Statistical Model for Organic Chromatographic Trace Analysis of Complex Samples. A Case Study: Plant Extracts

Abstract

The use of pooled plant extracts is described in the estimation of matrix interference in HPLC (UV and EC) determinations of organic compounds in plant extracts. An extract from freeze dried leaves of 134 different plant species was used for this purpose. It was split in different subgroups with solid extraction clean-up procedures. UV, EC and chromatographic data of the subgroups were used in the calculation of minimum concentrations of organic compounds which are still accurately determinable in plant samples with HPLC methods. The UV and/or EC characteristics of the compound must be known. The contribution of the solid phase extraction procedures and of the analytical system to the selectivity of the method can be estimated. Information is also supplied which allows rapid comparison of the selectivity of the UV and EC (single, or dual parallel) detectors for the determination of a specified compound.     

Mass spectrometric profiling of valepotriates possessing various acyloxy groups from Valeriana jatamansi

Valepotriates, plant secondary metabolites of the family Valerianaceae, contain various acyloxy group linkages to the valepotriate nucleus and exhibit significant biological activities. Identification of valepotriates is important to uncover potential lead compounds for the development of new sedative and antitumor drugs. However, making their structure elucidation by nuclear magnetic resonance (NMR) experiments is too difficult to be realized because of the overlapped carbonyl carbon signals of acyloxy groups substituted at different positions. Thus, the mass spectrometric profiling of these compounds in positive ion mode was developed to unveil the exact linkage of acyloxy group and the core of valepotriate. In this study, electrospray ionization tandem multistage mass spectrometry (ESI‐MS/MSⁿ) in ion trap and collision‐induced dissociation tandem MS were used to investigate the fragmentation pathways of four types of valepotriates in Valeriana jatamansi, including 5‐hydroxy‐5,6‐dihydrovaltrate hydrin (5‐hydroxy‐5,6‐dihydrovaltrate chlorohydrin), 5,6‐dihydrovaltrate hydrin (5,6‐dihydrovaltrate chlorohydrin), 5‐hydroxy‐5,6‐dihydrovaltrate and valtrate hydrin (valtrate chlorohydrin). The high‐resolution mass spectrum (HRMS) data of all the investigated valepotriates from quadrupole time‐of‐flight MS/MS were used as a supportive of the fragmentation rules we hypothesized from ion‐trap stepwise MSⁿ. As a result, the loss sequence of acyloxy groups and the abundance of key product ions, in combination with the characteristic product ions corresponding to the valepotriate nucleus, could readily differentiate the four different types of valepotriates. The summarized fragmentation rules were also successfully exploited for the structural characterization of three new trace valepotriates from V. jatamansi. The results indicated that the developed analytical method could be employed as a rapid, effective technique for structural characterization of valepotriates, especially for the trace compounds that could not be identified by NMR techniques. This study may also arouse interest for further structural analysis of other valepotriate‐containing type herbal medicines. Copyright © 2015 John Wiley & Sons, Ltd.     

Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology

A standardised LC-UV-MS micro-scale method for screening of fungal metabolites and mycotoxins in culture extracts is presented. The paper includes data for detection and dereplication of > 400 fungal metabolites to facilitate detection and identification when standards are not available. The data also shows the types of components that can be analysed by positive electrospray (ESI+) mass spectrometry (MS) along with common fragments and adducts of these, as well as giving suggestions on whether UV or ESI+-MS methods should be used. Examples of dereplication of penitrems and macro-cyclic ichothecenes, and detection of several novel compounds are shown. This was done by UV spectroscopy combined with accurate mass determination of adduct and fragment ions obtained by high-resolution orthogonal time-of-flight MS.     

Capillary electrophoresis in allergen preparation research and in production control

A CE method has been developed for the analysis of wasp venoms and pollen extracts of Phleum pratense and Betula verrucosa. Various electrolyte systems can be used but the best separation and reproducibility are attained in 0.15 mol/l phosphoric acid, pH 1.8. UV photometric detection at 190 nm is sufficiently sensitive for determination of the polypeptides and glycoproteins contained in these materials (the detection limit for phospholipase A₂ is 0.4 ng). The allergens from wasp venoms and pollen extracts yield characteristic electropherograms which can be used for control of the allergen production. Modification of capillary wall with poly(ethylhydroxy methacrylate) causes a decrease in the migration times and the separation efficiency is retained.     

