Quality assessment of Cortex Phellodendri by high‐performance liquid chromatography coupled with electrospray ionization mass spectrometry

A simple method based on liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (LC‐DAD‐ESI‐MS) was developed for the quality assessment of Cortex Phellodendri (CP), which was mainly derived from two species of Phellodendron chinense Schneid and Phellodendron amurense Rupr. Total 41 compounds, including 14 phenols, 24 alkaloids and three liminoidal triterpenes were identified or tentatively characterized from the 75% methanol extract of CP samples by online ESI‐MSⁿ fragmentation and UV spectra analysis. Among them, two phenols and six alkaloids were simultaneously quantified using HPLC‐DAD method. The validated HPLC‐DAD method showed a good linearity, precision, repeatability and accuracy for the quantification of eight marker compounds. Furthermore, the plausible fragmentation pathway of the representative compounds were proposed in the present study. The differences of the chemical constituents content and the comprehensive HPLC profiles between the two CP species using LC‐DAD‐ESI‐MS method are reported for the first time, indicating that the CP drugs from different resources should be used separately in the clinic. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of eight flavonoids in Sedum sarmentosum Bunge from different areas by UHPLC with triple quadrupole MS/MS

Sedum sarmentosum Bunge (SSB) is a traditional Chinese herbal medicine containing multiple components that has been extensively used clinically to treat chronic viral hepatitis and some inflammatory diseases. Total flavonoids are major pharmacologically active components of SSB. To gain a deeper understanding of SSB resources, we analyzed eight chemical constituents in 33 batches of SSB from 11 regions in China. An accurate, precise and sensitive ultra‐high‐performance liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry method was developed for the determination of eight flavonoids in SSB. Under the optimized chromatographic conditions, good separation for the eight target components was obtained on an Agilent Zobax SB C₁₈ (50 × 2.1 mm, 5 μm) column within 4 min. The established methods were validated with good linearity (r ≥ 0.9988), precision (RSD ≤ 2.68%), stability (1.43–3.28%) and repeatability (1.14–2.89%). Moreover, the average recoveries were 95.91–100.68%, and the RSDs were 1.50–3.80%. In addition, the analytical conditions of UPLC–ESI–MS/MS provided better sensitivity with a shorter analysis time when compared with the HPLC–DAD method. Hierarchical clustering analysis and principal component analysis were performed to estimate and classify these samples based on the contents of the eight chemical constituents. This study provided the theoretical basis and scientific evidence for the development and utilization of SSB resources.     

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.     

A fast isocratic liquid chromatography method for the quantification of xanthophylls and their stereoisomers

A fast isocratic liquid chromatography method was developed for the simultaneous quantification of eight xanthophylls (13‐Z‐lutein, 13’‐Z‐lutein, 13‐Z‐zeaxanthin, all‐E‐lutein, all‐E‐zeaxanthin, all‐E‐canthaxanthin, all‐E‐β‐apo‐8’‐carotenoic acid ethyl ester and all‐E‐β‐apo‐8’‐carotenal) within 12 min, compared to 90 min by the conventional high‐performance liquid chromatography method. The separation was achieved on a YMC C₃₀ reversed‐phase column (100 mm x 2.0 mm; 3 μm) operated at 20°C using a methanol/tert‐butyl methyl ether/water solvent system at a flow rate of 0.8 mL/min. The method was successfully applied to quantify lutein and zeaxanthin stereoisomers in egg yolk, raw and cooked spinach, and a dietary supplement. The method can be used for the rapid analysis of xanthophyll isomers in different food products and for quality control purposes.     

Separation of carotenol fatty acid esters by high-performance liquid chromatography

Employing isocratic and gradient-elution high-performance liquid chromatography (HPLC) a number of straight-chain fatty acid esters (decanoate, laurate, myristate, palmitate) of violaxanthin, auroxanthin, lutein, zeaxanthin, isozeaxanthin, and beta-cryptoxanthin, prepared by partial synthesis, have been separated on a C18 reversed-phase column. Several chromatographic conditions were developed that separated a mixture of di-fatty acid esters (dimyristate, myristate palmitate mixed ester, dipalmitate) of violaxanthin, auroxanthin, lutein, and zeaxanthin in a single chromatographic run. Hydroxycarotenoids such as lutein, zeaxanthin, and isozeaxanthin that are not easily separated by HPLC on C18 reversed-phase columns, can be readily separated after derivatization with fatty acids and chromatography of their esters. Chromatographic conditions for optimum separation of carotenoids from various classes are discussed.     

