Liquid Chromatography-diode Array Detection-electrospray Ionization Research Articles

Characterization and simultaneous quantification of multiple constituents in Aurantii Fructus Immaturus extracts by HPLC-DAD-ESI-MS/MS

AimTo develop a sensitive and accurate analytical method for the profiling, identification and simultaneous quantification of the chemical constituents in Aurantii Fructus Immaturus extracts. MethodsA high performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-MS/MS) method was established for the characterization and quantification of multi-constituents in Aurantii Fructus Immaturus extracts. Chromatographic separation was achieved on a C18 column using a detailed gradient program with 2% formic acid and acetonitrile as the mobile phase. Mass spectrometry was in the negative mode for identification, and a diode array detector was set at 283 nm for quantification. ResultsEighteen compounds were identified, or tentatively identified, by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data, including 12 flavonoids, 5 coumarins and a limonoid in Aurantii Fructus Immaturus extracts. Twelve of the major compounds were determined simultaneously by HPLC-DAD. The fragmentation pathways of the authentic references were also investigated for the structural identification of the constituents in the extracts. There have been no reports on the simultaneous and comprehensive analysis of both the major and minor compounds in Aurantii Fructus Immaturus extracts. ConclusionsThe method validation indicated that the developed analysis method could be employed as a rapid, effective technique for the structural characterization and simultaneous quantification of multi-constituents in this botanical extract. This work is expected to provide comprehensive information for the quality evaluation of Aurantii Fructus Immaturus extracts, which should be a valuable support for the further study and development of this raw material and its related medicinal products.

Simultaneous qualitative and quantitative determination of major polymethoxylated flavonoids in the leaves of Murraya paniculata by RRLC-DAD-ESI-MSn

A rapid-resolution liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (RRLC-DAD-ESI-MSn) method was established to identify and quantify the major bioactive constituents in the leaves of Murraya paniculata (L.) Jack. Seven of the main PMFs were determined by the RRLC method with detection wavelength at 330 nm in 30 minutes. The total PMFs in the samples ranged from 12.688 mg g−1 to 54.997 mg g−1. Meanwhile, sixteen PMFs could be identified from the complex extract of the leaves of M. paniculata by the RRLC-ESI-MS/MS method. The developed RRLC method was validated in terms of good linearity, precision and accuracy. The RRLC-ESI-MS/MS method was simple and useful for rapid identification of principal constituents in M. paniculata.

Isolation and identification of phenolic compounds from rum aged in oak barrels by high-speed countercurrent chromatography/high-performance liquid chromatography-diode array detection-electrospray ionization mass spectrometry and screening for antioxidant activity

Beverages, especially wines are well-known to contain a variety of health-beneficial bioactive substances, mainly of phenolic nature which frequently exhibit antioxidant activity. Significant information is available about the separation and identification of polyphenols from some beverages by chromatographic and spectroscopic techniques, but considerably poor is chemical data related to the polyphenolic content in rums. In this paper, a method involving the all-liquid chromatographic technique of high-speed countercurrent chromatography (HSCCC) combined with high-performance liquid chromatography coupled with diode-array detection and electrospray ionization mass spectrometry (HPLC–DAD–ESI-MS n ) has been successfully applied for separation and identification of phenolic compounds in an aged rum. Besides, the phenolic fraction (PF) was assayed for its antioxidant effects using three different free radical in vitro assays (DPPH , RO 2 and spontaneous lipid peroxidation (LPO) on brain homogenates) and on ferric reducing antioxidant power (FRAP). Results showed that PF potently scavenged DPPH and strongly scavenged peroxyl radicals compared to ascorbic acid and butylated hydroxytoluene (BHT); and almost equally inhibited LPO on brain homogenates subjected to spontaneous LPO when compared to quercetin. Moreover, PF also exhibited strong reducing power. This chemical analysis illustrates the rich array of phenols in the aged rum and represents a rapid and suitable method for the isolation and identification of phenolic compounds from mixtures of considerable complexity, achieving high purity and reproducibility with the use of two separation steps.

