Fingerprint Analysis of Euonymus alatus (Thuhb) siebold by LC–DAD and LC–ESI–MS

A liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry method was first developed for a chemical fingerprint analysis of Euonymus alatus (Thuhb) siebold (EAS) and rapid identification of major compounds in the fingerprints. Fingerprint profiles were found to be consistent for the herbs acquired from different locations, but the relative abundance of peaks was varied. Twelve peaks were chosen as the common peaks. Quercetin and rutin were detected by comparing the retention times, MS and UV spectra with the standards. The relative retention time and relative peak area of the 12 peaks in the fingerprint were calculated by setting the quercetin as the reference peak. The experimental data were used for similarity calculation and hierarchical clustering analysis. By comparing the UV and MS spectra data with those of the authentic standards and literature, five main peaks in the fingerprints were identified. Finally, five medicinal portions of the herb (leaf, fruit, stem, pterygium and root) were also analyzed by this method. It was found that there were similar chemical components in different parts of this herb but the contents were very different. The developed fingerprint assay was specific and could be readily utilized for comprehensive evaluation of EAS, as well as to distinguish different medicinal portions.     

Characterization and quantification of anthocyanins in selected artichoke (Cynara scolymus L.) cultivars by HPLC–DAD–ESI–MS n

The anthocyanin pattern of artichoke heads (Cynara scolymus L.) has been investigated by high-performance liquid chromatography–electrospray ionization mass spectrometry. For this purpose a suitable extraction and liquid chromatographic method was developed. Besides the main anthocyanins—cyanidin 3,5-diglucoside, cyanidin 3-glucoside, cyanidin 3,5-malonyldiglucoside, cyanidin 3-(3′′-malonyl)glucoside, and cyanidin 3-(6′′-malonyl)glucoside—several minor compounds were identified. Among these, two peonidin derivatives and one delphinidin derivative were characterized on the basis of their fragmentation patterns. To the best of our knowledge this is the first report on anthocyanins in artichoke heads consisting of aglycones other than those of cyanidin. Quantification of individual compounds was performed by external calibration. Cyanidin 3-(6′′-malonyl)glucoside was found to be the major anthocyanin in all the samples analyzed. Total anthocyanin content ranged from 8.4 to 1,705.4 mg kg⁻¹ dry mass.      

Simultaneous Chromatographic Fingerprinting and Quantitative Analysis of Flemingia philippinensis by LC–DAD

To evaluate the quality of Flemingia philippinensis, a validated reversed-phase high-performance liquid chromatographic method with diode-array detection has been developed for chromatographic fingerprint analysis and for quantification of genistin, genistein, and 5,7,3′,4′-tetrahydroxy-6,8-diprenylisoflavone, the three main bioactive isoflavones. In the fingerprint analysis, 21 chromatographic peaks were selected as characteristic peaks; among these 14 flavonoids were identified on the basis of their retention times and UV spectra. In quantitative analysis, the recovery of the method was in the range 94.5–107.7%, and good linearity (r ² > 0.9990) was observed for all the compounds over a relatively wide range of concentrations. The relative standard deviation of the method was less than 2.5% for intra-day and inter-day assays. The assay was successfully applied to analysis of the three active isoflavones in 19 samples. The results indicated the assay is reproducible and precise and could be used for convenient assessment of the quality of F. philippinensis.     

LC–DAD–MS Determination of the Major Constituents in Radix Angelicae sinensis

Column liquid chromatography with diode array and mass spectrometric detection was developed for the qualitative and quantitative analysis of the major constituents in Radix Angelicae sinensis. Sixteen compounds including phthalic acid, vanillic acid, ferulic acid, guaiacol, Z-6,7-epoxyligustilide, senkyunolide F, carvacrol, Z-ligustilide, Z-butylidenephthalide, E-6,7-dihydroxydihydroligustilide, senkyunolide I or senkyunolide H, coniferyl ferulate, sendanelolide, butylphthalide, E-ligustilide, E-butylidenephthalide except guaiacol and carvacrol were identified using online ESI–MS in comparisons with literature data and standard compounds. Nine compounds were quantified by LC–DAD simultaneously. For chromatographic analysis, a Merck C₁₈ column (5 μm, 250 mm × 4.6 mm) with a compatible guard column (C₁₈, 5 μm, 7.5 mm × 4.6 mm) was used. The mobile phase consisted of (A) 0.1% aqueous formic acid and (B) acetonitrile. Ten microliters of each sample solution were injected and eluted at a flow rate of 1 mL min^-1. The column temperature was maintained at 30 °C. The validation of this method proved good linear regression (r ² > 0.9992) within the test ranges, desirable repeatability with overall intra- and inter-day variations of less than 4.91% and well acceptable recoveries varied between 90.91 and 96.73% while the RSDs were below 3.23% (n = 3). The proposed method was successfully applied to the quantification of the nine components in sixteen samples from different localities in China. This assay provides a valid and an overall quality control of Radix Angelicae sinensis.     

Study on Absorption of Radix Astragali in Rabbit Plasma by Alternative Moving Window Factor Analysis and LC–DAD–MS

In the present paper, study on the multiple absorbed bioactive components of Radix Astragali in rabbit was performed by alternative moving window factor analysis (AMWFA) and LC–DAD–MS. AMWFA was used to confirm the common components both in its abstract fingerprint and plasma fingerprint. This method could be utilized not only to determine the number of common components in different systems but also to identify their corresponding spectra. As can be seen from the results, the method determines the common components in different chromatographic fingerprints more efficient and reliable, especially for none or only weak selective information of some chromatographic peak clusters. With the aid of AMWFA both its abstract fingerprint and plasma fingerprint, one could find that 25 chemical components in RA were absorbed into the rabbit body after oral solution of RA administration.     

Simultaneous determination of eight adulterants in weight management supplements and herbs by HPLC–DAD and LC–MS/MS

An efficient HPLC–DAD method was developed for simultaneous determination of eight adulterants in weight management supplements and herbs. The eight adulterants were phenolphthalein, sibutramine, nuciferine, and five anthraquinone compounds including aloe-emodin, rhein, emodin, chrysophanol, and physcion. The analytes were ultrasonically extracted with 70% (v/v) methanol aqueous solution followed by centrifugation. The supernatant was subjected to HPLC analysis. A Phenomenex Luna C18 column was applied for chromatographic separation. The mobile phase was consisted of methanol and aqueous solution of 0.05% (v/v) phosphoric acid–0.025% (m/v) sodium dodecyl sulfonate. The flow rate of mobile phase was 0.8?ml?min^?1 with gradient elution. Clenbuterol and ibuprofen were used as internal standards. The retention times and the characteristic UV spectrograms were used for qualitative analysis. Quantifications were based on the internal standard curves. Good linearities (r?>?0.9996) for all analytes were obtained with the intra- and inter-day precision (n?=?6) ranging from 0.76 to 5.9% and 0.90 to 8.1%, respectively. The average recoveries from the spiked samples with different matrices varied from 73.4 to 114%. Validations were subsequently performed using LC–MS/MS. The proposed method successfully determined the target adulterants in eight commercial weight management supplements and five weight reducing herbs with satisfactory results.     

Microwave-Assisted Extraction of Melamine Residues from Pet Food and Analysis by Ion-Exchange LC–DAD

Melamine is attracting much attention because of its toxicity. In the work discussed in this paper, microwave-assisted extraction in combination with ion-exchange high-performance liquid chromatography with diode-array detection was used for analysis of melamine in pet food. Trichloroacetic acid–N,N-dimethylformamide 10:1 was the best extractant, because of the strong polarity of melamine. Separation was performed on a 250 mm × 4.6 mm i.d., 5-μm particle, cation-exchange column; isocratic elution with a mixture of ammonium formate solution (pH 3.0) and acetonitrile was complete within 10 min. UV absorbance DAD detection was performed at 240 nm. Response was a linear function of melamine concentrations from 0.1 to 50 μg mL^?1, with a detection limit of 1.0 mg kg^?1. Intra-day and inter-day precision, as RSD, was <3% and the recovery of the assay was in the range 95.4–106.8%. In analysis of spiked pet food, the new method yielded satisfactory results. Because of its simplicity and accuracy this straightforward method is particularly suitable for routine melamine analysis.     

Determination of Drospirenone in Human Plasma by LC–Tandem-MS

A bioanalytical method has been developed and validated for determination of drospirenone in human plasma. The analytical method consists in the extraction of plasma sample with dichloromethane, followed by determination of drospirenone by LC–MS–MS using levonorgestrel as internal standard. Separation was achieved on a Peerless cyano column with an isocratic mobile phase consisting of methanol and ammonium formate buffer. Protonated ions formed by a Turboionspray in the positive mode were used to detect analyte and IS. The MS–MS detection was by monitoring the fragmentation for drospirenone and for levonorgestrel on a triple quadrupole mass spectrometer. The assay was calibrated over the range 5–100 ng mL^?1 with a correlation coefficient of 0.9998. Validation data showed intra-batch (n = 6) CV% ≤ 5.58 and RE (%) between ?3.34 and +6.27 and inter-batch (n = 18) CV% < 6.08 and RE (%) between ?1.84 and +6.73. Mean extraction recovery were 83.31–92.58% for three QC samples and 89.32% for IS. Plasma samples were stable for three freeze-thaw cycles, or 24 h ambient storage, or 1 and 3 months storage at ?20 °C. Processed samples (ready for injection) were stable up to 72 h at autosampler (4 °C). This method has been used for analyzing plasma samples from a bioequivalence study with 12 volunteers.     

Determination of Pitavastatin in Human Plasma by LC–MS–MS

A simple and rapid LC–MS–MS assay was developed and validated for the quantitative determination of pitavastatin in human plasma. Sample pretreatment involved simple protein precipitation by addition of acetonitrile. Separation was on an Agilent 1.8 μm Zorbax SB-C18 column (150 mm × 4.6 mm) at 25 °C using isocratic elution with methanol–0.1% formic acid in water (85:15, v/v) at a flow rate of 0.4 mL min^?1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 422.0 → 290.1 for pitavastatin, and m/z 330.1 → 192.1 for paroxetine (IS). LC–MS–MS was found to improve the quantitation of pitavastatin in plasma and was successfully applied in pharmacokinetic studies.     

Analysis of Nanafrocin in Foodstuffs of Animal Origin by LC–MS–MS

A new, rapid, and efficient method, multiple reaction monitoring liquid chromatography–tandem mass spectrometry, has been developed for analysis of nanafrocin in foodstuffs of animal origin. The researchers used a C₁₈ stationary phase coupled with triple-quadrupole tandem mass spectrometry in negative-electrospray mode. The limits of detection (LOD) and quantification (LOQ) were 0.005 and 0.01 mg kg^?1, respectively, in the matrixes. Detector response was found to be a linear function of concentration over the range 0.005–0.1 mg kg^?1 in each matrix. Mean overall recovery (n = 10) of nanafrocin varied from 71 to 101%. The results show that identification and quantification of nanafrocin residues in foodstuffs of animal origin can be successfully achieved by use of the proposed LC–MS–MS method.     

LC–DAD and LC–ESI–MS Chromatographic Fingerprinting and Quantitative Analysis for Evaluation of the Quality of Huang-Lian-Jie-Du-Tang

LC–DAD coupled with electrospray tandem mass spectrometry (LC–ESI–MS–MS) has been used to evaluate the quality of the traditional Chinese medicine Huang-Lian-Jie-Du-Tang (HLJDT). Twenty-five chromatographic peaks were obtained from a C₁₈ analytical column by gradient elution with acetonitrile and formate buffer (containing 0.5% formic acid) at a flow rate of 1.0 mL min^?1. The linearity, precision, and accuracy of the data obtained were acceptable. Thirteen components were identified by ESI–MS, and seven of these were quantitatively analyzed by LC–DAD. The method was used to analyze ten batches of HLJDT, and both chromatographic fingerprints and quantitative data were used to evaluate the quality of the HLJDT. It was concluded that this LC–DAD–ESI–MS method enables more fully validated and complete evaluation and monitoring of the quality of HLJDT.     

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min⁻¹. The established calibration curve was linear between 0.05–20 µg mL⁻¹, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C _max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.

     

Comparative Analysis of the Major Constituents in the Traditional Tibetan Medicinal Plants Saussurea laniceps and S. medusa by LC–DAD–MS

Liquid chromatography coupled with diode array detector and electrospray ionization mass spectrometry was developed for the qualitative and quantitative comparison of the main constituents in Saussurea laniceps (SL) and S. medusa (SM), two species of plants used under the name “Xuelianhua” in traditional Tibetan medicine. A method validation including linearity, limit of detection, precision and recovery was performed. The results showed that a good linearity with R ² > 0.99 was achieved, and the limit of detection of the quantified constituents was reported to be between 0.8 and 3.3 ng. The relative standard deviation value was below 3.82% for repeatability, and recovery studies for the quantified compounds were found to be within the range 90.92–103.12%. The unique properties of the present method were evaluated by analyzing twelve related herbal samples including five S. laniceps samples and seven S. medusa samples. Twenty-two compounds including phenolic acids, cumarins, lignanoids and flavonoids were identified by online ESI–MS and by comparison with literature data and standard compounds, and seven of them were quantified by LC–DAD simultaneously. The results demonstrated that the common constituents in the two herbs were protocatechuic acid, syringoside, chlorogenic acid, isoquercitroside, 1,5-dicaffeoylquinic acid, apigenin 7-O-β-d-glucoside, chrysoeriol 7-O-β-d-glucoside, acacetin 7-O-β-d-glucoside, apigenin and chrysoeriol. In the present study, it was found that the characteristic constituents were umbelliferone, scopoletin and their glucosides in S. laniceps, as well as arctiin and arctigenin in S. medusa. It was feasible to choose these characteristic compounds for the quality evaluation as well as chemical authentication of the two related herbs. The results also support discrimination between the two species when using them in folk medicine.     

LC–DAD–ESI-MS-MS for Characterization and Quantitative Analysis of Diterpenoids from Coleus forskohlii

Qualitative characterization and quantitative analysis of labdane diterpenoids from Coleus forskohlii have been achieved by liquid chromatography hyphenated with photodiode-array detection and tandem electrospray ionization mass spectrometry (LC–DAD–ESI-MS ⁿ ). By use of this method, thirteen forskolin-type diterpenoids were identified in the crude extract on the basis of their fragmentation mechanisms. Fragmentation rules were deduced from nine forskolin-type standards by ESI-MS in positive-ion mode. It was found that fragmentation behavior varied with the position and number of the substituents on the skeleton; this could be used for convenient identification of this type of compound. Six marker diterpenoids were also quantified, and the quality of both the cultivated and wild plants was evaluated.     

Determination of Amphotericin B in Human Cerebrospinal Fluid by LC–MS–MS

A simple, specific and sensitive column liquid chromatography–tandem mass spectrometry method has been developed for the determination of amphotericin B in human cerebrospinal fluid. Samples were prepared by dilution with methanol and quantitated by MS–MS detection in the positive mode. The determination was validated in the concentration range of 0.5–100 ng mL^?1 using 100 μL of human cerebrospinal fluid. The method was successfully used to support routine therapeutic drug monitoring.     

Quantitative Analysis of Triptolide in Human Whole Blood by LC–APCI-IT-MS-MS

In this study, the development and validation of an analytical method for triptolide in whole blood using high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization ion trap tandem mass spectrometry (LC–APCI-IT-MS-MS) is reported. This is the first report of the systematic development and validation of an LC–MS-MS method for the quantitation of triptolide in human whole blood using prednisolone as an internal standard (IS). Prior to LC–MS-MS analysis, liquid–liquid extraction with ethyl acetate was used to isolate them from the biological matrix. Validation parameters such as specificity/selectivity, limit of quantitation (LOQ), linearity, precision, accuracy and stability are evaluated for this method. The calibration curve was linear (r ² = 0.9973) in the concentration range of 0.5–100.0 ng mL⁻¹ in human whole blood with a lower limit of quantitation of 0.5 ng mL⁻¹. Intra- and inter-day relative standard deviations (RSDs) were less than 8.6 and 11.7%, respectively. Extraction recoveries of triptolide ranged from 81.5 to 88.1%. This assay can be used to determine trace triptolide in human whole blood.     

Determination of Eleutherosides in Dietary Supplements by LC–MS/MS

A simple and specific method for the simultaneous determination of eleutherosides B and E in powdered rhizomes of Eleutherococcus senticosus extract and in solid and liquid dietary supplements was developed and validated. E. senticosus extracts, often mixed with other plants or herbal extracts, are widely used in food supplements because of the tonic and adaptogenic activities referred to the eleutherosides B and E. In this study, samples were analyzed by a liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) method operated in single reaction monitoring (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values were fixed at 3 μg L^?1 and 10 μg L^?1, respectively, whereas linearity was established within 10–1,000 μg L^?1 range for both compounds. Good precision was obtained for both eleutherosides in terms of intra-day precision (RSD % lower than 4 %) and inter-day precision (RSD % lower than 6 %). Good percentage recoveries were obtained for both eleutherosides (91.5–103.6 %). Finally, the developed method was successfully applied to analyze a number of solid and liquid commercial dietary supplements containing E. senticosus extracts, also mixed with other herbal extracts.