Effects of Mobile Phase Ratios on the Separation of Plant Carotenoids by HPLC with a C30 Column

Plant products are dietary sources of lutein and zeaxanthin. Lutein and zeaxanthin have been implicated in the protection of age related macular degeneration (AMD) and in cardiovascular diseases. However, xanthophylls and unidentified components (λ_max = 423 and 468 nm) in plant products are often not separated well, and affect an accurate quantitative determination of lutein and zeaxanthin. A high performance liquid chromatography (HPLC) system equipped with a Bischoff C30 column and a mobile phase of methanol, methyl-tert-butyl ether (MTBE) and water was used to separate lutein, zeaxanthin and other unidentified components in plant products. Mobile phase A containing methanol, MTBE and water with a ratio of 60:33:7 by volume (1.5% ammonium acetate, NH₄Ac), combined with mobile phase B with a ratio of 8:90:2 by volume (1.0% NH₄Ac) is optimal for the separation. This method was successfully applied to the quantitative determination of lutein and zeaxanthin in extracts of plant products, such as chlorella, spirulina, celery and mallow.     

Analysis and quantification of flavonoidic compounds from Portuguese olive (Olea europaea L.) leaf cultivars

Twenty three samples of 18 Portuguese olive leaf cultivars were analysed by a reversed-phase HPLC/DAD procedure and eight flavonoidic compounds were identified and quantified (luteolin 7,4'-O-diglucoside, luteolin 7-O-glucoside, rutin, apigenin 7-O-rutinoside, luteolin 4'-O-glucoside, luteolin, apigenin and diosmetin). Luteolin 7,4'-O-diglucoside and luteolin 4'-O-glucoside were identified by HPLC/DAD/MS/MS - ESI. The studied olive leaf samples showed a common phenolic pattern, in which luteolin 4'-O-glucoside was almost always the major compound.     

An improved method of simultaneous determination of lutein and zeaxanthin in food

Based on an official standard method of lutein analysis, an improved high performance liquid chromatography (HPLC) method for simultaneously detecting lutein and zeaxanthin was developed as focusing on the sample preparation protocol. The optimal pretreatment conditions included a saponification in a water bath for 15?min at a constant temperature of 50?°C, using a 10?mL 60% (w/v) potassium hydroxide solution, followed by extraction using 100?mL mixture of n-hexane, ethyl ether and cyclohexane (40: 40: 20, v/v/v). A mixture of dichloromethane, acetonitrile and methanol (20: 30: 50, v/v/v) was validated to elute lutein and zeaxanthin on a C₃₀ column (4.6?×?250?mm, 5?µm). The resolution between lutein and zeaxanthin is ≥2.5. A millet sample was used for methodological verification and the results showed that the linear relations for lutein and zeaxanthin were good in ranges of 0.23–9.37?μg/mL and 0.30–12.02?μg/mL, respectively. The relative standard deviations of lutein and zeaxanthin were 1.40% and 5.09%, respectively, and their spiked recoveries were between 86.60% and 98.75%. The lutein and zeaxanthin results from this modified HPLC method are superior to those from the Chinese official method and ultrasonic extraction method.     

A rapid and reliable UPLC-MS/MS method for the identification and quantification of fourteen synthetic anti-diabetic drugs in adulterated Chinese proprietary medicines and dietary supplements

An ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed for simultaneous qualitative and quantitative analysis of 14 synthetic anti‐diabetic drugs in adulterated Chinese proprietary medicines (CPMs) and dietary supplements. The samples were prepared by ultrasonic extraction with methanol and separated on a C₁₈ column with mobile phase consisting of acetonitrile and water (both containing 0.10% formic acid). Gradient elution was applied with a flow rate of 0.20 mL/min. Two transitions from protonated molecules were monitored for each synthetic anti‐diabetic drug in positive mode of electrospray ionization (ESI). The two transitions, the peak area ratio of the two transitions and the retention time were used for identification. The more intensive transition was used for quantification. The analysis time was 6 min per sample. Satisfactory linear relationships were estimated between the peak area and the concentration with correlation coefficients higher than 0.995. The limit of detection ranged from 0.03 to 5.45 ng/mL. The relative standard deviation of intra‐day precision was below 7.6%, the RSD of inter‐day precision was below 15% and the relative error of accuracy was between –10 and 7.8%. The proposed method is rapid, selective, reliable and was successfully applied to the analysis of 30 real samples of 22 CPMs and eight dietary supplements from the local market in China. Copyright © 2010 John Wiley & Sons, Ltd.     

Applications of HPLC-MS in compound Ilex pubescens extract study

In this paper, high performance liquid chromatography (HPLC) along with mass spectrometry (MS) and HPLC along with a diode array detector (DAD) was used to study the compound Ilex pubescens extract. Two ionization techniques: electro spray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were used in this work. The liquid chromatograms obtained by DAD, total ion chromatograms (TIC) from positive-and negative-ion ESI-MS and the positive-and negative-ion APCI-MS were compared. The liquid chromatograms obtained by TIC from ESI-MS provided more information on chromatographic peaks than those obtained by DAD or TIC from APCI-MS. It is suggested that the fingerprints of the compound Ilex pubescens extract should be provided by the liquid chromatograms obtained by DAD together with TIC from the negative-ion ESI-MS. The molecular weights of the nine main components in an HPLC-DAD chromatogram were determined by the corresponding positive-and negative-ion ESI and the positive-and negative-ion APCI mass spectra information. In the liquid chromatogram obtained by TIC from the negative-ion ESI-MS, the molecular weights of 23 main components were determined based on the corresponding positive-and negative-ion ESI mass spectra information.     

Screening and analysis of the multiple absorbed bioactive components and metabolites in rat plasma after oral administration of Jitai tablets by high‐performance liquid chromatography/diode‐array detection coupled with electrospray ionization tandem mass spectrometry

Based on the serum pharmacochemistry technique and high‐performance liquid chromatography/diode‐array detection (HPLC/DAD) coupled with electrospray tandem mass spectrometry (HPLC/ESI‐MS/MS), a method for screening and analysis of the multiple absorbed bioactive components and metabolites of Jitai tablets (JTT) in orally dosed rat plasma was developed. Plasma was treated by methanol precipitation prior to liquid chromatography, and the separation was carried out on a Symmetry C₁₈ column, with a linear gradient (0.1% formic acid/water/acetonitrile). Mass spectra were acquired in negative and positive ion modes, respectively. As a result, 26 bioactive components originated from JTT and 5 metabolites were tentatively identified in orally dosed rat plasma by comparing their retention times and MS spectra with those of authentic standards and literature data. It is concluded that an effective and reliable analytical method was set up for screening the bioactive components of Chinese herbal medicine, which provided a meaningful basis for further pharmacology and active mechanism research of JTT. Copyright © 2010 John Wiley & Sons, Ltd.     

Use of TLC and UV–Visible Spectrometry for Fingerprinting of Dietary Supplements

We have studied dietary supplements containing sea-buckthorn (Hippophae rhamnoides), bilberry (Vaccinium myrtillus), and cranberry (Vaccinium vitis-idaea) by use of thin-layer chromatography (TLC), image analysis, and UV–visible spectroscopy. The results were subjected to multivariate techniques, for example cluster analysis (CA), principal-components analysis (PCA), and linear discriminant analysis (LDA). Evaluation of the discriminating power of TLC coupled with digital image analysis for characterization of the supplements was performed by use of chemometrics. Zero-order, first-order, and normalized zero-order UV–visible spectra of extracts of fruit and dietary supplements were digitized and used in the chemometric analysis. To increase the relevance of the study, the dietary supplement extracts were compared with extracts of the natural fruit. Results obtained by application of CA, PCA, and PCA–LDA to the chromatograms suggest the possibility of discrimination among dietary supplements on the basis of the raw material used for their preparation.

     

Forensic Identification of Dyes in Ballpoint Pen Inks Using LC–ESI–MS

The aim of this work was to develop a new technique using flow injection analysis combined with LC–ESI–MS which allows identification of dyes in ballpoint pen inks. A sample preparation procedure for the extraction of dyes from ballpoint pen strokes has been developed. The characteristic group of ions for each sample of 21 studied ballpoint pen inks corresponding to the present dyes has been determined using flow injection method. LC separation conditions for identified dyes have been optimized on reversed-phase sorbent based on silica gel. The best composition of the mobile phase for the dyes mixture LC separation was 0.1% aqueous formic acid and acetonitrile. Detection of dyes was carried out using mass spectrometry with electrospray ionization in positive and negative modes after reversed-phase liquid chromatography separation. Dye composition of ink was additionally confirmed comparing the data obtained from the literature. Flow injection analysis allows obtaining intensive ions of unknown strokes. It is difficult to get this information using only chromatographic methods, because dyes peak intensity can be low and noise of basic line high. Flow injection method allows distinguishing the analyzed 21 ballpoint pens by determining a characteristic set of dyes. The developed flow injection technique is very simple and quick. As a result, a novel approach for the identification of dyes in the ballpoint pen inks by flow injection analysis with LC–ESI–MS and UV detection without using standard dye samples has been established. It can be an effective alternative to the existing LC–DAD–MS and IR spectroscopy methods.     