High‐performance liquid chromatography‐diode array detection‐electrospray ionization multi‐stage mass spectrometric screening of an insect/plant system: the case of Spodoptera littoralis/Lycopersicon esculentum phenolics and alkaloids

High-performance liquid chromatography-diode array detection-electrospray ionization multi-stage mass spectrometry (HPLC-DAD-ESI-MS(n)) is considered to be a very valuable tool for the characterization of compounds found in trace amounts in natural matrices, as their previous isolation and clean-up steps can be avoided. Micro-scale separation increases the potential of this analytical technique, allowing the determination of compounds in reduced samples. Spodoptera littoralis represents a major challenge to Solanaceae plants, as it is one of the most deleterious pests. The S. littoralis/Lycopersicon esculentum system was studied for the first time concerning glycoalkaloids and phenolics. Using HPLC-DAD-ESI-MS(n) we were able to characterize 15 phenolic compounds in L. esculentum leaves. Nine of them are reported for the first time. Some differences were found between leaves of cerasiforme and 'Bull's heart' varieties. However, in the materials of S. littoralis (larvae, adults, exuviae and excrements) reared in both L. esculentum leaves no phenolics were identified. α-Tomatine was the main glycoalkaloid in the host plant. The glycoalkaloid composition of the different S. littoralis materials was distinct, with α-tomatine and dehydrotomatine being the main detected compounds in larvae and excrements. These results add knowledge to the ecological interaction in this insect/plant duo, for which it is hard to obtain considerable sample amounts.

Characterization and determination of the major constituents in Belamcandae Rhizoma by HPLC–DAD–ESI-MS n

Belamcandae Rhizoma, derived from the rhizome of Belamcanda chinensis (L.) DC., has been used as traditional Chinese medicine for the treatment of coughing and pharyngitis. However, there have been few studies dealing with the systematic analysis of the bioactive constituents in Belamcandae Rhizoma. In this work, high performance liquid chromatography–diode array detection–electrospray ionization multiple-stage mass spectrometry (HPLC-DAD-ESI-MS n ) combined with liquid chromatography–time of flight-mass spectrometry (HPLC–TOF/MS) was established for profiling and characterization of multi-constituent in Belamcandae Rhizoma. The ESI-MS n fragmentation behaviors of the authentic references were proposed for aiding the structural identification of components in the extract. Thirty-five flavonoids, including 30 isoflavones and five xanthones, were identified or tentatively identified by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data. Twelve of the identified compounds (neomangiferin, mangiferin, tectoridin, iristectorin B, iristectorin A, iridin, tectorigenin, iristectorigenin A, irigenin, irisflorentin, irilone and dichtomitin) were determined by HPLC–DAD using a C 18 column. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in herbal medicine. This work is expected to provide comprehensive information for the quality evaluation of Belamcandae Rhizoma, which would be a valuable reference for the further study and development of this herb and related medicinal products.

Determination of galacturonic acid content in pectin from fruit juices by liquid chromatographydiode array detection-electrospray ionization tandem mass spectrometry

Abstract A reversed-phase high-performance liquid chromatographic (HPLC) method is applied for the determination of galacturonic acid (GA) of pectins in different commercial fruit juices. The separation was carried out on a C18 column using precolumn derivatization with p-aminobenzoic acid (p-ABA) and UV detection at 304 nm. The identification of GA was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in positive ion mode. The concentration of GA in the samples analyzed ranged from 12.9 ± 0.5 to 49.4 ± 0.5 mgGA L−1. Amongst the samples analyzed, mango juice was found to be richest in GA content, and therefore a good source of pectins. Detection and quantification limits of the described methodology were 1.2 and 3.9 mg L−1, respectively. Quantitative GA recoveries in the beverages had a range between 90 and 98%. The results showed that the HPLC method proposed was precise and suitable for the identification and quantification of GA in commercial fruit juices.

