Application of analytical chemistry in the quality evaluation of Glycyrrhiza Spp

Licorice, one of the oldest traditional Chinese medications, is widely used in the treatment of various diseases. With the development of science and technology, an increasing amount of analytical techniques have been applied to the quality control of licorice. Herein, we summarize several of the quality control methods developed in recent years. These approaches include sample preparation processes, high-performance thin layer chromatography, gas chromatography, gas chromatography/mass spectrometry, high-performance liquid chromatography, and liquid chromatography/mass spectrometry (LC/MS). Among these various techniques, LC/MS has come forward as one of the main methods forquality control of licorice.     

Screening and characterization of natural antioxidants in four Glycyrrhiza species by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Licorice, derived from the dried roots and rhizomes of several species of genus Glycyrrhiza L. (Leguminosae family), has been traditionally used in herbal medicine for over 4000 years. In recent years, the interest in antioxidative constituents in licorice has greatly increased. In this work, a new method based on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) spiking test combined with HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis was developed to screen and identify the antioxidants in licorice. The results of the method validation indicated that the developed method was reliable and repeatable. Compared with DPPH on-line method, the HPLC-Q-TOF MS/MS method combined with DPPH spiking test offered much higher sensitivity and resolution. Using this method, 35 radical scavengers were screened from four Glycyrrhiza species (G. inflata, G. glabra, G. pallidiflora and G. uralensis), and 21 of them were unambiguously or tentatively identified by HPLC-Q-TOF MS/MS. Among the 21 identified flavonoids, 10 compounds had been reported to possess antioxidative activities in the previous studies, and the radical scavenging activities of the other 11 compounds were reported for the first time. The effects of six purified flavonoids on DPPH radical and lipid peroxidation were evaluated for validation of the developed method. The results indicated that HPLC-Q-TOF MS/MS coupled with DPPH treatment is an efficient and powerful method to discover the potential antioxidative compounds from the complex natural product mixtures. In this study, the identified components with free radical scavenging activity, would help to explain the therapeutic benefit of licorice in the treatment of human disease associated with oxidative stress.     

Quantitative determination of the group of flavonoids and saponins from the extracts of the seeds of Glinus lotoides and tablet formulation thereof by high-performance liquid chromatography

The total flavonoids and saponins of the seeds of Glinus lotoides in the crude extracts and tablet formulation thereof were quantified by reversed-phase high-performance liquid chromatographic (RP-HPLC) methods with UV detection. The saponins were analyzed after acid hydrolysis in 3 M HCI at 100 degrees C for 1 h. Vicenin-2 and mollugogenol B were isolated and used as reference substances for the quantification of total flavonoids and saponins, respectively. The identity and purity (> 97%) of the standards were confirmed by spectroscopic (UV, MS, and NMR) and chromatographic (HPLC) methods. The flavonoids and saponins of the crude extract of the seeds and tablet formulation were separated by RP-HPLC (Nucleosil RP-18 column, 250 mm x 4.6 mm) using linear gradient elution systems of acetonitrile-water-0.1 M H3PO4 for flavonoids and methanol-water for saponins. Satisfactory separation of the compounds was obtained in less than 30 and 25 min, for the flavonoids and saponins, respectively. The methods were validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). Repeatability (inter- and intra-day, n = 6 and 9, respectively) showed less than 2% relative standard deviation (RSD). The LOD and LOQ were found to be 0.075 and 0.225 mg/mL, respectively, for vicenin-2 and 0.027 and 0.082 mg/100 mL, respectively, for mollugogenol B. The content of flavonoids and saponins of six single tablets was between 95 and 103% for flavonoids and 94-98% for saponins. The validated HPLC methods were employed to standardize a fingerprint of a laboratory produced purified extract, which could be used as a secondary standard for the routine quality control. Accordingly, the purified extract was found to contain 21.3% flavonoids (vicenin-2, 10%) and 25.4% saponins (glinuside G, 14.2%).     

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MS(n) data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MS(n), non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools for rapid screening and structural assignment of saponins in plant extracts.     