Determination of zinc dialkyldithiocarbamates in latex condoms

A simple high-performance liquid chromatographic (HPLC) assay is developed for measuring zinc dialkyldithiocarbamate (DTC) levels in latex condoms. After extraction of 14 different brands of latex condoms in acetonitrile, aliquots of the extracts are subjected to a preliminary screening assay by treatment with cobalt chloride and measurement of UV absorption at 320 nm, which results in the identification of 6 DTC-containing samples. Prior to analysis by HPLC, zinc dimethyldithiocarbamate (ZDMC) or zinc diethyldithiocarbamate (ZDEC) is added to the extracts in order to block transmetalation reactions with the analytes of interest. A reversed-phase C(18) column, with gradient elution and UV detection at 260 nm, is used to measure the zinc DTCs. The limits of detection for ZDEC and zinc dibutyldithiocarbamate (ZDBC) are 5 and 10 micro g/mL. Levels of ZDBC and ZDEC range from not detectable to 3.31 and 1.79 mg/condom, respectively. Total protein and latex allergenic protein levels are determined and range from 98 to 776 and 0.01 to 14.04 micro g/unit, respectively, but are not related to the level of ZDBC or ZDEC. This methodology provides both screening and specific tools for the determination of unstable zinc DTC complexes in latex products.     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.     

Determination of iridoid glycosides by micellar electrokinetic capillary chromatography-mass spectrometry with use of the partial filling technique

A fast and easy method was sought for determination of the iridoid glycosides catalpol, ketologanin, verbenalin, loganin, 8-epi-loganic acid, geniposidic acid and 10-cinnamoyl catalpol in plant samples. The method involved micellar electrokinetic capillary chromatography (MEKC) coupled on-line to mass spectrometry. The partial filling technique and electrospray ionization were used. Seven iridoid glycosides could be separated with use of MEKC under basic conditions. However, 8-epi-loganic acid and geniposidic acid could not be detected simultaneously with the five neutral iridoid glycosides by mass spectrometry. Therefore, only the neutral iridoid glycosides were screened from plant samples. Catalpol, verbenalin, loganin and possibly 10-cinnamoyl catalpol were found in an examination of seven plant species in the genera Plantago, Veronica, Melampyrum, Succisa, and Valeriana. Aucubin, which was not included in the sample mixture used in method development because of overlapping with catalpol in MEKC, was also detected. The limits of detection for the iridoid glycosides, both at the UV and at the mass spectrometer, are given.     

Application of a high-performance liquid chromatographic fluorescence method for the rapid determination of alpha-tocopherol in the plasma of cattle and pigs and its comparison with direct fluorescence and high-performance liquid chromatography-ultraviolet detection methods

High-performance liquid chromatography is used to develop a sensitive method for the determination of tocopherol levels in the plasma of cattle and pigs. This method is compared with a similar method using UV detection and one using direct fluorescence determination of tocopherol. Finally a double injection technique used in conjunction with fluorescence detection is shown to enhance the rate of analysis of the tocopherol levels in bovine plasma extracts.     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal. Received: 23 July 1999 / Revised: 9 September 1999 / Accepted: 16 September 1999     

Selective immunoclean-up followed by liquid or gas chromatography for the monitoring of polycyclic aromatic hydrocarbons in urban waste water and sewage sludges used for soil amendment

A selective clean-up procedure using immunoaffinity solid-phase extraction was applied for the trace-level determination of polycyclic aromatic hydrocarbons (PAHs) in urban waste water and sewage sludges used for soil amendment. Anti-pyrene antibodies have been immobilized on a silica-based sorbent and the cross-reactivity of the antibodies towards structurally related compounds were allowed to extract the whole class of priority PAHs. The selectivity of the antibodies provided clean extracts from sludges and, therefore, the identification and quantification were shown to be easier using either liquid chromatography (LC) with UV diode array and fluorescence detection in series or gas chromatography-mass spectrometry (GC-MS), although some loss of up to 50% was observed for the clean-up. The identification of the PAHs by matching of UV and MS spectra was greatly improved. The procedure, including immunoclean-up and LC coupled to diode array and fluorescence detection, was validated using certified reference materials with native PAHs of concentrations in the range of 0.57-2.16 mg/kg (dry sludges).     