Rapid and Sensitive LC–MS/MS Analysis of Fatty Acids in Clinical Samples

Free fatty acids (FFAs), major cellular metabolites, play an important role during tumor pathogenesis. Enhanced de novo fatty acid synthesis in tissues is a characteristic feature of cancer. Therefore, measurement of FFA concentration in biological samples is beneficial for cancer research and clinical diagnosis. Herein, a rapid, stable, and sensitive detection methodology was established to simultaneously quantify 22 FFAs using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI–MS/MS). The HPLC–MS/MS system was run in negative ion mode for 15 min using multiple reaction monitoring. The lipids were extracted from colon tissues of colon cancer patients and then injected into the HPLC–MS/MS system for analysis. Colon samples were analyzed by inter-day repeatability and intra-day repeatability, with less than 5 % deviation for most fatty acids. This approach is successful to determine low picogram concentrations of each FFA molecule using milligrams of tissue, and provides a promising method for FFA microanalysis in clinical samples.

     

Application of Thin-Layer Chromatography to High-Performance Liquid Chromatographic Separation of Steroidal Hormones and Cephalosporin Antibiotics

Abstract

High-performance liquid chromatographic (HPLC) separation of steroidal hormones and cephalosporin antibiotics was investigated by adsorption chromatography and reversed-phase chromatography, respectively.

Prior to the HPLC separation of these pharmaceuticals, silica gel thin-layer adsorption chromatography of steroidal hormones and reversed-phase thin-layer partition chromatography of cephalosporin antibiotics with chemically bonded dimethylsilyl silica gel were performed in order to obtain suitable HPLC separation systems.

In the separation of steroidal hormones, the same binary mobile phase ratios of TLC did not give satisfactory results in HPLC. For the sharp separation in HPLC, solvent strength in the binary solvent mixture used for TLC had to be decreased.

The difference in solvent strength for efficient separation between TLC and HPLC might be attributed to the fact that in HPLC the solvent elution power acts in an isocratic manner while in TLC it acts in a gradient manner.

On the other hand, a correlation of mobility between TLC and HPLC separation for cephalosporin antibiotics was obtained, and the possibility of direct transfer of chromatographic systems from TLC to HPLC for separation of these antibiotics was confirmed.     

Identification and characterization of impurities in an insecticide,bifenthrin technical

The HPLC‐DAD and GC/MS methods were successfully used for the identification and characterization of the impurities in an agrochemical insecticide, bifenthrin technical. Three impurities ranging from 0.175%–0.541% were detected by the HPLC‐DAD method. The LC/MS technique with ESI or APCI source failed to detect the impurities detected by HPLC‐DAD, due to lack of ionization in ESI or APCI. The three impurities were enriched by prep‐HPLC, and then their structures were elucidated based on the GC/EIMS and CIMS data. The EI mass spectra of bifenthrin and its impurities displayed molecular ion and provided structure indicative fragment ions; the CIMS data further confirmed their molecular weight. The identity of the impurity 1 was further confirmed by the synthesis of the authentic sample followed by NMR and GC/MS data.     

Porous graphitic carbon chromatography/tandem mass spectrometric determination of cytarabine in mouse plasma

A high-performance liquid chromatography (HPLC) system using a porous graphitic carbon (PGC) stationary phase interfaced with an electrospray ionization (ESI) source and a tandem mass spectrometer (MS/MS) for the analysis of cytarabine (ara-C) in mouse plasma samples has been developed in support of a pharmacodynamic study. The graphitized carbon column was adopted for the separation of ara-C and endogenous peaks from mouse plasma samples under the reversed-phase phase mode in liquid chromatography. The retention characteristics of the PGC column and the ionization efficiencies of all analytes based on the experimental factors such as the composition of mobile phases were investigated. The potential of ionization suppression resulting from the endogenous biological matrices on the PGC column during HPLC/ESI-MS/MS was investigated using post-column infusion. The concentrations of ara-C in mouse plasma obtained by using PGC-HPLC/MS/MS and ion-pairing HPLC/MS/MS were found to be in good agreement in terms of analytical accuracy.     

Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS

The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD‐HPLC‐ESI‐TOF‐MS and IT‐MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF‐MS in order to obtain molecular formula and exact mass. Posterior analyses with IT‐MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode.     

检查中药中掺入吗啡的方法研究

我国每年出口到东南亚的中成药及保健品很多,价值逾亿。其中发现个别产品中掺有具有相似功效的化学药品。为此,新加坡卫生科学局颁布了《中成药管理办法》,其中规定自2004年1月1日起,只有经过一系列严格检验的产品方可进口。为此,中国实验室国家认可委员会组织了已通过国家实验室认可的若干实验室进行了此方面的能力比对。要求定性检查给定样品中是否掺入吗啡即为其中一例。文献主要报道了采用气相色谱-质谱和液相色谱-紫外检测方法测定吗啡含量,本文主要建立了HPLC／DAD和HPLC-MS方法,以检查样品中是否掺有吗啡。吗啡的结构式如右所示。