Simultaneous quantitation of 15 bioactive components in Yupingfeng granules by LC–MS/MS

Yupingfeng granules (YPFG) is commonly used in the treatment of immunological diseases, inflammations, and pulmonary diseases. Several studies have found that chromones, flavones, and saponins were the major bioactive compounds of YPFG. However, few studies have reported accurate quantification methods of these compounds. This study aimed to establish a simple and rapid method by using liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine 15 bioactive compounds in YPFG. The experimental parameters including extraction methods, extraction solvents, extraction time, solid–liquid ratio, and LC–MS/MS condition were optimized. The linearity, precision, repeatability, stability, and recovery of the established method were evaluated. The contents of 15 bioactive compounds in seven batches of YPFG samples were analyzed by the established method and the results were compared with the values determined by HPLC. The optimal extraction condition was to extract 0.1 g of YPFG by ultrasound with 50 mL 50% ethanol for 30 min. A Waters ACQUITY UPLCBEH C18 column using the 0.1% formic acid water solution and acetonitrile as mobile phase with a gradient elution was applied to the chromatographic separation. The linearity, precision, repeatability, stability, and recovery of the method were within acceptable ranges. Compared with HPLC analysis methods in Chinese Pharmacopoeia and literature, the established method was faster, simpler, more accurate, and more reliable. The method of simultaneous determination of 15 components in YPFG by LC–MS might provide a basis for the study of the bioactive compounds and the improvement of the quality standard of YPFG.     

Study on the photostability of guaiazulene by high-performance liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

The photostability of guaiazulene (1,4-dimethyl-7-isopropylazulene; GA), a natural azulenic compound used in cosmetic and health-care products, as well as in pharmaceutical preparations, was investigated in solution (methanol, ethanol, acetonitrile), by different techniques: gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry and UV detection (LC/APCI-MS and HPLC/UV). A solar simulator (xenon-arc lamp) was used as UV-A radiation source. The study involved: monitoring compound decomposition, identifying products of photodegradation (PPs), assessing the role of oxygen and evaluating the kinetics of the process. Minor PPs are volatile compounds and were characterized by GC/MS, while oligomeric polyoxygenated compounds, tentatively characterized on the basis of MS and MS/MS spectra, were found to be the main photoproducts. The photodegradation was found to be enhanced by the presence of oxygen; nevertheless, determination of the singlet oxygen quantum yield for GA gave a lower value than that for the reference standard Rose Bengal. The obtained results and the developed stability-indicating methods (GC/MS and LC/MS) are of interest for stability studies and/or quality control purposes of GA as raw material or cosmetic products.     

Ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry and chemometric analysis of licorice based on the simultaneous determination of saponins and flavonoids

Licorice is among the most popular herbal medicines and frequently used in traditional medicine, food products, and cosmetics. In China, only Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L. are officially used and are usually processed with honey prior to use. To maintain the quality of commercially available herbal products, a simple, rapid, and reliable ultra high performance liquid chromatography with triple quadrupole mass spectrometry was developed to investigate the major active constituents of commercially available licorice products. Nineteen components were accurately determined, including eight triterpenoid saponins, one triterpene, and ten flavonoids. Subsequently, multivariate statistical analysis methods were employed to further explore and interpret the experimental data. The results indicated that liquiritin apioside may be considered as a candidate index for the quality control of licorice as well as 18β‐glycyrrhizic acid and liquiritin. In addition, both 18β‐glycyrrhizic acid and licorice‐saponin G2 can be used for discrimination between crude and honey‐processed licorice. Furthermore, using 18β‐glycyrrhizic acid and liquiritin as markers, this work revealed that the quality of licorice products may have declined in recent years. This highlights the need for additional effort focused on good agricultural practice during the processing of licorice. In summary, this study provides a valuable reference for the quality assessment of licorice.     

Characterization and identification of multiple constituents in Yinhuang granules by high-performance liquid chromatography with diode-array and time-of-flight mass spectrometry detection

A fast high-performance liquid chromatography (HPLC) method with diode-array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) has been developed for the analysis of multi-constituent in Yinhuang granules, a well-known combined herbal remedy prepared from the extract mixtures of Flos Lonicerae and Radix Scutellariae. The fast HPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6×50 mm, 1.8 μm) and 0.2% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution in 17 min, which is five times faster than the performance of conventional columns packed with 5.0 μm particles. With various fragmentor voltages in TOF/MS, accurate mass measurements (<5 ppm error) for molecular ions and characteristic fragment ions represented reliable identification criteria for different constituents. A total of 28 compounds, including nine phenolic acids, three iridoid glycosides and nine saponins from Flos Lonicerae and seven flavonoids from Radix Scutellariae, were identified or tentatively characterized in the extract of Yinhuang granules. The established fast HPLC-DAD-TOF/MS method turns out to be useful and efficient for quality control of this commonly used Chinese herbal preparation.     

