Simultaneous determination of phenolic acids and flavonoids in Lycium barbarum Linnaeus by HPLC–DAD–ESI-MS

A high-performance liquid chromatography–diode array detection–mass spectrometry method with electrospray ionization mode (HPLC–DAD–ESI-MS) was developed for simultaneous determination of phenolic acids and flavonoids in fruits of Lycium barbarum Linnaeus, a widely used traditional Chinese herb possessing vital biological activity. Both phenolic acids and flavonoids were extracted with 50% ethanol and purified using a polymeric solid phase extraction cartridge followed by HPLC–DAD–ESI-MS analysis. By employing a Vydac C18 column, a total of 52 phenolic acids and flavonoids were separated within 70 min using a gradient mobile phase of 0.5% (v/v) formic acid in water and acetonitrile–water (94:6, v/v) with flow rate at 1 mL/min, column temperature at 30 °C and detection wavelength at 280 nm. Of 52 compounds, 15 phenolic acids and flavonoids were positively identified based on both absorption and mass spectra, with the remaining 37 tentatively identified by comparison of absorption spectra with reported values in the literature. Internal standards 3-hydroxybenzoic acid and hesperidin were used for quantitation of phenolic acids and flavonoids, respectively. Among the 15 positively identified compounds, quercetin-rhamno-di-hexoside was present in largest mass fraction (438.6 μg/g), followed by quercetin-3-O-rutinoside (281.3 μg/g), dicaffeoylquinic acid isomers (250.1 μg/g), chlorogenic acid (237.0 μg/g), quercetin-di-(rhamnohexoside) (117.5 μg/g), quercetin-di-(rhamno)-hexoside (116.8 μg/g), kaempferol-3-O-rutinoside (97.7 μg/g), isorhamnetin-3-O-rutinoside (72.1 μg/g), p-coumaric acid (64.0 μg/g), caffeic acid (23.7 μg/g) and vanillic acid (22.8 μg/g).     

Phytochemical analysis of an antiviral fraction of Radix astragali using HPLC–DAD–ESI–MS/MS

Radix astragali (Huangqi in Chinese) is a well-known traditional Chinese medicinal herb that has been used clinically in China for centuries to cure various diseases. To profile the antiviral constituents of this herb, a high-performance liquid chromatography–diode array detector–electrospray ionization–tandem mass spectrometry (HPLC–DAD–ESI–MS/MS) analytical method was developed to separate and determinate the active part of the extract of Radix astragali, which showed potent inhibition of several viruses. By comparing their retention time and MS data with those obtained from the authentic compounds and the published data, a total of 18 compounds, comprising 11 flavonoids and 7 saponins, were identified. This study provides an approach to rapidly characterize bioactive constituents in traditional Chinese medicines (TCMs).     

Identification and determination of the major constituents in Traditional Chinese Medicinal formula Danggui-Shaoyao-San by HPLC–DAD–ESI-MS/MS

Danggui-Shaoyao-San (DSS), a famous traditional Chinese medicine formula consisting of six herbal medicines (Paeonia lactiflora, Angelica sinensis, Ligusticum chuanxiong, Poria cocos, Atractylodis macrocephalae and Rhizoma Alismatis), has been used as a classical gynecological remedy in China for centuries. However, its active substances have remained unknown. In this paper, an HPLC/DAD/ESI-MS/MS method was developed for the qualitative and quantitative analysis of the major constituents in DSS. The ESI-MS/MS fragmentation behavior of the reference compounds was proposed for aiding the structural identification of components in DSS extract. Forty-one compounds including monoterpene glycosides, phenolic acids, phathalides, sesquiterpenoids and triterpenes were identified or tentatively characterized by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data, and 14 of them (gallic acid, albiflorin, paeoniflorin, ferulic acid, benzoic acid, senkyunolide I, coniferyl ferulate, senkyunolide A, 3-butylphthalide, Z-ligustilide, Z-butylidenephthalide, atractylcnolide II, atractylcnolide I and levistolide A) were determined by HPLC–DAD using a C₁₈ column and gradient elution of acetonitrile/water–formic acid (100:0.1, v/v). The linearity, precision, accuracy, LOD and LOQ were validated for the quantification method, which proved sensitive, accurate and reproducible. The study might provide a basis for the quality control of DSS extracts and preparations.     