Chemical Composition and Bioactivity of Pleiogynium timorense (Anacardiaceae)

A liquid chromatography-diode array detection-electrospray ionization mass spectrometric (HPLC-PDA-ESI/MS/MS) method was used for the analysis of the phenolic composition of the ethanolic extract obtained from the leaves of Pleiogynium timorense (DC.) Leenh. Twenty compounds were detected and tentatively characterized. In addition, further phytochemical investigations of the extract resulted in the isolation of twelve major phenolic compounds. Evidence of the structures of these compounds was obtained based on the interpretation of the UV, 1H NMR, 13C NMR and DQF-COSY spectral data. The antioxidant effect of the ethanolic extract was examined in vitro using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and super oxide anion radical scavenging assays. DPPH radical scavenging activity was observed for the extract, with an IC50 of 21.9 microg/mL, while its super oxide anion scavenging activity was less pronounced, with an IC50 of 123.5 microg/mL The ethanolic extract showed significant hypoglycemic, antioxidant and anti-inflammatory properties. This study suggests that the ethanolic extract of Pleiogynium timorense is a potential source of antioxidant compounds, relatively non-toxic, and have possible beneficial health effects.

Simultaneous Determination of Five Active Constituents in the Aerial Parts of Saururus chinensis by HPLC-DAD

The chemical profile of five main bioactive constituents including rutin, quercetin 3-O-β-D-glucuronopyranoside, quercetin 3-O-β-D-glucopyranosyl(1→2)-O-α-L-rhamnopyranoside, quercitrin and sauchinone in extract of Saururus chinensis was studied using high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS). The HPLC-DAD conditions were optimized for the simultaneous analysis of these five compounds. This method was validated in terms of specificity, linearity (R2 > 0.9987), precision (<6.4% RSD), and recoveries (93.9 − 103.0%). The limits of detection of these compounds were ranged from 38.7 to 648.4 ng. In addition, the seasonal variation of the chemical composition of these main compounds in S. chinensis was investigated using this validated method, which resulted in a significant difference in the contents of these compounds according to the harvest time.

Rapid Identification of two Species of Peucedanum by High-Performance Liquid Chromatography-Diode Array Detection-Electrospray Ionization Tandem Mass Spectrometry

The fragmentation behaviors of the angular- and linear-type coumarins from Peucedanum praeruptorum Dunn and P. decursivum (Miq.) Maxim were simultaneously investigated by HPLC-DAD/ESI-MS(n). For more structural identification, the fragment ions were analyzed and some possible fragmentation pathways were proposed. Different positions and numbers of the substituent also led to different fragment behaviors. Two types of coumarins from P. praeruptorum and P. decursivum were structurally elucidated by these techniques. In addition, UV spectra were applied to support the MS analysis. This is the first time that the two types of coumarins from herbal extracts have been differentiated by HPLC-DAD/ESI-MS(n). The method further illustrated the importance of the ESI-MS(n) technique in the identification of different types of coumarins and was applied for the rapid differentiation of the two herbs.

Analysis of Biologically Active Constituents in Centella asiatica by Microwave-Assisted Extraction Combined with LC–MS

Centella asiatica (L.) Urban is a traditional herbal medicine used in Asiatic countries, and is commonly used to treat various wounds, leprosy, tuberculosis and lupus diseases. In this work, a new method based on microwave assisted extraction followed by liquid chromatography–diode array detection–electrospray ionization multistage tandem mass spectrometry analysis has been developed for the identification and quantification of biologically active constituents in C. asiatica, including asiatic acid, asiaticoside and madecassoside. The separation was performed on an Agilent Eclipse C18 column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.1% aqueous acetic acid within 50 min. Detection was performed at 205 nm. The calibration curves showed good linearity (r2 > 0.9992). The limits of detection ranged from 1.2 to 1.6 μg mL−1. The intra- and inter-day precision was less than 3% and the recovery of the assay was in the range of 95.4–106.8%. The method was successfully applied to the quantification of the three constituents in different samples of C. asiatica. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of C. asiatica. The samples were also analyzed on a liquid chromatography–electrospray ionization–time-of-flight mass spectrometry system to confirm the identification results.

Metabolic analysis of four phenolic acids in rat by liquid chromatography–tandem mass spectrometry

A liquid chromatography-diode array detection-electrospray ionization ion trap mass spectrometry (LC–DAD–ESI-MS n ) method was established for the analysis of danshensu, caffeic acid, ferulic acid and isoferulic acid in rat plasma, bile, urine and feces after oral administration or intravenous injection. Liquid–liquid extraction was employed for the preparation of biosamples, and the chromatographic separation was carried out using an Agilent Zorbax Extend C 18 reversed phase column and acetonitrile-0.1% formic acid as the mobile phase. Totally nineteen metabolites were detected and identified as prototype, methylated, hydroxylated, sulfated and glucuronized conjugates. The metabolism of the individual phenolic acids in biosamples was investigated, and the metabolic pathway was proposed. By comparing the metabolism of different compounds which shared similar structures, we were able to find that methylation was the main pathway of danshensu metabolism, and the double bond on the side chain was critical for the drug excretion via bile and the formation of glucuronized conjugates. The results proved that the established method was simple, sensitive and reliable, which could be used to detect and identify the structures of metabolites and to better understand their in vivo metabolism.

Chromatographic fingerprint analysis and characterization of furocoumarins in the roots of Angelica dahurica by HPLC/DAD/ESI-MS n technique

A high performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry (HPLC/DAD/ESI-MS n ) method was used for the chromatographic fingerprint analysis and characterization of furocoumarins in the roots of Angelica dahurica for the first time. Nine “common peaks” were identified by comparing with the retention time, UV and MS spectra of reference furocoumarins. The software “Similarity Evaluation System for Chromatographic Fingerprint of TCM” was used to evaluate the similarities of 13 batches of Baizhi samples collected from Henan, Zhejiang, Sichuan and Anhui provinces of China. The results indicated that the samples from different batches had similar HPLC fingerprints, and the method could be applied for the quality control of the roots of Angelica dahurica. In addition, a total of 20 furocoumarins were identified or tentatively characterized by HPLC/DAD/ESI-MS n technique, and their fragmentation patterns in an electrospray ion trap mass spectrometer were also summarized.

Simultaneous analysis of coumarins and secoiridoids in Cortex Fraxini by high-performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry

A high-performance liquid chromatography–diode array UV detection–electrospray ionization tandem mass spectrometry (HPLC–DAD–ESI-MS) method was developed and validated for the simultaneous analysis of seven major constituents in Cortex Fraxini, including esculin, esculetin, fraxetin, fraxin, escuside, oleuropein and ligustroside. The contents of the seven constituents were determined by using HPLC–DAD, and the chemical structures of these constituents were identified by using HPLC–DAD–ESI-MS method. The separation was performed on an Agilent Zorbax SB-C 18 column (250 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.3% aqueous acetic acid within 40 min. Detection was performed at 254 nm. The calibration curves showed good linearity ( r 2 > 0.9992). The limits of detection (LOD) ranged from 1.07 to 3.19 ng/ml and limits of quantification (LOQ) ranged from 2.79 to 12.75 ng/ml, respectively. The intra- and inter-day precision was less than 5% and the accuracy was ranging from 96.49% to 103.55%. The recovery of the assay was in the range of 97.61–104.36%. The method was successfully applied to the quantification of the seven constituents in different samples of Cortex Fraxini. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of Cortex Fraxini.

Studies on lignan constituents from Schisandra chinensis (Turcz.) Baill. fruits using high‐performance liquid chromatography/electrospray ionization multiple‐stage tandem mass spectrometry

A reversed-phase high-performance liquid chromatography-diode array detector-electrospray ionization multiple-stage tandem mass spectrometry (RP-HPLC-DAD-ESI-MS(n)) method has been developed for the detection and analysis of lignan constituents in the methanol extract from the fruits of Schisandra chinensis (Turcz.) Baill. RP-HPLC-DAD-ESI-MS(n) and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MS(n)) have been applied to investigate the characteristic product ions of four lignan reference compounds. Then, the logical fragmentation pathways of the lignans have been proposed. By comparing the retention time (t(R)) of HPLC, the ESI-MS(n) data and the structures of analyzed compounds with the data of reference compounds and in the literature, 11 peaks in HPLC have been unambiguously identified and another 5 peaks have been tentatively identified or deduced. Also, in the present paper, the extracted ion chromatograms (EIC) have been used to analyze the lignan isomers. The experimental results demonstrate that RP-HPLC-DAD-ESI-MS(n) is a specific and useful method for the identification of the lignan constituents and their isomers.

Identification of tanshinones and their metabolites in rat bile after oral administration of TTE-50, a standardized extract of Salvia miltiorrhiza by HPLC–ESI–DAD–MS n

TTE-50 is a standardized extract of Salvia miltiorrhiza which mainly consisted of tanshinones. A sensitive and specific method using liquid chromatography–diode array detection-electrospray ionization (ESI) ion trap mass spectrometry was established for the study of the constituents and metabolites of TTE-50 in rat bile sample after oral administration. The bile samples were extracted with ethyl acetate (EtOAc) of three-fold volume for three times. The chromatographic separation was carried out on a Zorbax Extend-C 18 column with a gradient elution program whereas acetonitrile–water was used as mobile phase. Mass spectra were acquired in positive ionization mode and data-dependant scan was used for the identification of the tanshinones and metabolites in the bile samples. Identification and structural elucidation of the tanshinones and their metabolites in bile samples were performed by comparing their retention-times and full scan MS n spectra with those of reference compounds and data in the literatures. Sixteen tanshinones in TTE-50 along with seventeen phase I metabolites were identified simultaneously. The metabolic modification could take place in the C-4 side chain of tanshinone IIA, from methyl to primary alcohol, then to aldehyde group was proposed for the first time. The established method was valuable for the study of the metabolism of complex system such as herbal extracts or traditional Chinese medicine (TCM) formula.

Fingerprint analysis of the fruits of Cnidium monnieri extract by high-performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry

A method incorporating high-performance liquid chromatography (HPLC) with electrospray ionization (ESI) and tandem mass spectrometry (MS), with parallel analysis by HPLC with UV detection using a diode-array detector (DAD) was developed for the qualitative characterization of coumarin and chromone constituents in the fruits of Cnidium monnieri. The chromatographic separations were performed on a Diamonsil™ C18 column (4.6 mm × 200 mm, 5 μm) with water with 50 mM ammonium acetate and 2% acetic acid (A) and acetonitrile (B) as the mobile phase. According to the characteristic UV spectra, the information of molecular weight and structure provided by ESI–MS/MS, 13 coumarin and 7 chromone components were detected and identified. This method is rapid and reliable for identification of the constituents in the complex herbal system, and the fragmentation patterns proposed could be extended to the similar compounds.

Identification and analysis of absorbed and metabolic components in rat plasma after oral administration of ‘Shuangdan’ granule by HPLC–DAD–ESI-MS/MS

A valid method using liquid chromatography–diode array detection–electrospray ionization (ESI) ion trap mass spectrometry was established for the study of the absorbed and metabolic components of a Chinese medicine ‘Shuangdan’ granule in rat plasma after oral administration. The plasma samples were acidified with 1 M hydrochloric acid and extracted with ethyl acetate (EtOAc) of two-fold volume for three times. The chromatographic separation was carried out on a Zorbax SB-C 18 column with a linear gradient whereas 0.1% acetic acid/water/acetonitrile was used as mobile phase. Mass spectra were acquired in both negative and positive modes. More than 20 components including 16 components from ‘Shuangdan’ granule and 5 metabolites were simultaneously identified by comparing their mass spectra and retention behavior with reference compounds or literature data. The results proved that the established method could be used to identify the structure of active components responsible for the pharmacological effects of ‘Shuangdan’ granule.