Liquid chromatography/tandem mass spectrometry of unusual phenols from Yucca schidigera bark: comparison with other analytical techniques

Qualitative and quantitative analyses of phenolic compounds are of interest for both medicinal and food plants. In the present work, the phenolic fraction from Yucca schidigera, a plant bearing the GRAS (Generally Recognized as Safe) label approved by the US Food and Drug Administration, was studied. Crude extracts of Y. schidigera bark were investigated by liquid chromatography/UV spectrophotometry with diode-array detection, liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), in order to develop and optimize simple and rapid techniques to determine both stilbenes and yuccaols for the purposes of quality control of collected material. With optimal LC and MS conditions, stilbenes and yuccaols were quantified with all the proposed methods and the results were compared. Sensitivity was evaluated and the results indicated that MS/MS detection in the multiple reaction monitoring mode is easily applicable to this plant and allows the rapid and direct identification and quantification of these peculiar compounds in crude plant extracts.     

Identification of flavonoids using liquid chromatography with electrospray ionization and ion trap tandem mass spectrometry with an MS/MS library

Searchable MS/MS spectra libraries, constructed using the results of liquid chromatography coupled with electrospray ionization (ESI) tandem mass spectrometry (LC/MS/MS) with data-dependent acquisition on an ion trap mass spectrometer, are presented with regard to the identification and confirmation of a variety of closely related flavonoids in a set of biological samples. Flavonoids were found to exhibit a maximum amount of structurally specific MS/MS spectra at 45% of normalized collision energy on the instrument used, without wideband activation. These MS/MS spectra were then searched automatically against a 297-substance MS/MS library that contains many previously acquired spectra of standard flavonoids. The possible applications of this powerful technique to biological samples are also discussed. Daidzein and genistein were identified through the MS/MS spectra library while searching through LC/MS/MS data for plant and microbial extracts. Moreover, these compounds proved completely distinguishable from other flavonoids of closely related structures in the MS/MS spectra library, using the NIST MS search program. The applicability of the library-searchable spectra at low concentrations was demonstrated by successful identification of daidzein and genistein at 0.05 and 0.5 microg/mL, respectively.     

Determination of flavonoids and saponins in Gynostemma pentaphyllum (Thunb.) Makino by liquid chromatography-mass spectrometry

Gynostemma pentaphyllum (Thunb.) Makino, a traditional Chinese herb possessing antitumor and antioxidant activities, has been shown to contain several functional components like saponins and flavonoids. However, their identities remain uncertain. The objectives of this study were to develop an appropriate extraction, purification and HPLC-MS method to determine saponins and flavonoids in G. pentaphyllum. Both flavonoids and saponins were extracted with methanol, followed by purification with a C18 cartridge to elute the former with 50% methanol and the latter with 100% methanol. A total of 34 saponins were separated within 40 min by a Gemini C18 column and a gradient mobile phase of acetonitrile and 0.1% formic acid in water, in which 18 saponins were identified by LC-MS with ESI mode and Q-TOF (LC/MS/MS). Similarly, a total of eight flavonoids were separated within 45 min by the same column and a gradient solvent system of methanol and 0.1% formic acid in water, with identification being carried out by a post-column derivatization method and LC-MS with ESI mode. The amounts of flavonoids in G. pentaphyllum ranged from 170.7 to 2416.5 μg g⁻¹, whereas saponins were from 491.0 to 89,888.9 μg g⁻¹.     

Rapid determination of flavonoids in licorice and comparison of three licorice species

A simple, sensitive and fast method for the simultaneous quantitation of 15 flavonoids in licorice based on an ultra high performance liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry had been established and validated in this study. The analysis was performed on an ACQUITY HSS T3 column with gradient elution using a mobile phase consisted of A (0.1% formic acid in water)/B (acetonitrile) at a flow rate of 0.4 mL/min. Satisfactory separation of these compounds was obtained in less than 9 min. All calibration curves showed good linearity (r² = 0.9940) during the test ranges. The precision, repeatability, accuracy, limit of detection and limit of quantification were also fully investigated. The validated method was successfully applied for the simultaneous quantitation of 15 flavonoids in 106 licorice samples which contained 83 batches of G. uralensis, 14 batches of G. glabra and 9 batches of G. inflata. Furthermore, multivariate statistical analysis (using principal components analysis) was performed to classify the samples based on the contents of the 15 analyzed compounds. The results showed that all of these licorice samples were rich in flavonoids, although their contents were obviously various, and the proposed method could serve as a prerequisite for quality control of licorice products.     

液相色谱-电喷雾电离质谱与电子轰击质谱联用筛选百合中的甾体皂甙

 利用高效液相色谱 电喷雾电离质谱联用仪 (HPLC/ESI MS)、电子轰击质谱 (EI MS)和半制备型高效液相色谱 ,从卷丹百合中筛选出了两种甾体皂甙 ,其中一种为含有 3个糖基与提果皂甙元的甾体皂甙 ,另一种为含有 3个糖基和薯蓣皂甙元的甾体皂甙。结果表明 :在线的HPLC/ESI MS能够准确快速地提供糖甙类化合物的分子质量和糖链部分的有益信息 ,但对甙元部分提供的信息极少 ;离线的EI MS只需极少量 (1mg～ 2mg)的纯品就能准确地提供甙元部分的有益信息 ,但很难获得糖甙的分子离子峰与糖链部分的信息 ,两者有机地结合起来能快速地从植物中筛选甾体皂甙。     

Liquid chromatography/mass spectrometry analysis of PHY906, a Chinese medicine formulation for cancer therapy

PHY906 is a Chinese medicine formulation prepared from four medicinal herbs for adjuvant cancer chemotherapy. In this paper, liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) was used to clarify the chemical composition of PHY906. The aqueous extract of PHY906 was separated on a Waters Atlantis C(18) column, and was eluted with acetonitrile/0.05% (v/v) formic acid. The separated compounds were identified with pure standards, or tentatively characterized by analyzing their mass spectra recorded in both negative and positive ion polarity modes. Further structural information was obtained from in-source fragmentation. Based on the LC/MS analysis, we proposed the structures for 64 bioactive compounds, including flavonoids, triterpene saponins, and monoterpene glycosides. All the compounds identified from PHY906 were further assigned in the four individual herbs, and some of them are reported for the first time.     

Analysis of phenolic and triterpenoid compounds in licorice and rat plasma by high‐performance liquid chromatography diode‐array detection,time‐of‐flight mass spectrometry and quadrupole ion trap mass spectrometry

High‐performance liquid chromatography with diode‐array detection (HPLC/DAD), time‐of‐flight mass spectrometry (HPLC/TOFMS) and quadrupole ion trap mass spectrometry (HPLC/QITMS) were used for separation and identification of several compounds in licorice and rat plasma after oral administration of the herbal extract. Three phenolic compounds and one triterpenoid in licorice extract were unambiguously identified by comparing with the standard compounds. A formula database of known compounds in licorice was established, against which the other 42 compounds were identified effectively based on the accurate extract masses and formulae acquired by HPLC/TOFMS. In order to differentiate the isomers, tandem mass spectrometry was also used. The deduced fragmentation behaviors in QITMS were used to distinguish seven groups of isomers in licorice. By means of the three detectors, 46 compounds in licorice were identified. After oral administration of the extract, 25 compounds in rat plasma were detected and identified by comparing and contrasting the compounds measured in licorice with those in the plasma samples by HPLC/TOFMS. It is concluded that a rapid and effective method based on three analytical techniques was established, which is useful for identification of multiple compounds in licorice in vitro and in vivo. The result should be very useful for the quality control and curative mechanism study of licorice. Copyright © 2009 John Wiley & Sons, Ltd.     