Identification of multiple constituents in the traditional Chinese medicine formula GuiZhiFuLing-Wan by HPLC–DAD–MS/MS

GuiZhiFuLing-Wan (GFW) has been used in China for centuries to improve blood stagnation. In this paper, a HPLC–DAD–MS/MS method was established for the efficient and rapid identification of the chemical constituents in extract of GuiZhiFuLing-Wan. Separation was performed on an Alltima C₁₈ analytical column by gradient elution with CH₃CN/H₂O–CH₃COOH as mobile phase at a flow rate 1.0 ml/min. 27 potentially bioactive compounds including monoterpene glycosides, galloyl glucoses, acetophenones, phenylallyl compounds and triterpenoids were identified or tentatively characterized by online ESI/MS/MS and the comparison with literature data and authentic compounds. After the identification, six different brands of GFW commercial products in various dosage forms were evaluated. The results demonstrated that capsule of GFW was superior to the other two dosage forms, honeyed pill and concentrated pill in administration. The points that should be paid more attention during the manufacturing process of GFW were also analyzed. The method can be the basis for the quality control of this commonly used herbal formula.     

Simultaneous analysis of bioactive metabolites from Ziziphus jujuba by HPLC–DAD–ELSD–MS/MS

A high-performance liquid chromatography (HPLC) with diode array detector (DAD), evaporative light scattering detector (ELSD) and electrospray ionization mass spectrometry (ESI-MS) was established for the simultaneous determination of nine representative metabolites of the alkaloid, flavonoid and triterpenoid classes from Ziziphus jujuba var. spinosa. The optimal chromatographic conditions were obtained on an ODS column and the mobile phase was composed of water and methanol with 0.1% formic acid using a gradient elution system. Using the developed methods, all of the validation parameters were successfully obtained. In addition, effectiveness of diverse extraction methods was compared to each other for the development of standard analytic method. The verified method was successfully applied to the quantitative determination of representative metabolites in commercial samples of Z. jujuba and Z. mauritiana from different markets in Korea, China and Myanmar. The analytical results showed that the contents of the nine analytes vary significantly with sources and species, thus demonstrating its potential for the detection of this plant.     

Quantitative determination of hippuric and benzoic acids in urine by LC–MS/MS using surrogate standards

An LC–MS/MS method for simultaneous determination of hippuric acid (HA) and benzoic acid (BA) in monkey urine after direct injection was developed. Since HA and BA are endogenous compounds in urine, surrogate standards (¹³C₆-hippuric and ¹³C₆-benzoic acid) were employed to generate calibration curves. l-Phenylalanine-ring-D5 served as an internal standard. Multiple reaction monitoring in the negative ionization mode with an APCI source was used for detection of all components in the assay. The developed method is intended for determination of HA and BA in the range of 0.25–250 and 0.1–100 μg/ml, respectively. Weighted (1/x) quadratic regression (r² > 0.99) was used to generate calibration curves. Precision and accuracy of the method were assessed by analyzing 3 quality control samples (concentrations at low, medium, and high range of calibration curve) prepared in monkey urine. Stability for 48 h at room temperature and after 3 freeze–thaw cycles was also evaluated.     

HPLC–DAD–MS/MS profiling of standardized rosemary extract and enhancement of its anti-wrinkle activity by encapsulation in elastic nanovesicles

The anti-wrinkle activity of defatted rosemary extract (DER) was assessed, and its effect was optimized by encapsulation in transferosomes (TFs). DER was standardized to a rosmarinic acid content of 4.58 ± 0.023 mg% using reversed-phase high performance liquid chromatography (Rp-HPLC), and its components were identified by HPLC-diode array detection-tandem mass spectrometry. In vitro free radical scavenging assays showed DER had high free radical scavenging activity against 2,2-diphenyl-2-picryl hydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and superoxide radicals. DER also inhibited bleaching of β-carotene with high Fe(III) and Fe(II) chelating ability. In vivo anti-wrinkle activities of topically applied DER (20, 50, and 100 mg) and a TF formulation (TF4, 20 mg of DER) were evaluated in UVB-irradiated mice using a wrinkle scoring method, metalloproteinase (MMP) expression, and histopathology. Among the nanovesicles, TF4 was the most deformable, and had an acceptable size and encapsulation efficiency and enhanced permeation of DER through rat skin compared with unencapsulated DER. DER (50 and 100 mg) and TF4 significantly inhibited MMP-2 and MMP-9 expression and improved wrinkle scores. DER and TF4 moderately decreased epidermal thickness without pigmentation. DER is a potent natural antioxidant for combating skin aging. Moreover, encapsulation of DER in TFs will enhance its skin permeation and anti-wrinkle activity.     

Simultaneous quantification of marker components in Ojeok-san by HPLC–DAD

A systematic high-performance liquid chromatography–diode array detection (HPLC–DAD) method was developed for the rapid, accurate, and simultaneous quantification of eight marker compounds, paeoniflorin, 6-gingerol, decursin, glycyrrhizin, cinnamic acid, hesperidin, poncirin and magnolol, in Ojeok-san, a traditional Korean herbal medicine. These compounds were separated in less than 50 min using a Dionex C₁₈ column with a gradient elution system of water and methanol at a flow rate of 1 ml/min. All calibration curve of standard components showed excellent linearity (R ² > 0.9922). Limits of detection (LOD) and limits of quantification (LOQ) ranged from 0.01 to 0.11 μg/ml and 0.03 to 0.34 μg/ml, respectively. The relative standard deviations (RSDs) of data of the intra- and inter-day experiments were less than 2.15 and 2.60%, respectively. The accuracy of recovery test ranged from 94.27 to 107.68% with RSD values 0.15–3.61%. The results of validation showed that the HPLC method was stable and very accurate for the quantification of eight marker components in Ojeok-san.     

Simple and reproducible HPLC–DAD–ESI-MS/MS analysis of alkaloids in Catharanthus roseus roots

Catharanthus roseus is one of the most important medicinal plants worldwide. The leaves of this species are the only source of the indolomonoterpenic alkaloids vincristin (leurocristine) and vinblastin (vincaleucoblastine), whose anticancer activity represents powerful therapeutics to many diseases, such as Hodgkin lymphoma. Usually, the remaining plant parts go to waste. Here we describe a phytochemical study on this species roots. Alkaloids in aqueous extracts, the usual form of consumption of this matrix, were studied using HPLC–DAD–ESI-MS/MS, which allowed the identification of 19-S-vindolinine, vindolinine, ajmalicine and an ajmalicine isomer, tabersonine, catharanthine, serpentine and a serpentine isomer. Quantification of the identified compounds revealed that serpentine and its isomer were predominant (64.7%) over the other alkaloids, namely vindolinine and its isomer (23.9%), catharanthine (7.7%) and ajmalicine (3.8%). The used procedure revealed to be simple, sensitive and reproducible.     

Determination of tramadol and metabolites by HPLC-FL and HPLC–MS/MS in urine of dogs

Tramadol is a centrally acting analgesic drug used in veterinary and human clinical practice. Its metabolism has been largely characterized in human being but is still long to be comprehended in several animal species, especially in the dog. The aim of the present study was to develop and validate a new analytical procedure to investigate HPLC the metabolization/elimination process tramadol in urine of dogs by HPLC-FL or HPLC–MS/MS. A single oral dose of tramadol (4 mg/kg) was administered to 4 male Beagle dogs and the urine was naturally collected. This matrix either hydrolyzed than un-hydrolyzed was extracted with different blends of solvents to detect the total or free form of the analytes, respectively.     

Supercritical fluid CO2 extraction and simultaneous determination of eight annonaceous acetogenins in Annona genus plant seeds by HPLC–DAD method

Annonaceous acetogenins (ACGs) isolated from Annonaceae plants exhibited a broad range of biological bioactivities such as cytotoxic, antitumoral, antiparasitic, pesticidal and immunosuppresive activities. However, their structures were liable to change at more than 60 °C and their extraction yields were low using traditional organic solvent extraction. In the present study, all samples from Annona genus plant seeds were extracted by supercritical carbon dioxide under optimized conditions and a high-performance liquid chromatography (HPLC) method was established for simultaneously determining eight ACGs. All of the eight compounds were simultaneously separated on reversed-phase C₁₈ column (250 mm × 4.6 mm, 5 μm) with the column temperature at 30 °C. The mobile phase was composed of (A) methanol and (B) distilled water, the flow rate was 1.0 ml/min and the detection wavelength was set at 220 nm. All calibration curves showed good linear regression (γ > 0.9995) within the test range. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 0.87–2.53% and 1.91–3.42%, respectively, and overall recoveries of 95.81–105.39% for the eight compounds analyzed. The established method can be applied to evaluate the intrinsic quality of Annonaceae plant seeds. The determination results recover the content-variation regularities of various ACGs in different species, which are helpful to choose the good-quality Annonaceae plant seeds for anticancer lead compound discovery.     

