Identification and determination of corticosteroids in adrenal gland extracts for pharmaceutical use by HPLC

Summary Some HPLC procedures with isocratic or gradient elution are reported for the identification and determination of most of the characteristic components of cortical extracts. The proposed solvent systems were: A) for normal phase chromatography, mixtures of chloroform-methanol-water on silica columns. B) For reversed phase chromatography, mixtures of methanol-water or acetonitrile-water or tetrahydrofuran-water on octadecyl silica columns of different brands. With these systems it was possible to identify and determine, in addition to the principal corticosteroids, some minor components of the cortical extracts as the 20β-dihydroderivatives of compounds F, E, A, B, the 17-ketosteroids adrenosterone, 11β-hydroxyandrostendione and androstendione and finally, progesterone and 17-OH progesterone. In reversed phase chromatography it was also possible, by monitoring the effluent at 205 nm, to reveal the 5α- and 5β-tetrahydroderivatives of the main corticosteroids and to separate them from most of the steroidal components of the adrenal extracts; in these conditions it was also possible to reveal some characteristic, unknown components of the cortical extracts. Some results of quantitative analysis of cortical extracts are also reported, comparing different analytical procedures. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Pesticide residue analysis in foodstuffs applying capillary gas chromatography with atomic emission detection State-of-the-art use of modified multimethod S19 of the Deutsche Forschungsgemeinschaft and automated large-volume injection with programmed-temperature vaporization and solvent venting

Atomic emission detection (AED) provides high element-specific detection of all compounds amenable to gas chromatography (GC). The heteroatoms nitrogen, chlorine, phosphorus, sulfur, bromine and fluorine, which are important elements in pesticide residue analysis, are of major interest. A main drawback of AED is its lower sensitivity with respect to other selective detection methods used in pesticide residue analysis such as electron-capture and nitrogen-phosphorus detection. This holds true especially for the important nitrogen trace. For this reason, more sensitive detection can be achieved by injection of larger volumes or higher concentrations of sample extracts, because matrix compounds were usually registered only in the carbon, hydrogen and oxygen traces. This paper focuses on recent developments from the authors' laboratory in order to demonstrate the feasibility of screening analyses with the identification of pesticide residues down to the 0.01 ppm concentration level in plant foodstuffs. This has been achieved by means of automated large volume injection with programmed-temperature vaporization and solvent venting as well as careful optimization of make-up and reactant gases with AED. Clean up follows the principle of multimethod S19 of the Deutsche Forschungsgemeinschaft in a reduced procedure. After elimination of lipids and waxes by gel permeation chromatography, extracts from 10 g of the food samples were concentrated to 200 μl, of which 12.5 μl were introduced into the GC-AED system. Two analyses were usually performed with the element traces of sulfur, phosphorus, nitrogen and carbon in the first run and chlorine and bromine in the second run. Fluorine and oxygen were not detected in any screening analyses. The method has proved to be of great value especially with “problem foodstuffs”. The limits of detection were determined for 385 pesticides and are presented together with their retention data.     

Determination of vanillin and related flavor compounds in natural vanilla extracts and vanilla-flavored foods by thin layer chromatography and automated multiple development

Summary Thin layer chromatography on silica gel high performance layers and automated multiple development was used to separate the polar aromatic flavor compounds vanillin, ethyl vanillin, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol, vanillic acid, coumarin, piperonal, anisic acid, and anisaldehyde commonly found in extracts of natural and artificial vanilla flavors. The ratio of 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde and vanillic acid to vanillin in natural vanilla extracts was used to confirm the authenticity of extracts purchased in the United States of America and the United Kingdom. Natural vanilla extracts purchased in Mexico and Puerto Rico were identified as counterfeit products based on changes in the above ratio and the presence of synthetic flavor compounds such as ethyl vanillin and coumarin. It is also demonstrated that the proposed method is suitable for the determination of natural and synthetic vanilla flavors in solvent extracts from food, beverage and confectionery products. The main advantage of thin layer chromatography for the analysis of vanilla extracts and food stuffs flavored with vanilla is its high sample throughput since sample preparation requirements are minimal and multiple samples can be separated simultaneously.     