Capillary electrophoretic determination of sanguinarine and chelerythrine in plant extracts and pharmaceutical preparations

Capillary electrophoresis was employed to determine the principal quaternary benzo[c]phenanthridine alkaloids, sanguinarine and chelerythrine, in two plant extracts and one oral hygiene product. Phosphate-Tris buffer of pH 2.5 was used as a background electrolyte, limits of detection were 3 micromol/l(-1) (sanguinarine) and 2.4 micromol,l(-1) (chelerythrine) using UV detection at 270 nm. The method, which correlated well with HPLC, is suitable for serial determination of sanguinarine and chelerythrine in plant products and pharmaceuticals.     

Identification and quantification of base and nucleoside markers in extracts of Ganoderma lucidum, Ganoderma japonicum and Ganoderma capsules by micellar electrokinetic chromatography

The present paper describes the development of a micellar electrokinetic chromatographic method for the determination of nucleoside (adenosine, uridine) and base (uracil) markers in aqueous extracts of Ganoderma medicinal preparations. The markers were successfully separated within 10 min using an 80 mM borate buffer, with 25 mM sodium dodecyl sulfate adjusted to pH 9.0, an operating voltage of 22 kV, temperature of 20 degrees C and a hydrodynamic injection time of 5 s. Separations were carried out in a fused-silica capillary with peak detection by direct UV at 254 nm. Following semi-validation of the method, with each analyte showing a good linear relationship over a 0.2 to 20 ppm concentration range (correlation coefficients from 0.9986 to 0.9998), the amounts of the three markers in the various forms of Ganoderma were easily determined using a relatively simple extraction procedure.     

Determination of Aztreonam by liquid Chromatography with UV and Amperometric Detection

Abstract

A comparative study of UV and amperometric detection of aztreonam after HPLC separation is presented. At pH 2.0 and a detection potential of +1.15 V (vs. Ag/AgCl), the detection limits with amperometric detection are about two times higher (3–5 ng) than those obtained with UV detection (1–3 ng) for aztreonam and its main decomposition products, the E-isomer and open-ring aztreonam. With the advantage of specificity for the aminothiazole group of the aztreonam molecule, amperometry can be used as an alternative or complementary mode to UV detection for the determination of aztreonam in injectable formulations and in human serum.     

Determination of tenoxicam in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of tenoxicam in plasma has been developed. Tenoxicam was extracted from buffered plasma (pH 3 or 4, respectively) with dichloromethane and the evaporated extracts were analysed on a C18 reversed-phase column using a methanol-phosphate buffer mobile phase and with UV detection at 371 nm. The detection limit was 20 ng/ml using a 0.5-ml sample. The method is selective with respect to the 5'-hydroxy metabolite, which is present in plasma after multiple administration of tenoxicam; this metabolite may also be determined using this procedure.     

Use of an on-line,precolumn photochemical reactor in high-performance liquid chromatography of naphthodianthrones in Hypericum perforatum preparations

A method has been developed for the determination of naphthodianthrones in Hypericum perforatum L. extracts and phytopharmaceutical preparations by high-performance liquid chromatography combined with on-line, precolumn photochemical conversion followed by photodiode-array detection. The chromatographic separation was performed on a C18 column under isocratic reversed-phase conditions. An on-line, precolumn photochemical reactor equipped with a knitted PTFE reaction coil around a visible light source was used in order to transform the light sensitive naphthodianthrones, protohypericin and protopseudohypericin, very easily into the non-protoforms, hypericin and pseudohypericin, respectively. Two UV chromatograms (photochemical reactor "on" and "off") were compared and were quite useful in characterizing the sample. Validation studies demonstrated that this HPLC method is simple, rapid, reliable and reproducible. The time-consumptive manual irradiation of the samples is omitted by this automated on-line irradiation step. The developed method was successfully applied to the quality control of Hypericum perforatum L. extracts and its phytopharmaceutical preparations.     

Simultaneous determination of inorganic anions, carboxylic and aromatic carboxylic acids by capillary zone electrophoresis with direct UV detection

Co-electroosmotic capillary zone electrophoresis (CZE) with direct UV detection was developed for simultaneous determination of inorganic anions, carboxylic and aromatic carboxylic acids. These solutes were separated using a 30 mM phosphate buffer containing 1.0 mM tetradecyltrimethylammonium bromide (TTAB) and 20% (v/v) acetonitrile at pH of 6.5 and directly detected by UV at 190 nm. Calibration curves were linear in the range 0.01-2.0 mM, depending of the solutes. The detection limits ranged from 1.0 to 8.0 microM and the relative standards deviations (n=5) in range from 1.9 to 3.6% for the peak area. The proposed method was used to determine inorganic anions and carboxylic and aromatic acids in soil and plant tissue extracts.     

Quality control of medicinal herbs Fructus gardeniae, Common Andrographis Herb and their preparations for their active constituents by high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry

Dehydroandrographolide, andrographolide and geniposide are the main active constituents of many herbal medicines, e.g., Fructus gardeniae, Common Andrographis Herb. They are used as the markers to control the quality of such herbal medicines and their herbal preparations. In this paper, a simple and sensitive high-performance liquid chromatographic (HPLC) method coupled with photodiode array detection (DAD) and electrospray mass spectrometry (ESI/MS) were developed to determine the three compounds simultaneously in extracts of medicinal herbs and herbal preparations produced by different companies. The extracts were separated on a C₁₈ reversed phase HPLC column, with a gradient solvent system, the time for the separation of the three target analytes was 10 min. The abundance ions were recorded using selected ion monitoring (SIM) mode with m/z 297.3, 297.3 and 411.1 for dehydroandrographolide, andrographolide and geniposide, respectively. The limit of detection for dehydroandrographolide, andrographolide and geniposide were 20, 30 and 150 ng mL⁻¹, respectively. The proposed method was successfully applied to the determination of the contents of the compounds in related to medicinal herbs and preparations.     

Flexible and powerful strategy for qualitative and quantitative analysis of valepotriates in Valeriana jatamansi Jones using high‐performance liquid chromatography with linear ion trap Orbitrap mass spectrometry

Valeriana jatamansi Jones is an important medicinal plant and its quality is closely related to its region of origin. In the current study, we utilized a flexible and powerful strategy for comprehensive evaluation of the quality diversity for 15 regions in China. The method was based on a hybrid linear ion trap‐Orbitrap mass spectrometry platform. For structure characterization, fragmentation patterns were detected by analyzing a series of standard compounds using data dependent multistage mass spectrometry acquisition. A fragment ion database for valepotriates was established, and the acquired data were high throughput filtered by fragment ion search for compound identification. For quantitative purposes, we normalized the mass spectrometry data of 15 samples using SIEVE 2.0 and the differences in composition were analyzed using principal component analysis combined with hierarchical clustering analysis. The results identified a total of 92 compounds from Valeriana jatamansi Jones. Samples from Dali, Kunming, and Baoshan have better qualities and concentrations of the main active constituents. To verify our strategy, we compared the valtrate, acevaltrate, and baldrinal contents using high‐performance liquid chromatography with diode array detector. We developed and validated a comprehensive qualitative and quantitative analytical method to achieve quality control of Valeriana jatamansi Jones.     

Screening method for detecting cross-contamination residues of tiamulin in swine feeds

A method was developed for the determination of tiamulin (TML), 14-deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy]mutilin hydrogen fumarate, a semisynthetic derivative of the naturally occurring antibiotic pleuromutilin produced by the fungus Pleurotus mutilis. This drug, with high activity against Gram-positive bacteria, some Gram-negative bacteria, and several strains of mycoplasms is administered to animals in food, drinking water, or by injection; however, its chemical structure causes problems in analysis of feeds. Although the molecule is charged below pH 8, attempts to analyze TML-containing extracts on ion-exchange columns or other polar stationary phases have failed. Additionally, TML shows no fluorescence activity and only poor UV activity. The present method consists of organic solvent extraction followed by liquid chromatography with UV detection. A low wavelength (208 nm) was used for detection. Limits of detection and quantitation, as well as data for recovery and repeatability obtained during characterization of the method, are described. The applicability of the optimized method was tested by analyzing commercial blank feeds processed after TML-medicated feeds.     

Reversed-phase ion-pair liquid chromatographic procedure for the simultaneous analysis of S-adenosylmethionine, its metabolites and the natural polyamines

A method using reversed-phase ion-pair liquid chromatography with dual detection was developed for the simultaneous determination of the S-adenosylmethionine (SAM) analogues and the natural polyamines. The separation is obtained with a gradient elution and by adjusting the concentration of octanesulfonic acid used as ion-pairing agent, the ionic strength of the eluent, the pH and the acetonitrile content of the eluents. The SAM analogues are analyzed by UV detection at 254 nm and the polyamines by fluorescence detection after post-column derivatization with o-phthalaldehyde. The method allows the determination of the SAM analogues and the polyamines in one single run by direct injection of tissue extracts. The procedure is applied to the study in rats and in hepatoma tissue culture cells of the biochemical effects of alpha difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase.