Detection of fortification of ginkgo products using nanoelectrospray ionization mass spectrometry

We report here a negative ionization nanoelectrospray ionization mass spectrometry (nanoESI-MS) technique that simultaneously detects active components, terpenes and intact flavonol glycosides, and toxic ginkgolic acids in ginkgo products. Unlike the conventional methods that hydrolyze flavonol glycosides to flavonoids for analysis, this technique directly detects intact flavonol glycosides, enabling differentiation of these natural glycosides from the synthetic flavonoids. Thus, it allows the detection of fortification of ginkgo products, alleviating a common problem encountered by the conventional methods. Analysis of 14 commercial ginkgo products using this technique demonstrates large variations and deviation from the well-accepted standardized ginkgo extract. Four products showed evidence of fortification with synthetic surrogates. Two products were found to have toxic ginkgolic acids that exceed the 5 microg g(-1) limit by as much as 60000 fold. These results emphasize the importance of appropriate monitoring of ginkgo product quality.     

Multiresidue determination of organophosphorus pesticides in ginkgo leaves by accelerated solvent extraction and gas chromatography with flame photometric detection

A rapid and simple method using accelerated solvent extraction and solid-phase extraction cleanup was developed and validated for the determination of 15 organophosphorus pesticides in ginkgo leaves by capillary gas chromatography with flame photometric detection. The pesticides were extracted at 100 degrees C under 1500 psi pressure in <20 min. The average recovery from 10 g ginkgo leaves, fortified at 3 levels ranging from 0.05 to 1.00 mg/kg, was 95.2% with a relative standard deviation of 4.6%. The limits of detection ranged from 1.11 x 10(-3) mg/kg (dimethoate) to 4.44 x 10(-3) mg/kg (dichlorvos). The proposed method showed acceptable accuracy and precision while minimizing environmental concerns, time, and labor. Furthermore, the method could be easily applied to the monitoring of these 15 organophosphorus pesticides in ginkgo leaves.     

A sensitive method for the detection and quantification of ginkgo flavonols from plasma

Extracts from Ginkgo biloba leaves (family Ginkgoaceae) have antioxidant and free radical scavenging effects, largely attributed to the flavonols, which are a major class of functional components in ginkgo extracts. In order to facilitate analysis of systemic exposure to ginkgo-derived products in animals and/or humans, we developed a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based method that is capable of routinely monitoring plasma levels of ginkgo flavonols. We used an initial acidic hydrolysis step to convert the plasma ginkgo flavonol conjugates into their aglycone forms [quercetin (QCT), kaempferol (KMF) and isorhamnetin (ISR)] prior to EtOAc-based extraction and subsequent LC/MS/MS-based analysis. Comparative studies showed that the use of a mobile phase containing an extremely low concentration of HCOOH (0.01 per thousand) dramatically improved the electrospray ionization efficiency of the analytes in the negative ion mode; the efficiencies were approximately 4-, approximately 8- and approximately 20-fold higher for QCT, KMF and ISR, respectively, versus the results obtained using an electrolyte-free mobile phase, or approximately 2-, approximately 3- and approximately 4-fold higher, respectively, versus the results obtained using a mobile phase containing the more commonly utilized concentration of HCOOH (1 per thousand). In addition, use of the low concentration of HCOOH also decreased undesired matrix effects. These favorable effects have been referred to as 'LC-electrolyte effects'. Due to structural differences in the B-ring substituent, different types of precursor-to-product ion pairs (m/z 301 --> 151 for QCT, 285 --> 187 for KMF, and 315 --> 300 for ISR) were used for the selected reaction monitoring of the analytes. In addition, the chromatographic conditions were optimized on the basis of an initial scouting of matrix effects on analyte ionization. Despite the absence of an internal standard, the validation results consistently demonstrated that our bioassay is valid, reproducible, and reliable. The newly developed assay provided lower limits of quantification of 1.3, 1.3 and 0.4 pg on-column for QCT, KMF and ISR, respectively, which is more sensitive than any previously reported method for determining ginkgo flavonols. Finally, the assay suitability was demonstrated in a pilot pharmacokinetic measurement of a pharmaceutical ginkgo product in a beagle dog. This newly developed method should prove useful for wide-scale monitoring of ginkgo flavonol plasma concentrations for both pharmaceutical investigations and clinical applications.     

高频感应炉燃烧–红外吸收法测定银杏及银杏叶中的硫含量

采用高频感应炉燃烧–红外吸收法测定银杏叶、银杏果肉、银杏壳和银杏仁中的硫含量。样品以艾士卡试剂为熔融剂,在800℃马弗炉内熔融1 h,冷却后测定硫含量。硫的质量分数在0.40%~4.00%范围内与吸收峰面积呈良好的线性关系,线性相关系数为0.990 4,检出限为0.000 9%。测定结果的相对标准偏差为4.04%(n=11),平均加标回收率为101.03%。将红外吸收法与电感耦合等离子体原子发射光谱法及硫酸钡重量法进行比对试验,3种方法测定结果相一致。利用该方法测定了不同区域银杏叶中硫的含量,结果表明,风景区和居民区银杏叶干燥基全硫的含量较低,重工业区硫含量较高。对同一地区的银杏果肉、银杏壳、银杏仁中干燥基全硫的含量进行比较,结果表明银杏果肉硫含量最高,银杏壳次之,银杏仁最低。该方法灵敏度高,重现性好,可用于银杏及银杏叶中硫含量的准确测定。     

Comparative Analysis of Volatile Terpenes and Terpenoids in the Leaves of Pinus Species—A Potentially Abundant Renewable Resource

Pinaceae plants are widely distributed in the world, and the resources of pine leaves are abundant. In the extensive literature concerning Pinus species, there is much data on the composition and the content of essential oil of leaves. Still, a detailed comparative analysis of volatile terpenes and terpenoids between different species is missing. In this paper, headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry was used to determine the volatile terpenes and terpenoids of typical Pinus species in China. A total of 46 volatile terpenes and terpenoids were identified, and 12 common compounds were found, which exhibited a great diversity in the leaves of Pinus species. According to the structures and properties of the compounds, all those compounds can be classified into four categories, namely monoterpenes, oxygenated terpenes, terpene esters, and sesquiterpenes. The results of principal component analysis and cluster analysis showed that the leaves of the six Pinus species could be divided into two groups. The species and contents of volatile terpenes and terpenoids in the leaves were quite different. The results not only provide a reference for the utilization of pine leaves resource, but also bring a broader vision on the biodiversity.     

高效液相色谱-串联质谱法同时测定银杏保健茶中的16种黄酮类功效成分

建立了银杏保健茶中16种黄酮类物质的液相色谱-串联质谱(LC-MS/MS)测定方法。16种黄酮成分分别为儿茶素、牡荆素、葛根素、大豆苷元、水飞蓟宾、槲皮素、木犀草素、芹菜素、柚皮素、橙皮素二氢查尔酮、山柰酚、橙皮素、异鼠李素、黄芩素、川陈皮素、桔皮素。实验优化了液相色谱条件和质谱参数。采用C₁₈柱分离,流动相为乙腈-水(含0.1%甲酸)梯度洗脱,流速0.25 mL/min,以电喷雾离子源正离子多反应监测(MRM)模式进行MS/MS检测。16种黄酮类物质在各自的线性范围内具有良好的线性关系,相关系数大于0.996,低、中、高3个添加水平的平均回收率在70.9%~100.0%之间,相对标准偏差小于10%。通过检测发现实际样品中9种黄酮物质含量较高,分别是:山柰酚、槲皮素、橙皮素、牡荆素、木犀草素、儿茶素、芹菜素、柚皮素、异鼠李素,占总量的99.6%,此9种物质可作为银杏保健茶的质量控制指标。本法简便、快速、准确可靠,可用于控制银杏保健茶的质量。     

Simultaneous determination of ginkgo flavonoids and terpenoids in plasma: Ammonium formate in LC mobile phase enhancing electrospray ionization efficiency and capacity