Liquid chromatography with tandem mass spectrometry method for trace level analysis of migrated secondary alkanesulfonate from poly(tetramethylene terephthalate) into food simulants

Secondary alkanesulfonate has been widely used as an antistatic additive in polymers for producing anti‐dust food packaging containers. Currently, no reported method exists for accurate quantification of secondary alkanesulfonate in ethanolic and acidic food simulants. A new liquid chromatography with tandem mass spectrometry method was developed to quantify the migrated amount of secondary alkanesulfonate at trace levels in food simulants from a poly(tetramethylene terephthalate) containing secondary alkanesulfonate. The poly(tetramethylene terephthalate) samples loaded with the antistatic additive were exposed to various food simulants. The collected extracts were directly analyzed by liquid chromatography with tandem mass spectrometry in electrospray ionization negative mode. As secondary alkanesulfonate is a mixture containing C14 to C17 chain lengths, it was separated on a Poroshell 120 EC‐C8 column adopting methanol water gradient program. The migration of secondary alkanesulfonate ranged from 58 to 329 ppb; which is well within the allowed permissible regulatory limits and the adopted method was validated by conducting spiking studies and acceptable recoveries were obtained. The developed method was not only sensitive to detect lower levels of the migrated antistatic additive, but it also avoided more cumbersome sample preparation methodologies like sample enrichment and other derivatization approaches.     

Evaluation of Polyvinyl Acetate for Food Packaging by Studying Interactions Using HPLC

Abstract

Polyvinyl acetate was evaluated for food packaging by studying interactions with food nutrients like ascorbic acid, niacin, phenylalanine and caffeine. The polymer was immobilized on silica support and food based solvent like water used as a mobile phase. Thermodynamic parameters such as enthalpy of sorption, Gibb's free energy and activity coefficient data were used to determine mechanism, magnitude and kind (weak or strong) of interactions. Possibility of migration of the nutrients into the polymer and its suitability as a packaging matrix was concluded from the activity coefficient values.     

Layer-by-layer functionalized porous Zinc sulfide nanospheres-based solid-phase extraction combined with liquid chromatography time-of-flight/mass and gas chromatography-mass spectrometry for the specific enrichment and identification of alkaloids from Crinum asiaticum var. sinicum

The current widely utilized polymer or C8, C18 end-capped material-based sorbents for solid-phase extraction could not capture alkaloids well only based on “like dissolves like” principle. In this paper, a layer-by-layer functionalized porous Zinc sulfide nanospheres-based solid-phase extraction (SPE) combined with liquid chromatography time-of-flight/mass spectrometry (LC-TOF/MS) and gas chromatography-mass spectrometry (GC-MS) was developed for the specific enrichment and identification of alkaloids from complex matrixes, Crinum asiaticum var. sinicum crude extracts. The functionalized porous Zinc sulfide nanospheres were prepared by the amidation reaction of poly-(acrylic acid) (PAA) homopolymer with amino groups onto the porous ZnS nanospheres. Tandem LC-TOF/MS spectrometry presented that the almost all of the twenty-three main peaks in elution fraction from the SPE could be inferred as alkaloids with ion of mass according to the nitrogen rule and hit formula with Peak View1.2@software from AB SCIEX, and seven alkaloids including two new found chemical entities were directly identified from their GC-MS spectra and retention indices. We believe that this SPE protocol can also be utilized in the future to selectively enrich alkaloids from extracts of other plant species.     

Ultrafast UPLC‐ESI‐MS and HPLC with monolithic column for determination of principal flavor compounds in vanilla pods

In this study, two novel chromatographic methods based on monolithic column high‐performance liquid chromatography (HPLC) and ultra‐performance liquid chromatography (UPLC) were developed for the ultrafast determination of principal flavor compounds namely vanillin, vanillic acid, p‐hydroxybenzoic acid, and p‐hydroxybenzaldehyde in ethanolic extracts of Vanilla planifolia pods. Good separation was achieved within 2.5 min using Chromolith RP18e column (100 mm×4.6 mm) for HPLC and Acquity BEH C‐18 (100 mm×2.1 mm, 1.7 μm) column for UPLC. Both methods were compared in terms of total analysis time, mobile phase consumption, sensitivity, and validation parameters like precision, accuracy, LOD, and LOQ. Further, system suitability test data including resolution, capacity factor, theoretical plates, and tailing factor was determined for both the methods by ten replicate injections. Monolithic column based HPLC gave better results for most of the selected parameters while UPLC was found to be more eco‐friendly with low mobile phase consumption and better sensitivity. Both methods may be used conveniently for the high throughput analysis of large number of samples in comparison to traditional particulate column.     

高效液相色谱/电喷雾质谱测定减肥保健品中的盐酸西布曲明

盐酸西布曲明为减肥处方药,禁止添加到保健食品中;探索了利用高效液相色谱检测盐酸西布曲明的条件,提高了检测的抗干扰能力和检测限;探索了最佳质谱条件,提高了定性分析的可靠性.所采用的方法可用于准确检测保健食品中的盐酸西布曲明.     

HPLC determination of carotenoids in human serum by gradient elution technique

Summary Polarities of the carotenoids in human serum are very different; many nonpolar carotenoid hydrocarbons (e.g. -carotene, lycopene) and highly polar hydroxycarotenoids (e. g. -cryptoxanthin, zeaxanthin, lutein) can be found among them.Gradient elution chromatography was used for the separation of -carotene, lycopene, -cryptoxanthin, lutein and zeaxanthin in serum samples applying amino and cyano packings (Chromsil-NH₂, Chromsil-CN). The effects of different stationary phases on the selectivity were compared. The method is particularly suitable for the direct determination of serum levels of carotenoid in serum extracts.     

A Coupled HPLC/Radioimmunoassay for Analysis of Zidovudine Metabolites in Mononuclear Cells

Abstract

Reverse-phase HPLC, with the ion-pairing agent tetrabutyl ammonium phosphate, was used to separate zidovudine (ZDV) and its 5′-phosphorylated metabolites in extracts from peripheral blood mononuclear cells incubated with 2 μM ZDV. Because intracellular concentrations were too small to be visualized using UV detection, 1 ml fractions were collected and assayed for ZDV and metabolites using a commercial radioimmunoassay (RIA). Resolution of components was satisfactory, with a total chromatography time of 32 minutes. Interassay variability of peak areas was less than 14%. Comparison of UV detected chromatograms to RIA detected chromatograms from a standard mixture of ZDV and metabolites showed no significant difference between corresponding relative peak areas. The ion-pairing agent elevated baseline concentrations as measured by RIA. Quantitation was therefore performed by concurrent measurement of total phosphorylated ZDV using an established procedure, followed by comparison of relative peak areas. Results indicate that ZDV 5′-triphosphate, the active metabolite, is a major component of total phosphorylated ZDV, with peak heights significantly above baseline in extracts from less than 10⁷ mononuclear cells. Therefore, it should be possible to reliably quantitate this metabolite in cells from HIV-infected patients using only 10 to 20 ml of blood.     

SPE/HPLC/UV studies on acrylamide in deep-fried flour-based indigenous Chinese foods

A fast and cost-effective method using HPLC/UV has been developed for determination of acrylamide in deep-fried flour-based leaven dough foods available in Hong Kong. The samples were purified by a simple solid-phase extraction method which combined Oasis HLB and Bond Elut-Accucat cartridges. The aqueous sample solution was centrifuged at 14,500 ×g and 0 °C for 15 min to successfully remove the fat in the samples. A gradient elution program and a mobile phase of 4.0% v/v acetonitrile in water allowed sufficient retention and well resolved acrylamide from the food matrices in the sample extracts. Acrylamide was detected at UV wavelengths of 210 and 225 nm. The amounts of acrylamide in eight food samples were 27-198 μg/kg when 1-g samples were analyzed. The recoveries of acrylamide were larger than 78.0% and the precisions were 2.1-10.9% (n = 3). Our proposed method is especially relevant for analyzing acrylamide in those oily food matrices.     

Determination of nanogram/kilogram levels of polycyclic aromatic hydrocarbons in foods by HPLC with fluorescence detection

Methodology was developed for the determination of eight polycyclic aromatic hydrocarbons (PAH) in five food categories including meat/fish, dried dairy products, cereals, leafy vegetables and vegetable/marine oils. The eight PAH were fluoranthene, benzo[a]anthracene, benzo[k]fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene, 7,12-dimethylbenzo[a]anthracene, dibenzo[ah]anthracene and dibenzo[ai]pyrene. Samples were digested with alcoholic KOH followed by partitioning into solvents such as cyclohexane or isooctane. Lipids and other interferences were removed by solvent partitioning with dimethylformamide or dimethylsulfoxide/water. Additional cleanup involving column chromatography on silica gel, Florisil or Sephadex LH-20 was employed as required. Reversed-phase chromatography with gradient elution and fluorescence detection was employed for the determinations. Confirmation was carried out by GC-MS/SIM. Detection limits ranged from 2-90 ng/kg depending on the PAH and food analyzed. Results of a small survey indicated that the meat/fish category had the highest levels (low microgram/kg, on average) of the foods studied.     

Determination of polycyclic aromatic sulfur heterocycles in diesel particulate matter and diesel fuel by gas chromatography with atomic emission detection

The sulfur content of diesel fuel is of environmental concern because sulfur can facilitate the formation of diesel particulate matter (DPM) and sulfur dioxide (SO2) in the exhaust can poison catalytic converters. The US Environmental Protection Agency (EPA) has established more stringent regulations to reduce the sulfur content of diesel fuels in the near future. In this study, various types of organosulfur compounds in DPM extracts and the corresponding fuels have been determined by gas chromatography with atomic emission detection. The diesel fuels used have sulfur contents of 2284 and 433 ppm, respectively, and are labeled as high-sulfur and low-sulfur diesel fuels. The compounds identified are mainly polycyclic aromatic sulfur heterocycles (PASHs). In the fuels tested, trimethylbenzothiophenes (TMBTs), dibenzothiophenes (DBTs), and 4-methyldibenzothiophene (4-MDBT) were the most abundant sulfur compounds, while larger PASH compounds were more abundant in DPM extracts. The high-sulfur diesel fuel contained a larger proportion of PASHs with one or two rings (lighter PASHs). In DPM, the concentrations of total organic sulfur and individual PASHs are higher for the high-sulfur diesel fuel, and the relative percentage of one or two-ring PASHs is higher as well. The influence of engine load on the DPM composition was also examined. With increasing load, the PASH concentration in DPM decreased for lighter PASHs, increased for heavier PASHs, and had a bell-shaped distribution for PASHs in between.     

Bioactive Phenolic Compounds from Primula veris L.: Influence of the Extraction Conditions and Purification

Our experiments may help to answer the question of whether cowslip (Primula veris L.) is a rich source of bioactive substances that can be obtained by efficient extraction with potential use as a food additive. A hypothesis assumed that the type of solvent used for plant extraction and the individual morphological parts of Primula veris L. used for the preparation of herbal extracts will have key impacts on the efficiency of the extraction of bioactive compounds, and thus, the health-promoting quality of plant concentrates produced. Most analysis of such polyphenolic compound contents in extracts from Primula veris L. has been performed by using chromatography methods such as ultra-performance reverse-phase liquid chromatography (UPLC−PDA−MS/MS). Experiments demonstrated that the most effective extraction agent for fresh study material was water at 100 °C, whereas for dried material it was 70% ethanol. The richest sources of polyphenolic compounds were found in cowslip primrose flowers and leaves. The aqueous and ethanol extracts from Primula veris L. were characterized by a quantitatively rich profile of polyphenolic substances, and a high antioxidative potential. Selective extraction with the use of mild conditions and neutral solvents is the first step to obtaining preparations from cowslip primrose with a high content of bioactive substances.     

Evaluation of comprehensive two-dimensional gas chromatography with flame photometric detection: Potential application for sulfur speciation in shale oil

Flame photometric detection in the sulfur channel has been evaluated for sulfur speciation and quantification in comprehensive two-dimensional gas chromatography [GC × GC-FPD(S)] for S-compound speciation in shale extracts. Signal non-linearity and potential quenching effects were reportedly major limitations of this detector for analysis of sulfur in complex matrices. However, reliable linear relationships with correlation coefficient >0.99 can be obtained if the sum of the square root of each modulation slice in GC × GC is plotted vs. sulfur concentration. Furthermore, the quenching effects are reduced due to essentially complete separation of S-containing components from the hydrocarbon matrix. An increase of S/N of up to 150 times has been recorded for benzothiophene and dibenzothiophene in GC × GC-FPD when compared to GC-FPD due to the modulation process. As a consequence, 10 times lower detection limits were observed in the former mode. The applicability of the method was demonstrated using shale oil sample extracts. Three sulfur classes were completely separated and the target class (thiophenes) was successfully quantified after the rest of the sample was diverted to the second detector by using a heart-cut strategy. Based on the proposed method, 70% of the sulfur in the shale oil was assigned to the thiophenes, 24% to benzothiophenes, and 5% to dibenzothiophene compounds.     

Quantitation of acrylamide in food products by liquid chromatography/mass spectrometry

A simple and inexpensive liquid chromatography/mass spectrometry (LC/MS) method was developed for the quantitation of acrylamide in various food products. The method involved spiking the isotope-substituted internal standard (1-C13 acrylamide) onto 6.00 g of the food product, adding 40 mL distilled/deionized water, and heating at 65 degrees C for 30 min. Afterwards, 10 mL ethylene dichloride was added and the mixture was homogenized for 30 s and centrifuged at 2700 x g for 30 min, and then 8 g supernatant was extracted with 10, 5, and 5 mL portions of ethyl acetate. The extracts were combined, dried with sodium sulfate, and concentrated to 100-200 microL. Acrylamide was determined by analysis of the final extract on a single quadrupole, bench-top mass spectrometer with electrospray ionization, using a 2 mm id C18 column and monitoring m/z = 72 (acrylamide) and m/z = 73 (internal standard). For difficult food matrixes, such as coffee and cocoa, a solid-phase extraction cleanup step was incorporated to improve both chromatography and column lifetime. The method had a limit of quantitation of 10 ppb, and coefficients of determination (r2) for calibration curves were typically better than 0.998. Acceptable spike recovery results were achieved in 11 different food matrixes. Precision in potato chip analyses was 5-8% (relative standard deviation). This method provides an LC/MS alternative to the current LC/MS/MS methods and derivatization gas chromatography/mass spectrometry methods, and is applicable to difficult food products such as coffee, cocoa, and high-salt foods.     

Multivariate optimization and HS-SPME/GC-MS analysis of VOCs in red, yellow and purple varieties of Capsicum chinense sp. peppers

Peppers are used not only in cookery, but also in many other applications, like cosmetic, pharmaceutical and nourishing industry. The chemical composition of peppers is quite complex and several volatile and non-volatile substances contribute to their flavor, which is an important sensorial propriety. In this work a headspace/solid phase microextraction/gas chromatography coupled to mass spectrometry method was developed to evaluate the profiles of volatile compounds that contribute to the aroma of red, yellow and purple varieties of Capsicum chinense sp. peppers. The optimization of the extraction conditions was carried out using multivariate strategies such as factorial design and response surface methodology. The GC-MS analysis allowed the tentative identification of 34 compounds, with similarities higher than 85%, in accordance with the NIST mass spectral library. The data obtained by the analysis of volatile compounds, according to the proposed method, were treated with PCA chemometrics tool in order to group different varieties of C. chinense sp. peppers with similar VOC profiles. Amongst the most abundant VOCs, hexyl ester of pentanoic acid, dimethylcyclohexanols, humulene and esters of butanoic acid were found. Principal component analysis turned possible to visualize the grouping tendencies of the studied varieties of pepper, as well as the identification of the volatile compounds responsible for discriminating the three groups. Considering the fact that many species of peppers are used as human food, the significance of this work is further emphasized by its applicability to the study of food quality indicators, and as a tool for investigations on the composition of the pepper sources.     

Analysis of Biogenic Amines Using Immunoassays,HPLC, and a Newly Developed IC-MS/MS Technique in Fish Products—A Comparative Study

In this study, comparative analyses were carried out with ion chromatography mass-spectrometry (IC-MS/MS) which has no derivatization step, high-performance liquid chromatography (HPLC) technique, as well as two quantitative and two semi-quantitative immunoassays. The results demonstrated that HPLC and quantitative immunoassay methods were well-correlated with IC-MS/MS in determining histamine in various types of fish products. The best correlation was observed with the HistaSure ELISA Fast Track kit (R² = 0.9903). More than half of the values (68%) obtained by two methods were also statistically similar. The results of semi-quantitative test kits also supported histamine values estimated by quantitative methods, with some exceptions. The best results were found for HistaSure Lateral Flow in supporting the quantitative techniques. Therefore, these methods are found suitable for monitoring histamine in fish products in terms of food safety. Good correlations were also observed HPLC and IC-MS/MS in determining cadaverine, putrescine, and tyramine with the highest value observed for tyramine as R² = 0.9785. However, no correlation was observed for other biogenic amines, and the majority of the results were significantly different from each other for these amines (p < 0.05). The differences may be caused by the drawbacks reported previously for HPLC. However, further studies are required to confirm the possible effects. This study provides a comparative evaluation of several methods in terms of their suitability in determining biogenic amines in fish products for both monitoring and regulatory purposes.     

Quantitative HPLC Determination and Stability Studies of Pyridostemin in Extracts and Water Dispersible Granule Formulations of Stemona curtisii

A reversed-phase high-performance liquid chromatographic method has been developed and validated for the determination of pyridostemin, the major pesticidal alkaloid found in Stemona curtisii. This methodology was applied to the investigation of plant extracts and water dispersible granule formulations. Stability indicating procedures have also been carried out. The chromatographic separation was on a C₁₈ column with a mixture of acetonitrile–water–triethylamine (30:70:0.12, v/v/v), using UV detection at 300 nm. Validation procedures showed that the method was specific, accurate and precise. The response was linear over a range of 5–25 μg mL⁻¹ with recoveries in the range of 98.28–102.85%. The RSD for intra- and inter-day precision were <0.72 and <1.29%, respectively. Extraction of plant material with dichloromethane gave a significantly higher pyridostemin content in the crude extracts when compared with extractions in methanol. Partial purification of the crude extracts by silica gel column chromatography was used to concentrate the mixture about fourfold. Degradation behavior of pyridostemin in the partially purified extracts followed first-order kinetics. The main pathways for its decomposition were base hydrolysis and oxidation.     

Screening and analysis of bioactive compounds with biofingerprinting chromatogram analysis of traditional Chinese medicines targeting DNA by microdialysis/HPLC

Biofingerprinting chromatogram analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein, cell, etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophoraflavescens Ait. (Sophora) to bind to ct-DNA, respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated.