An ultrafiltration high-performance liquid chromatography coupled with diode array detector and mass spectrometry approach for screening and characterising tyrosinase inhibitors from mulberry leaves

Tyrosinase is a key enzyme in melanin synthesis. Its inhibitor may be used to efficiently treat hyperpigmentation and widely applied in cosmetic products and food supplements. In the present study, a new assay based on ultrafiltration high-performance liquid chromatography coupled with diode array detector and mass spectrometry (HPLC–DAD–MS) was developed for the rapid screening and identification of ligands for tyrosinase. Experiments were carried out to select the optimal binding conditions, tyrosinase concentration, and incubation time. Non-specific binding to the denatured tyrosinase was also investigated. Twelve compounds with tyrosinase binding activity were found in mulberry leaf extracts. The identities of these compounds were characterised by HPLC–DAD–MSⁿ. Particularly, two compounds, namely, quercetin-3-O-(6-O-malonyl)-β-d-glucopyranoside and kaempferol-3-O-(6-O-malonyl)-β-d-glucopyranoside, were identified as new tyrosinase inhibitors. The screening results were verified by tyrosinase inhibition assays. Experimental results proved that the proposed method could rapidly screen tyrosinase inhibitors in complex mixtures.     

Qualitative and quantitative determination of polyacetylenes in different Bupleurum species by high performance liquid chromatography with diode array detector and mass spectrometry

Polyacetylenes are main toxic ingredients in Bupleurum longiradiatum, a poisonous plant that has ever been misused as substitutes for Chaihu (Bupleuri Radix). For the first time, a high-performance liquid chromatography method coupled with diode array detector and mass spectrometry (HPLC-DAD-MS) was developed for qualitative and quantitative analysis of nine polyacetylenes in Bupleurum species. All references, including two new polyacetylenes, were isolated from B. longiradiatum and purified using a semi-preparation HPLC chromatography. The analysis was performed on a TSKgel ODS-100V C18 column (3 μm, 150 mm x 4.6 mm i.d.) using a gradient system of acetonitrile and water, with diode array detection (254 nm). The method was validated for linearity, precision, accuracy, limit of detection and quantification. A total of 27 Bupleurum samples were examined with this method, which showed a great variety in the distribution and contents of the polyacetylenes. It was found that polyacetylenes (1-8) were the main ingredients in B. longiradiatum, while a few kinds of polyacetylenes (5-8) were also identified in B. smithii, B. smithii var. parvifolium, B. bicaule and B. angustissimum. However, no polyacetylenes (1-9) were detected in the authentic Chaihu samples and the other Bupleurum species. The results indicated that the toxic B. longiradiatum could readily be distinguished from other medicinal Bupleurum species based on the polyacetylene profiles, and the guidelines and quality control of polyacetylenes for Chaihu are useful. The acute toxicity of the ethanol extract of B. longiradiatum and its fractions was also investigated.     

Direct identification of phenolic constituents in Boldo Folium (Peumus boldus Mol.) infusions by high-performance liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry

A very simple and direct method was developed for the qualitative analysis of polyphenols in boldo (Peumus boldus Mol., Monimiaceae) leaves infusions by high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (HPLC-MSⁿ). The phenolic constituents identified in infusions of the crude drug Boldo Folium were mainly proanthocyanidins and flavonol glycosides. In the infusions, 41 compounds were detected in male and 43 compounds in female leaf samples, respectively. Nine quercetin glycosides, eight kaempferol derivatives, nine isorhamnetin glycosides, three phenolic acids, one caffeoylquinic acid glycoside and twenty one proanthocyanidins were identified by HPLC-DAD and ESI-MS for the first time in the crude drug. Isorhamnetin glucosyl-di-rhamnoside was the most abundant flavonol glycoside in the male boldo sample, whereas isorhamnetin di-glucosyl-di-rhamnoside was the main phenolic compound in female boldo leaves infusion. The results suggest that the medicinal properties reported for this popular infusion should be attributed not only to the presence of catechin and boldine but also to several phenolic compounds with known antioxidant activity. The HPLC fingerprint obtained can be useful in the authentication of the crude drug Boldo Folium as well as for qualitative analysis and differentiation of plant populations in the tree distribution range.     

Optimization for detection of flunarizine by high performance liquid chromatography/electrospray/mass spectrometry

This paper describes a newly developed high performance liquid chromatography/electrospray/mass spectrometry (HPLC/ES/MS) method for the determination of flunarizine (FZ) in artificial cerebrospinal fluid. The optimization for the detection of FZ in biological fluid by LC/ES/MS was investigated. The effects of solvent composition, the addition of modifier and flow rate on the detection of FZ by ES/MS were examined. The detection limit of this method (0.8 nM) proved to be much better than previously reported methods. Satisfactory accuracy (98.2–106.0%) of this newly developed method was obtained. The application of this method was demonstrated by analyzing FZ in rat microdialysis samples.     

高效液相色谱-电喷雾串联质谱法测定饲料中的N-氨基甲酰-L-谷氨酸

建立了高效液相色谱-电喷雾串联质谱(HPLC-ESI-MS/MS)测定饲料中N-氨基甲酰-L-谷氨酸(NCG)含量的方法。饲料样品经甲醇提取、混合型强阴离子交换反相固相萃取(PXA)柱净化、HPLC分离后,采用ESI-MS/MS在正离子多反应监测(MRM)模式下进行检测,以碎片离子m/z 148.0和m/z 84.0进行定性,以碎片离子m/z 130.0进行定量。NCG的检出限(S/N >3)为24 μg/kg,定量限(S/N >10)为80 μg/kg,在20~1000 μg/L的质量浓度范围内峰面积与含量的线性关系良好,相关系数为0.9999。对饲料中NCG在80、200、500 mg/kg等3个添加水平下的回收率进行了测定,分别为104.0%、103.5%、95.3%,相对标准偏差分别为7.5%、6.3%、5.8%。结果表明,该方法操作简单,净化效果好,快速,灵敏度和准确度高,符合对饲料样品中NCG检测分析的要求。     

Qualitative and quantitative determination of complicated herbal components by liquid chromatography hybrid ion trap time-of-flight mass spectrometry and a relative exposure approach to herbal pharmacokinetics independent of standards

To date, the pharmacokinetic research of herbal medicines (HMs) is still in its infancy and is facing critical technical challenges on the qualitative and quantitative analysis of complicated components from biological matrices. Additionally, the lack of authentic standards constitutes another bottleneck on assessing herbal pharmacokinetics. This present work contributes to the development of a powerful technical platform for both qualitative and quantitative pharmacokinetic analysis of herbal components, and a strategy of relative exposure that provides a practicable pharmacokinetic assessment independent of authentic standards, based on the use of liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LC–IT-TOF/MS). Taking schisandra lignans extract (SLE) as an example, the LC–IT-TOF/MS assay was initially applied to the global qualitative analysis of components contained in SLE per se and in the rat plasma post SLE dosing. Afterwards, this study focused on validating the quantitative performance of LC–IT-TOF/MS assay by comparison with a well-established LC–Q/MS assay. For the absolute quantification of five lignans components with authentic standards, both assays showed very similar analytical figures of merit such as linearity, precision, accuracy, and pharmacokinetic parameters. Compared with LC–Q/MS, the prominent advantage of LC–IT-TOF/MS assay is its much higher sensitivity. Moreover, a ‘relative exposure approach’ (REA) that entails the use of sequentially diluted original herbal preparations to prepare the ‘mixed calibration curves’ was developed to assessing herbal pharmacokinetics independent of specific authentic compounds for each component. Such an approach was found capable of providing virtually identical pharmacokinetic parameters as that from the typical pharmacokinetic assay calibrated by authentic standards, except for the absolute plasma concentrations. The presently developed methodology and approach will find its wide use in, but not limited to, the qualitative and quantitative pharmacokinetic analysis of herbal medicines.     

Qualitative and quantitative determination of the major coumarins in Zushima by high performance liquid chromatography with diode array detector and mass spectrometry

A method, high-performance liquid chromatography coupled with diode array and electrospray tandem mass spectrometry (HPLC-DAD-ESI-MS), was developed to qualitatively identify and quantitatively determine the 10 major active coumarins of Zushima. The analysis was performed by using a ZORBAX SB-C18 analytical column (250 mm x 4.6 mm ID, 5 microm) at gradient elution of 0.5% aqueous formic acid and acetonitrile with diode array detection (325 nm). The method was validated for linearity, precision, accuracy, limit of detection and quantification. The proposed method was successfully applied for the qualitative and quantitative analysis of 10 coumarins in five different species of Zushima which had great variation on the contents of investigated coumarins.     

Qualitative and quantitative analysis of diterpenoids in Salvia species by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

An on-line ultra-high-performance liquid chromatography (UHPLC) coupled with photodiode-array detection and quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) method has been optimized and established for the qualitative and quantitative analysis of the important diterpenoids in the methanol extracts of 12 Salvia species. Specific marker components were identified for the classification of the Salvia samples by principal component analysis. The accurate mass measurement within 3 ppm error for all the protonated molecules and subsequent fragment ions offers higher quality structural information for interpretation of fragmentation pathways of various groups of diterpenoids. Thus, a total of 21 diterpenoids from different Salvia species were separated within 10 min, and were unequivocally or tentatively identified via comparisons with authentic standards and literature. This UHPLC-QTOF-MS/MS method was validated to be sensitive, precise and accurate with the limit of detections at 3.0–16 ng/ml, and the overall intra-day and the inter-day variations less than 3%. The recovery of the method was in the range of 96.2–101.8%, with relative standard deviation (RSD) less than 3.0%. The results demonstrated that the qualitative and quantitative differences in diterpenoids were not only useful for chemotaxonomy in some Salvia species but also for the standardization and differentiation of large numbers of similar samples.     

Analysis of alkaloids in Macleaya cordata (Willd.) R. Br. using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry

A method for the analysis of alkaloids in Macleaya cordata (Willd.) R. Br. using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS) was developed. Using protopine (PRO), allocryptopine (ALL), sanguinarine (SA), and chelerythrine (CHE) as the model components, different columns for the separation and different mobile phases for the signal intensities of alkaloids in ESI/MS were investigated, respectively. The results showed that good separation and high signal intensities can be obtained on a high carbon loading (17%) reversed-phase C(18) column with 30 mM formic acid in mobile phase for the analysis of alkaloids. Under the optimal separation condition and UV detection (284 nm), linearity of the six alkaloids was obtained over concentration range from 0.05 to 100.00 microg/ml. The limit of detection (LOD) was 1.62, 1.87, 1.79, 1.76, 1.10, and 0.94 ng/ml for SA, CHE, PRO, ALL, dihydrosanguinarine (DHSA), and dihydrochelerythrine (DHCHE), respectively. The LODs with ESI/MS detection were lower three orders of magnitude than those obtained with UV detection. The proposed method could be used to control quality of the raw materials of the herb more comprehensively.     

Characterization and identification of isomeric flavonoid O-diglycosides from genus Citrus in negative electrospray ionization by ion trap mass spectrometry and time-of-flight mass spectrometry

Flavonoid O-diglycosides are important bioactive compounds from genus Citrus. They often occur as isomers, which makes the structural elucidation difficult. In the present study, the fragmentation behavior of six flavonoid O-diglycosides from genus Citrus was investigated using ion trap mass spectrometry in negative electrospray ionization (ESI) with loop injection. For the flavonoid O-rutinosides, [M − H − 308]⁻ ion was typically observed in the MS² spectrum, suggesting the loss of a rutinose. The fragmentation patterns of flavonoid O-neohesperidosides were more complicated in comparison with their rutinoside analogues. A major difference was found in the [M − H − 120]⁻ ion in the MS² spectrum, which was a common feature of all the flavonoid O-neohesperidosides. The previous literature for naringin located the loss of 120 Da to the glycan part, whereas the present study for naringin had shown that the [M − H − 120]⁻ ion was produced by a retro-Diels-Alder reaction in ring C, and this fragmentation pattern was confirmed by the accurate mass measurement using an orthogonal time-of-flight mass spectrometer. Combined with high performance liquid chromatography (HPLC) and diode array detection (DAD), the established approach to the structural identification of flavonoid O-diglycosides by ion trap mass spectrometry was applied to the analysis of extracts of two Chinese medicines derived from genus Citrus, namely Fructus aurantii and F. aurantii immaturus. According to the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by multistage mass spectrometry (MSⁿ) spectra, 13 flavonoid O-glycosides in F. aurantii and 12 flavonoid O-glycosides in F. a. immaturus were identified rapidly.     

Identification and characterization of supercritical fluid extracts of Rhizoma Chuanxiong by high performance liquid chromatography ion trap mass spectrometry

The main constituents, senkyunolide A, Z-ligustilide, neocnidilide, 3-butylphthalide, and ligustilide dimers, in supercritical CO₂ fluid extracts of Rhizoma Chuanxiong, a popular Chinese traditional medicine, have been identified and characterized by high performance liquid chromatography tandem mass spectrometry. Separations were carried out on an Agilent (ECLIPSE XDB) C18 analytical column by gradient elution with 0.25% acetic acid and methanol (containing 0.25% acetic acid). An Agilent 1100 series LC/MSD XCT system was operated under positive ESI and auto MS/MS modes for mass spectrometric analysis. Collision-induced dissociation (CID) fragmentations of these phthalides have been investigated and elucidated. Phthalides have primarily undergone two ESI CID pathways: side-chain cleavage with losses of alkenes and ring-opening with eliminations of H₂O followed by losses of CO. Direct neutral loss of CO has not been observed. Sodium adduct ions have demonstrated completely different CID pathways. __________ Translated from Journal Instrumental Analysis (in press, in Chinese)     

Rapid simultaneous determination of dexamethasone and betamethasone in milk by liquid chromatography tandem mass spectrometry with isotope dilution

A simple, sensitive and reliable analytical method for the rapid simultaneous determination of dexamethasone and betamethasone in milk by high performance liquid chromatography–negative electrospray ionization tandem mass spectrometry (HPLC–NESI-MS/MS) with isotope dilution was developed. Samples were directly purified through C₁₈ cartridge. Then the eluate was dried under nitrogen and residues were dissolved in mobile phase. Samples were analyzed by HPLC–MS/MS on a Hypercarb graphite column with a mixture of acetonitrile–water–formic acid as mobile phase. The samples were quantified using dexamethasone-D₄ as an internal standard. The procedure was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), trueness, precision, linearity and stability. The method is demonstrated to be suitable for the determination of dexamethasone and betamethasone in milk. The total time required for the analysis of one sample was about 35 min.     

Metabolic profiling of Actaea species extracts using high performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry

Despite persistent questions about the safety of black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa L.), its products continue to be one of the most popular botanical supplements in the United States market. Black cohosh products have been associated with cases of liver toxicity, but subsequent evaluation found some products to be adulterated with other related plants from the same genus. US FDA regulations require that black cohosh products be unadulterated, and correct identification of different species of Actaea is a key first step for their good manufacturing practice. We have developed a phytochemical method to distinguish four different groups of Actaea, including: species other than A. racemosa, Asian species, A. racemosa, and North American species other than A. racemosa. Using HPLC-TOF-ESI-MS technique and principal component analysis, we identified 15 chemical markers (1-3, 5-6, 8-10, 12, 16-21). Three marker compounds were unambiguously identified using authentic standards, and 12 marker compounds were tentatively identified by comparison of fragmentation patterns with previously reported data. The presence of these marker compounds is critical for discrimination among the four groups of closely related plants. The use of metabolic profiling to distinguish black cohosh from related species of Actaea has broader implications in the identification of markers to help authenticate other important medicinal plants.     

Screening and identification of multi-component in Qingkailing injection using combination of liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

An approach for screening and identification of multi-component in complex traditional Chinese medicine systems with a combinative LC/MS (MSⁿ) technique was described in this paper. The chemical profile of Qingkailing injection, a well-known traditional Chinese formula in China, was studied using the established method as for an application. Benefit from combining the accurate mass measurement of LC/TOF-MS to generate elemental compositions and the complementary multilevel structural information provided by LC/ion trap MSⁿ, 33 components in Qingkailing injection were identified in all. The three isomers of chlorogenic acid, isochlorogenic acid and neochlorogenic acid which are derived from Flos Lonicerae, one of the medicinal materials in Qingkailing, were differentiated by verifying their MS³ fragmentation data. All the components identified were surveyed and classified according to their medicinal materials derivation. This study is expected to provide an effective and reliable pattern for comprehensive and systematic characterization of the complex traditional Chinese medicine systems.     

The high throughput analysis of N-methyl carbamate pesticides in wine and juice by electrospray ionization liquid chromatography tandem mass spectrometry with direct sample injection into a short column

We developed a new analysis method for the N-methyl carbamate pesticides in juice and wine. The juice and wine were diluted with ultra pure water, and determined by electrospray ionization tandem mass spectrometry (ESI LC/MS/MS) with direct sample injection into a short column. The new method, including sample preparation and determination, is simple and rapid, and allows simultaneous determination of nine N-methyl carbamate pesticides in juice and wine within analysis time that is much shorter as compared with the traditional method. The average recoveries from juice and wine fortified at the level of 0.1 ppm ranged from 59.6 to 126.7% with the coefficients of variation ranging from 0.4 to 5.1% for intra-day (n = 5 × 3 days) and from 0.5 to 22.6% for inter-day (n = 15). At the fortified level of 0.5 ppm, the recoveries ranged from 69.3 to 127.2% with the coefficients of variation ranging from 0.4 to 6.9% for intra-day (n = 5 × 3 days) and from 0.5 to 22.6% for inter-day (n = 15). The method is considered to be satisfactory for the monitoring of the carbamate pesticides residues in juice and wine, suggesting that the present method is applicable to other pesticide residues in foods.     

液相色谱-二极管阵列检测/质谱联用在活性染料结构鉴定中的应用

基于多年运用液相色谱-二极管阵列检测/质谱(HPLC-DAD/MS)联用技术分析活性染料的实验积累,总结了HPLC-DAD/MS联用方法在商品活性染料结构鉴定中的重要应用。商品染料不需要纯化即可通过单次进样达到复杂组分的分离并得到光谱和质谱信息;结合衍生物质量差值法可推定染料的活性基种类和数目;采用二极管阵列检测器的全光谱扫描功能可以得到染料的发色体类别等有用的信息。HPLC-DAD/MS联用方法简便、快速、准确,尤其适用于多组分拼混的商品染料的结构鉴定,有较大的实用价值。通过对多组分商品染料活性灰、活性深黑、海洋蓝中关键组分的结构分析实例,介绍了HPLC-DAD/MS联用分析技术在未知商品活性染料结构分析中的应用。     

The high throughput analysis of N-methyl carbamate pesticides in fruits and vegetables by liquid chromatography electrospray ionization tandem mass spectrometry using a short column

We developed a new analysis method for the nine N-methyl carbamate pesticides in fruits and vegetables using ESI LC/MS/MS with direct sample injection into a short column. After extraction of the pesticides with ethyl acetate from sample, the extract is evaporated to dryness and redissolved in ultra pure water before injection into LC/MS/MS. The method needs no cleanup steps. The average recoveries from fruits and vegetables fortified at the level of 0.01 μg/g ranged from 56.0 to 119.1% with the coefficients of variation ranging from 0.2 to 7.6% for intra-day (n = 5 × 3 days) and from 0.8 to 18.4% for inter-day (n = 15). At the fortified level of 0.5 μg/g, the recoveries ranged from 67.7 to 119.3% with the coefficients of variation ranging from 0.5 to 7.8% for intra-day (n = 5 × 3 days) and from 0.9 to 14.8% for inter-day (n = 15). The method is considered to be satisfactory for the monitoring of the carbamate pesticide residues in fruits and vegetables, suggesting that the present method is applicable to other pesticide residues in foods.     

Simultaneous extraction and analysis by high performance liquid chromatography coupled to diode array and mass spectrometric detectors of bixin and phenolic compounds from annatto seeds

This study was designed to identify and quantify the carotenoids and phenolic compounds from annatto seeds using high performance liquid chromatography coupled to diode array and mass spectrometer detectors (HPLC-DAD-MS/MS). Furthermore, using response surface methodology, an optimized procedure for simultaneous extraction of these compounds was established. In addition to bixin, known to be the main carotenoid in annatto seeds, hypolaetin and a caffeoyl acid derivative were identified as the main phenolic compounds. The optimized procedure involved 15 extractions using acetone:methanol:water (50:40:10, v/v/v) as solvent, a solid-liquid ratio of 1:9 (m/v) and an extraction time of 5 min. Validation data indicated that the HPLC method proposed provided good linearity, sensitivity, procedure accuracy, system precision and suggested its suitability for the simultaneous analysis of phenolic compounds and carotenoids in annatto seeds.     

超快速液相色谱-四极杆/线性离子阱质谱同时检测猪组织中9种β-受体阻断剂残留

建立了同时检测猪组织中9种β-受体阻断剂(BBs)残留的超快速液相色谱-四极杆/线性离子阱质谱方法。均质试样经β-葡糖醛苷酶/芳基硫酸酯酶水解,乙腈提取,硅藻土与BondElut分散固相萃取填料双重快速净化,以0.1%(v/v)甲酸水溶液-甲醇为流动相使用KinetexTMC18-XB色谱柱(150 mm×2.1 mm,2.6μm)超快速液相色谱分离,优化多反应监测(MRM)离子对后,采用预设定多反应监测(sMRM)-信息依赖性采集(IDA)-增强子离子扫描(EPI)模式检测,在线EPI谱库定性分析,内标法定量。结果表明,9种BBs在线性范围内的线性关系良好(r≥0.995);定量限(LOQ,S/N≥10)均达到0.5μg/kg;3个添加水平(0.5、1.0和5.0μg/kg)下的回收率为87.5%~111.8%;RSD为4.0%~12.5%。该方法快速、准确、灵敏,可有效用于猪组织样品中多种BBs残留的同时测定。