Extracts from Ginkgo biloba leaves confer their therapeutic effects through the synergistic actions of flavonoid and terpenoid components. We herein describe the development of an LC-MS/MS-based method for simultaneous determination of flavonoids (quercetin, kaempferol, and isorhamnetin) and terpenoids (bilobalide, ginkgolides A, B, C, and J) in acid-hydrolyzed plasma by circumventing cross-interference between the flavonoids and terpenoids identified. Notably, inclusion of ammonium formate (0.2 mM) in the mobile phase generated beneficial LC-electrolyte effects, including increased ESI efficiency and capacity, with the result that the newly developed procedure exhibits the highest analytical performance reported to date for ginkgo-associated studies. The method yields high sensitivity, negligible matrix interference and cross-interference, wide linear dynamic ranges, high sample throughput, and quite small initial sample size. The assay utility to dog pharmacokinetic measurements of commercial ginkgo products yielded the most comprehensive data on systemic exposure to the ginkgo compounds to date. The newly developed multi-analyte procedure should be widely useful.     

PURIFICATION OF GINKGO LEAVES EXTRACT WITH MACROPOROUS ADSORBENT BASED ON UREA—FORMALDEHYDE CONDENSED POLYMERS

The relationship between the adsorption properties for the active components of ginkgo leaves and the structure of the adsorbents based on urea-formaldehyde condensed polymers was investigated.The results revealed that these adsorbents showed very high adsorpton selectivity for both flavonol glycosides and terpene lactones contained in ginkgo leaves.Thus,an adsorption separation procedure for purification of ginkgo leaves extracts was developed.     

Sample preparation and determination of ginkgo terpene trilactones in selected beverage, snack, and dietary supplement products by liquid chromatography with evaporative light-scattering detection

Ginkgo biloba leaf extract has been widely used in dietary supplements and more recently in some foods and beverages. Sample preparation procedures for determination of ginkgo terpene trilactones (including bilobalide and ginkgolides A, B, C, and J) in various sample matrixes were developed in this study. Ginkgo leaves and capsules were extracted with 5% KH2PO4 aqueous solution under sonication. Tea bags were extracted with boiling water, whereas drink samples were taken directly from the bottles. After filtration and the addition of NaCl to approximately 30% (w/v), the terpene trilactones in aqueous solutions were quantitatively extracted with ethyl acetate-tetrahydrofuran (4 + 1, v/v). Puff samples (a cereal-based fried snack item) were first defatted by using hexane or by using supercritical fluid extraction and then extracting under sonication with methanol-acetic acid (99 + 1, v/v). After evaporation of the organic phase, the terpene trilactones were redissolved in methanol and determined on a C18 reversed-phase column by liquid chromatography (LC) with evaporative light-scattering detection. The method of standard additions and gas chromatography with flame ionization detection were used for method validation. For most samples, the relative standard deviation was <10%. The identities of target compounds in ginkgo leaves and drink samples were confirmed by LC/electrospray ionization-tandem mass spectrometry.     

PURIFICATION OF GINKGO LEAVES EXTRACT WITH MACROPOROUS ADSORBENT BASED ON UREA-FORMALDEHYDE CONDENSED POLYMERS

I INTRODUCTIONGinkgo biloba L. is one of the important economic trees, which is widely distributed inChina. In recent years, medicinal and healthful preparations based on the extractS from ginkgoleaves have been rapidly developed for the treatment of peripheral circulatory and cerebralinsufficiencyl']. Such eXtracts contain flavonol glycosides and terpene lactones as thepharmacologically active compounds. Organic-solvent extraction is one of the earliest methodsto prepare the extracts o…     

Simultaneous determination of terpenes and cannabidiol in hemp (Cannabis sativa L.) by fast gas chromatography with flame ionization detection

Hemp (Cannabis sativa L.) has become widely used in several sectors due to the presence of various bioactive compounds such as terpenes and cannabidiol. In general, terpenes and cannabidiol content is determined separately, which is time consuming. Thus, a fast gas chromatography with flame ionization detection method was validated for simultaneous determination of both terpenes and cannabidiol in hemp. The method enabled a rapid detection of 29 different terpenes and cannabidiol within a total analysis time of 16 min, with satisfactory sensitivity (limit of detection = 0.03–0.27 µg/mL, limit of quantitation = 0.10–0.89 µg/mL). The inter‐ and intraday precision (RSD) was <7.82 and <3.59%, respectively. Recoveries at two spiked concentration levels (low, 3.15 µg/mL; high, 20.0 µg/mL) were determined on both apical leaves (78.55–101.52%) and inflorescences (77.52–107.10%). The reproducibility (RSD) was <5.94 and <5.51% in apical leaves and inflorescences, respectively. The proposed and validated method is highly sensitive, robust, fast, and accurate for determination of the main terpenes and cannabidiol in hemp and could be routinely used for quality control.     

超高效液相色谱串联质谱法测定银杏中氨基甲酸酯类农药残留

建立了银杏叶中15种氨基甲酸酯类农药残留量的超高效液相色谱一串联质谱测定方法。样品经乙腈提取,TPT固相萃取柱净化,通过XTerra MS Cl8色谱柱分离,供串联质谱测定。15种氨基甲酸酯类农药在0．02-0．5mg／L范围内线性良好,相关系数为0．9962-0．9999;在0．004,0．01,0．02,0．04mg／kg4个添加水平下,其平均回收率为65．22％-96．66％,相对标准偏差为1．43％-10．06％（n=5）。该方法净化效果好、灵敏度高、重现性好,可满足银杏叶中15种氨基甲酸酯类农药残留量的检验要求。     

GC-MS联用分析三个品种杨梅树叶的挥发油组分

采用气相色谱-质谱联用技术对木叶、黑炭、东魁3个品种杨梅树叶中的挥发油进行分析比较.3种杨梅树叶挥发油中共检出14种相同成分,这些共有成分的相对含量分别占木叶梅叶、黑炭梅叶和东魁梅叶挥发油总相对含量的53. 3%、48. 4%、55. 15%.3种杨梅树叶挥发油中均舍有多种萜烯,对杨梅果实具有一定抗菌保鲜作用.东魁杨梅树叶挥发油萜烯含量最高,这可能是东魁杨梅果实保鲜期较长的原因;木叶杨梅叶含的特殊香味成分的含量较高,这可能是木叶梅果实香气最浓郁的原因.     

Within-Plant Variation in Rosmarinus officinalis L. Terpenes and Phenols and Their Antimicrobial Activity against the Rosemary Phytopathogens Alternaria alternata and Pseudomonas viridiflava

This study investigated within-plant variability of the main bioactive compounds in rosemary (Rosmarinus officinalis L.). Volatile terpenes, including the enantiomeric distribution of monoterpenes, and phenols were analyzed in young and mature foliar, cortical and xylem tissues. In addition, antimicrobial activity of rosmarinic acid and selected terpenes was evaluated against two rosemary pathogens, Alternaria alternata and Pseudomonas viridiflava. Data showed that total concentration and relative contents of terpenes changed in relation to tissue source and age. Their highest total concentration was observed in the young leaves, followed by mature leaves, cortical and xylem tissues. Rosmarinic acid and carnosic acid contents did not show significant differences between leaf tissues of different ages, while young and mature samples showed variations in the content of four flavonoids. These results are useful for a more targeted harvesting of rosemary plants, in order to produce high-quality essential oils and phenolic extracts. Microbial tests showed that several terpenes and rosmarinic acid significantly inhibited the growth of typical rosemary pathogens. Overall, results on antimicrobial activity suggest the potential application of these natural compounds as biochemical markers in breeding programs aimed to select new chemotypes less susceptible to pathogen attacks, and as eco-friendly chemical alternatives to synthetic pesticides.     

Antibacterial activities of essential oils extracted from leaves of Murraya koenigii by solvent-free microwave extraction and hydro-distillation

Solvent-free microwave extraction (SFME) for the isolation of essential oil from leaves of Murraya koenigii L. (Rutaceae) has been compared with the conventional hydro-distilled oil (HD) in terms of yield, composition, antioxidant activity, and antibacterial activity against Listeria innocua. The yield of essential oil obtained from 30 min of SFME was similar to that of HD for 180 min. By GC-MS analysis, the major compounds of the essential oil extracted by SFME, which were obtained in somewhat lower amounts than in the essential oil obtained by HD, were alpha-copaene (44.3%), beta-gurjunene (25.5%), isocaryophyllene (12.1%), beta-caryophyllene (8.7%) and germacrene D (2.9%). The content of oxygenated terpenes, slightly higher for the SFME-essential oil (2.3%) than the HD-essential oil (1.4%), were much lower than that of nonoxygenated terpenes in both oils. DPPH radical scavenging activities of both essential oils were relatively low (10%-24%). Complete inhibition of growth of L. innocua was observed with both SFME and HD essential oils, at 400 and 600 microg/mL (minimum inhibitory concentration), respectively. The SFME-essential oil at 300 microg/mL provided 92% inhibition, indicating its potential as a natural antimicrobial agent.     