Higher-throughput screening for Caco-2 permeability utilizing a multiple sprayer liquid chromatography/tandem mass spectrometry system

In the current drug discovery environment, higher-throughput analytical assays have become essential to keep pace with the screening demands for drug metabolism and pharmacokinetics (DMPK) attributes. This has been dictated by advances primarily in chemical procedures, notably combinatorial and parallel syntheses, which has resulted in many-fold increases in the number of compounds requiring DMPK evaluation. Because of its speed and specificity, liquid chromatography/tandem mass spectrometry (LC/MS/MS) has become the dominant technology for sample analysis in the DMPK screening assays. For higher-throughput assays, analytical speed as well as other factors such as method development, data processing, quality control, and report generation, must be optimized. The four-way multiplexed electrospray interface (MUX), which allows for the analysis of four LC eluents simultaneously, has been adopted to maximize the rate of sample introduction into the mass spectrometer. Generic fast-gradient HPLC methods that are suitable for approximately 80% of the new chemical entities encountered have been developed. In-house-written software programs have been used to streamline information flow within the system, and for quality control by automatically identifying analytical anomalies. By integrating these components together with automated method development and data processing, a system capable of screening 100 compounds per week for Caco-2 permeability has been established.     

Description and validation of a staggered parallel high performance liquid chromatography system for good laboratory practice level quantitative analysis by liquid chromatography/tandem mass spectrometry.

The Aria LX4 staggered parallel high performance liquid chromatography (HPLC) system is evaluated for application to good laboratory practice (GLP) level quantitative analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This system consists of four fully independent binary HPLC pumps, a modified autosampler, and a series of switching and selector valves all controlled by a single computer program. The system improves sample throughput without sacrificing chromatographic separation or data quality. Validation results for four different compounds, each analyzed on a separate channel of the Aria system, show precision and accuracy equivalent to that required of a single-channel system. The results show that sample throughput can be increased nearly four-fold without requiring significant changes in current analytical procedures. The flexibility and ease of use of the Aria system suggest that it should be possible to quickly implement it in any analytical LC/MS/MS environment.     

Determination of in vitro permeability of drug candidates through a caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry

Studying the permeability of compounds across a Caco-2 cell monolayer is an established in vitro model to screen for oral absorption and to evaluate the mechanism of transport. This assay can also be used to evaluate compounds as potential P-glycoprotein substrates and/or inhibitors. The traditional methods of sample analysis (high-performance liquid chromatography (HPLC) with a UV or fluorescence detector) limit the throughput and sensitivity of this assay. Data are presented here describing the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the analysis of samples derived from the Caco-2 cell studies. During the analysis an automatic switching valve was used to divert the flow from the HPLC column to waste for the first minute, preventing the early eluting salts from entering and contaminating the LC/MS interface. This approach allows the rapid and accurate determination of drug transport across the Caco-2 cell monolayer. The high sensitivity and specificity of LC/MS/MS make this technique an ideal candidate for the low concentration and high throughput routine analysis of Caco-2 cell solutions, especially if multiple compounds are administered and analyzed simultaneously. Thus, the use of LC/MS/MS will increase the value of the Caco-2 cell assay as an in vitro screening tool.     

Analysis of urinary nucleosides. I. Optimisation of high performance liquid chromatography/electrospray mass spectrometry

In order to optimise the analysis of urinary nucleosides by high performance liquid chromatography/mass spectrometry (HPLC/MS), the HPLC separation of these compounds was performed at different 'flow rates' and 0.2mL/min was found to give both a better separation and ionisation. The ionisation conditions were optimised to give the best intensity of the molecules quasi-molecular ions. The ion distribution profile and ionisation in both positive and negative mode were examined and the detection of the protonated molecule in positive mode chosen for further analysis. The limits of detection of the method developed are reported and representative LC/MS and LC/MS/MS spectra shown. Typical urinary nucleoside chromatograms are presented.     

Coupling techniques in LC/MS and SFC/MS

Summary Techniques and applications of analytical instruments combining a chromatographic technique, including liquid chromatography and supercritical fluid chromatography, with mass spectrometry (LC/MS and SFC/MS), that have appeared over the past five years, are reviewed and discussed. It is shown that still many different methods co-exist and have both specific advantages and limitations. SFC/MS appears easier to run for many compounds so far analysed by conventional LC/MS methods. On the other hand, new LC/MS methods that use fast atom bombardment or electrospray ionization have the greater potential for the investigation of polar biopolymers.