC, including three gradient steps and column temperature. Under optimal UPLC conditions, a total of 23 phenolic compounds in the crude methanol extracts of A. minor were well separated on a Waters Acquity UPLC BEH C(18) column (100×2.1?mm, 1.7?μm particle size) within 16.5?min, and the compounds were unequivocally or tentatively identified via comparisons with authentic standards and literature. In this study, a total of six major phenolic compounds were quantified in A. minor and the method was validated to be sensitive, precise and accurate within the LOD from 1.24 to 5.27?μg/mL, and the overall intra- and inter-day variations in detection were less than 3.76%. The recovery of the method ranged from 97.9 to 103.8% with RSDs that were less than 5.8%. These results demonstrate that this approach has the potential for quality control of A. minor and other Tibetan herbal medicines.     

HPLC‐DAD methodology for the quantification of organic acids,furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo‐diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250×4.6 mm, 5 μm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan‐3‐ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans‐resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n=5) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rosé and fortified wines.     

超高效液相色谱-高分辨质谱联用结合整合过滤策略全面分析茶树花中化学成分

茶树花与茶鲜叶同为茶树的生物产出,但茶树花往往被视为茶叶生产过程中的废物被舍弃,造成了茶树花资源的极大浪费.目前对于茶树花中化学成分的分析主要集中在氨基酸、茶多酚等单一类型化学成分上,对于茶树花中多类化学成分的同时分析仍鲜见报道.研究者对于茶树花中所含化学成分的种类和含量不完全清楚,成为制约茶树花深度开发与利用的重要原...     

Liquid chromatographic analysis of vitamin B6 in reconstituted infant formula: collaborative study

A liquid chromatographic (LC) method was validated for the determination of total vitamin B6 in infant formula. Total vitamin B6 was quantified by converting the phosphorylated and free vitamers into pyridoxine. Pyridoxine was determined by ion pair reversed-phase LC with fluorescence detection. The method was subjected to an AOAC collaborative study involving a factory-manufactured, milk- and soy-based infant formula. Each was spiked at 3 concentrations in the range of 0-1 microg/g and sent as blind duplicate to participant laboratories. Nine laboratories returned valid data which were statistically analyzed for outliers and precision parameters. The repeatability relative standard deviation (RSD(r)) ranges were 2.0-4.0 and 3.5-5.9% for fortified milk- and soy-based formulas, respectively. The reproducibility relative standard deviation (RSD(R)) ranges were 8.2-8.4 and 6.7-11.2% for fortified milk- and soy-based formulas, respectively. HORRAT values ranged from 0.42 to 0.53, indicating that the precision of the method is acceptable. The mean RSD(r):RSD(R) values were 0.60 and 0.55 for milk- and soy-based formulas, respectively. As expected, RSDs for the unfortified samples were higher, but their HORRAT values (0.81 and 2.06) helped define a realistic limit of quantitation as 0.05 microg/g. Recovery data were quantitative and varied between 81.4 and 98.0% (mean = 89.8%) for each of 6 spiked materials.     

Identification and comparative determination of rhodionin in traditional tibetan medicinal plants of fourteen Rhodiola species by high-performance liquid chromatography-photodiode array detection and electrospray ionization-mass spectrometry

Using the HPLC/PDA/ESI/MS method, a comparative analysis of rhodionin (RH) was undertaken in order to conduct a qualitative and quantitative study in 38 batches of fourteen species of Rhodiola for quality control purposes. Alongside of this RH analysis, a simultaneous determination of salidroside (SA), tyrosol (TY), and gallic acid (GA) was carried out. Rhodiola plants are a popularly used ethnodrug from the Qinghai-Tibetan plateau of China. The identity of RH was unambiguously determined based on the quasimolecular ions in negative ESI-MS mode. This method was validated in respect to sensitivity, linearity, precision, repeatability and recovery using optimized chromatographic conditions. The linear calibration curve was acquired with R(2)>0.999, and the limit of detection (S/N=3) was estimated to be 43.75 microg/g. The relative standard deviations (RSDs) of the intra- and inter-day precisions were 0.75% and 0.50%, respectively. The repeatability was evaluated by a replicated analysis of samples with the RSD value found within 0.67%. The recovery rates varied within the range of 98.79-100.08% with RSD less than 1.10%. In the present study, the content of RH was quantified within 0.4192-4.7260 mg/g for 16 batches of R. crenulata. It was also found in eight other species plants. The results demonstrated that RH is a useful characteristic standard compound for quality evaluation and chemical differentiation among species of Rhodiola. The study also indicated that the analytical procedure is precise, reproducible and a potential tool for both quality assessment and species identification.     

Rapid quantitative analysis of 18 chemical constituents in HuangQi JianZhong Tang based on UHPLC–MS

A rapid, highly sensitive, and specific analytical method using ultra-high-performance liquid chromatography-triple quadruple MS was developed to quantitatively measure 18 chemical constituents in Huangqi Jianzhong Tang (HQJZ), a famous traditional Chinese medicine formula. Chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) with a gradient mobile phase (A: 0.1% aqueous formic acid and B: acetonitrile) at a flow rate of 0.25 mL/min. The chromatographic peaks of 18 components were identified by comparing with the reference compounds. Multiple reaction monitoring was employed for quantitative analysis of the components. Seven batches of HQJZ samples were analyzed with a good linear regression relationship (R², .9978–.9993), precisions [relative standard deviation (RSD), 0.90%–3.60%], repeatability (RSD, 2.50%–4.00%), stability (RSD, 1.00%–4.00%), and recovery (96.10%–104.30%). Based on this established method, the present study offered a highly sensitive, specific, and rapid determination method for identification of 18 compounds, which greatly promoted the systemic quality control of HQJZ.     

Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high‐performance liquid chromatography with diode array detection and tandem mass spectrometry

The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High‐performance liquid chromatography coupled with time‐of‐flight mass spectrometry and high‐performance liquid chromatography with electrospray multistage tandem ion‐trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p‐coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high‐performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system.     

Determination of pesticides in olives by gas chromatography using different detection systems

A multiresidue method has been developed and optimized for the quantitative analysis of 32 pesticides in olives. The extraction was based on homogenization with light petroleum using a high speed homogenizer. A gel permeation chromatography (GPC) clean-up process with ethyl acetate/cyclohexane (1:1) as mobile phase was applied to the extracts to separate the low-molecular mass pesticides from the high-molecular mass fat constituents of the oil. The target compounds were quantified in the final extract by gas chromatography (GC) using thermoionic specific detection (TSD) and electron-capture detection (ECD). In the case of positive samples, the amounts found were confirmed by GC-MS/MS. The obtained recovery (with mean values between 70 and 121, 71 and 114, and 82 and 134% for ECD, TSD and MS/MS systems, respectively) and RSD values (repeatability, n=10) below 16% in all cases confirm the usefulness of the proposed method for the analysis of this complex sample. Diuron, terbuthylazine and endosulfan sulfate were the most frequently detected residues in olive samples collected during the harvest 2004-2005. Finally, in order to know the proportion of pesticides that are transferred to the oil during olive oil production in olive mills, obtained results in some of the sampled olives applying the proposed method were compared to levels found in the corresponding olive oil, which was obtained by means of the Abencor method.     

Fast separation and determination of carnosic acid and rosmarinic acid in different rosemary (Rosmarinus officinalis) extracts by capillary zone electrophoresis with ultra violet-diode array detection

Summary Carnosol, carnosic acid, rosmarinic acid and other not identified phenolic compounds were separated by capillary zone electrophoresis (CZE) using a 40-cm long capillary and a 20 mM tetraborate buffer (pH 9.0), within 3 min. A UV-diode array detector was employed to collect spectra of phenolic compounds. The effect of some separation parameters on peak resolution and migration time of phenolic species present in a refined rosemary extract was studied. The repeatability of the method was also investigated: the intraday relative standard deviation on total peak area was less than 4%, while the intraday relative standard deviation on migration time was less than 0.6%. Moreover the CZE method showed good sensitivity (0.0007 μg mL¹ for carnosic acid and rosmarinic acid). Carnosic acid and rosmarinic acid have been quantified in different commercial extracts of rosemary. Finally, the optimized method was also applied to evaluate the recovery of these two compounds when different organic solvents were employed during the extraction procedure.