Blending Technology Based on HPLC Fingerprint and Nonlinear Programming to Control the Quality of Ginkgo Leaves

The breadth and depth of traditional Chinese medicine (TCM) applications have been expanding in recent years, yet the problem of quality control has arisen in the application process. It is essential to design an algorithm to provide blending ratios that ensure a high overall product similarity to the target with controlled deviations in individual ingredient content. We developed a new blending algorithm and scheme by comparing different samples of ginkgo leaves. High-consistency samples were used to establish the blending target, and qualified samples were used for blending. Principal component analysis (PCA) was used as the sample screening method. A nonlinear programming algorithm was applied to calculate the blending ratio under different blending constraints. In one set of calculation experiments, the result was blended by the same samples under different conditions. Its relative deviation coefficients (RDCs) were controlled within ±10%. In another set of calculations, the RDCs of more component blending by different samples were controlled within ±20%. Finally, the near-critical calculation ratio was used for the actual experiments. The experimental results met the initial setting requirements. The results show that our algorithm can flexibly control the content of TCMs. The quality control of the production process of TCMs was achieved by improving the content stability of raw materials using blending. The algorithm provides a groundbreaking idea for quality control of TCMs.     

双波长吸光光度法测定植物中黄酮含量

研究了双波长吸光光度法测定芹菜叶、银杏叶植物中黄酮含量的方法，与原比色法相比，具有较高的准确度和可靠性。     

QCM sensor array for monitoring terpene emissions from odoriferous plants

Abstract  An e-nose comprised of six sensing channels each coated with a molecularly imprinted polymer (MIP) selectively interacting with alpha-pinene, thymol, estragol, linalool, and camphor, respectively, was designed. When applying it for continuous online surveillance of terpenes emitted from basil and peppermint leaves as a criterion of freshness, it very appreciably reproduced the emanation patterns from these plants as shown by GC-MS. Chromatography yielded a variety of terpenes in a concentration range below 70 ppm. Trend lines obtained from the e-nose were corroborated by GC-MS and also appreciably fit the usual conduct of these plants as observable by the human nose. Graphical abstract        

Selective dissolution and one step separation of terpene trilactones in ginkgo leaf extracts for GC-FID determination

Under ultrasonication, the ginkgo terpene trilactones, ginkgolides and bilobalide, in ginkgo extracts can be selectively dissolved in 10% aqueous NaH(2)PO(4) solution at a temperature of 50-60 degrees C and separated from the solution by extraction with a mixture of ethyl acetate/tetrahydrofuran in a capped vial. After derivatization, these terpene trilactones can be quantified using GC-FID. This method has a detection limit of 10 ng, and the RSD was 6% (n=5). Twelve commercial GBE products in powder, liquid, tablet and capsule forms were analyzed. The total time required for analyzing these samples from sample preparation to final data processing was less than 6 h, and the total organic solvent consumption was less than 40 ml. This procedure proves to be a simple, fast, safe, and effective method for all types of Ginkgo biloba extracts (GBE) including the "complex" or "advanced" formulas.     

Supercritical fluid chromatographic analysis of polyprenols in Ginkgo biloba L

A supercritical fluid chromatographic (SFC) procedure for the quantitation of three major polyprenols present in the leaves of Ginkgo biloba was developed. In contrast to previously reported high-performance liquid chromatographic (HPLC) methods, the SFC method does not require extensive pre-purification for polyprenol analysis. The SFC analytical procedure described shows a very broad range of linearity and detects many known polyprenol isoprenologs with baseline separation. Dodecaprenol was used as the internal standard. The coefficient of variation of the method was 5.8% for the quantitation of C₈₅, C₉₀ and C₉₅ polyprenols. The SFC assay results showed that the content of polyprenols in ginkgo leaves were higher than the previously published values. In addition, the chromatogram of the highly concentrated leaf extract revealed the presence of an isoprenolog (C₁₂₀) not previously detected by HPLC methods.