Quality assessment of traditional herbal formula,Hyeonggaeyeongyo-tang through simultaneous determination of twenty marker components by HPLC–PDA and LC–MS/MS

Simultaneous analysis of 20 marker components (gallic acid, cimifugin, geniposide, paeoniflorin, ferulic acid, nodakenin, narirutin, naringin, neohesperidin, arctiin, baicalin, oxypeucedanin hydrate, wogonoside, baicalein, arctigenin, glycyrrhizin, wogonin, pulegone, decursin, and decursinol angelate) for quality assessment of the traditional herbal formula, Hyeonggaeyeongyo-tang (HYT) was carried out by using high-performance liquid chromatography (HPLC) with photodiode array detection (PDA) and liquid chromatography–mass spectrometry with tandem mass spectrometry (LC–MS/MS). The coefficient of determination showed excellent linearity of more than 0.9999 for all analytes. The recovery of 20 marker components was 93.92 to 102.66% with relative standard deviation (RSD) < 3.00% and RSD value of precision was ≤ 3.44%. The amounts of 20 marker components using HPLC–PDA and LC–MS/MS were determined to be 0.18–14.60 and 0.01–1.76 mg/freeze-dried g, respectively.     

Analysis of the constituents of aqueous preparations of Stachys recta by HPLC–DAD and HPLC–ESI-MS

In the present study a method based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of phenolic constituents in three aqueous preparations of the herbal medicinal drug Stachys recta. The developed assay was simple and effective and permitted the quality control of S. recta decoctions and infusion. Overall, 30 constituents were detected and identified, belonging mainly to three classes of compounds: caffeoylquinic acids, phenylethanol glycosides and flavonoids. 15 of them were quantified having a lower limit not less than 0.02% of the lyophilized extracts. Only seven of them were previously reported in this species, while 23 were identified for the first time as constituents of S. recta. HPLC–DAD–ESI-MS analysis provided evidence for the certain identification of the main constituents and in some cases of their isomers. Eight constituents were isolated and their structure elucidated by HPLC–ESI-MS and 1D- and 2D-NMR spectroscopy. Among the investigated preparations, the infusion seems to be the best method to extract the native constituents of the plant, while decoction is a more aggressive treatment and causes partial degradation of some acylated flavonoids.     

Microsomal metabolism of calycosin,formononetin and drug–drug interactions by dynamic microdialysis sampling and HPLC–DAD–MS analysis

A dynamic microdialysis sampling method with liquid chromatography–diode array detection and time-of-flight mass spectrometry (LC–DAD–TOF/MS) analysis was developed to investigate rat microsomal metabolisms of calycosin and formononetin, and their drug–drug interactions. Two hydroxylated metabolites from calycosin, and three hydroxylated or 4′-O-demethylated derivatives from formononetin were detected and identified after co-incubation with microsomes. Calibration curves offered linear ranges of two orders of magnitude with r² > 0.999 for calycosin, formononetin and daidzein. The quantitative LC method provides a range of 0.028–0.034 μg/mL for limits of detection, overall precision less than 5% and accuracy less than 3% by RSD. Besides, calycosin and formononetin were found to produce the depressive effect on the CYP450 enzyme reaction, and inhibit phase I enzyme reaction of each other when they are concurrent. Dynamic microdialysis sampling with LC–DAD–TOF/MS analysis developed in this work is a powerful tool for in vitro metabolism studies of drugs and metabolic interactions.     

LC–MS/MS determination of carbamathione in microdialysis samples from rat brain and plasma

A selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of S-(N, N-diethylcarbamoyl) glutathione (carbamathione) in microdialysis samples from rat brain and plasma. S-(N, N-Diethylcarbamoyl) glutathione (carbamathione) is a metabolite of disulfiram. This metabolite may be responsible for disulfiram's effectiveness in the treatment of cocaine dependence. Chromatographic separations were carried out on an Alltech Altima C-18 (50 mm long × 2.1 mm i.d., 3 μm particles) analytical column at a flow rate of 0.3 ml/min. Solvent A consisted of 10 mM ammonium formate, methanol, and formic acid (99:1:0.06, v/v/v). Solvent B consisted of methanol, 10 mM ammonium formate and formic acid (99:1:0.06, v/v/v). A 20 min linear gradient from 95% aqueous to 95% organic was used. Tandem mass spectra were acquired on a Micromass Quattro Ultima “triple” quadrupole mass spectrometer equipped with an ESI interface. Quantitative mass spectrometric analysis was conducted in positive ion mode selected reaction monitoring (SRM) mode looking at the transition of m/z 407–100 and 175 for carbamathione and m/z 392–263 for the internal standard S-hexyl glutathione. The simultaneous collection of microdialysate from blood and brain was used to monitor carbamathione concentrations centrally and peripherally. Good linearity was obtained over a concentration range of 0.25–10,000 nM. The lowest limit of quantification (LLOQ) was determined to be 1 nM and the lowest limit of detection (LLOD) was calculated to be 0.25 nM. Intra- and inter-day accuracy and precision were determined and for all the samples evaluated, the variability was less that 10% (R.S.D.).     

Determination of nucleosides and nucleobases in different species of Cordyceps by capillary electrophoresis–mass spectrometry

In present study, a capillary electrophoresis–mass spectrometry (CE–MS) method was developed for the simultaneous analysis of 12 nucleosides and nucleobases including cytosine, adenine, guanine, cytidine, cordycepin, adenosine, hypoxanthine, guanosine, inosine, 2′-deoxyuridine, uridine and thymidine in natural and cultured Cordyceps using 5-chlorocytosine arabinoside as an internal standard (IS). The CE separation conditions and MS parameters were optimized systematically for achieving good CE resolution and MS response of the investigated compounds. The optimum CE electrolyte was 100 mM formic acid containing 10% (v/v) methanol. The optimum MS parameters were as follows: 75% (v/v) methanol containing 0.3% formic acid with a flow rate of 3 μL/min was selected as the sheath liquid; the flow rate and temperature of drying gas were 6 L/min and 350 °C, respectively. The optimized CE–MS method was successfully applied for the simultaneous determination of 12 nucleosides and nucleobases in natural and cultured Cordyceps. On the basis of quantitative results, the total content of nucleosides is much higher in cultured Cordyceps (9138 ± 4823 μg/g for cultured C. sinensis; 3722 ± 1446 μg/g for C. militaris) than in natural ones (2167 ± 412 μg/g). However, the hypoxanthine (131 ± 47 μg/g) and inosine (335 ± 90 μg/g) are much higher in natural C. sinensis. Cordycepin, which is abundant in cultured C. militaris (2276.5 ± 842.6 μg/g), is only found in natural C. sinensis with very low content and cannot be detected in the cultured ones.     

Rapid simultaneous identification and determination of the multiple compounds in crude Fructus Corni and its processed products by HPLC–MS/MS with multiple reaction monitoring mode

Context: Fructus Corni, a traditional Chinese medicines, is derived from the dry ripe sarcocarp of Cornus officinalis Sieb. et Zucc (Cornaceae). Gallic acid, 5-hydroxymethylfurfural, morroniside, sweroside, loganin, cornin, 7-O-methyl-morroniside and cornuside are the active constituents of Fructus Corni used in many traditional Chinese medicines. This paper describes a sensitive and specific assay for the determination of the eight bioactive compounds in crude and processed Fructus Corni extracts.

Materials and methods: In this paper, the eight components were determined by high performance liquid chromatography coupled with triple quadrupole mass spectrometry (MS/MS). Quantization was based on multiple reaction monitoring using the precursor production combination for determination of the eight analytes. The analysis was performed on an Agilent Zorbax Extend C₁₈ column (100?mm × 3.0?mm, 3.5 μm), and an electrospray ionization (ESI)-tandem interface in the positive and negative ion polarity mode was employed prior to mass spectrometric detection.

Results: With the optimized conditions, the eight bioactive compounds were detected properly within 10?min. The developed method showed good precision and reproducibility with the limits of detection ranged from 0.0042 to 12.7875?ng/mL and the average recoveries ranged from 97.08 to 103.7%.

Discussion and conclusion: This newly established method is validated as simple, reliable and accurate. It can be used as a valid analytical method for intrinsic quality control of crude and processed Fructus Corni.