GC-MS characterization and quantification of sterols and cholesterol oxidation products

Summary A GC-MS method has been studied for characterization and quantification of phytosterols, cholesterol and cholesterol oxidation products. Baseline separations have been achieved between cholesterol, cholesterol 5-6-epoxide, 5-cholestene-3-ol-7one (7-keto-cholesterol), cholestene-3-5-6-triol, 5-cholestene-3-25-diol (25-hydroxycholesterol), 5-cholestene-3-20-diol (20-hydroxycholesterol), 5-cholestene-3-7-diol (7-hydroxycholesterol) and 5-cholestene-3-19-diol (19-hydroxycholesterol) as well as between -cholestane, cholesterol, stigmasterol, campesterol and -sitosterol. Excellent linearity of response has been obtained permitting reliable quantification. The characterization of each derivatized sterol has been performed by mass-spectrometry. The results confirm the utility of combined gas chromatography-mass spectrometry in the analysis and characterization of sterols and cholesterol oxidation products.     

Interfacial Modification and Structural Transitions Induced by Guest Molecules Solubilized in U‐Type Nonionic Microemulsions

Abstract

Alcohols and polyols are essential components (in addition to the surfactant, water, and oil) in the formation of U‐type self‐assembled nano‐structures, (sometimes called L‐phases or U‐type microemulsions). These microemulsions are characterized by large isotropic regions ranging from the oil side of the phase diagram up to the aqueous corner. The isotropic oily solutions of reverse micelles (“the concentrates”) can be diluted along some dilution lines with aqueous phase to the “direct micelles” corner via a bicontinuous mesophases (i.e., two structural transitions). This dilution takes place with no phase separations or occurrence of liquid crystalline phases. The structural transitions were determined by viscosity, conductivity, and pulsed gradient spin echo NMR (PGSE NMR), and are not visible to the eye. Two guest nutraceutical molecules (lutein and phytosterols) were solubilized, at their maximum solubilization capacity, in the reversed micellar solutions (L₂ phase) and were further diluted with the aqueous phase to the aqueous micellar corner (L₁ phase). Structural transitions (for the two types of molecule) from water‐in‐oil to bicontinuous microstructures were induced by the guest molecules. The transitions occurred at an earlier stage of dilution, at a lower water content (20 wt.% aqueous phase), than in the empty (blank) microemulsions (transitions at 30 wt.% aqueous phase). The transitions from the bicontinuous microstructure to the oil‐in‐water microemulsions were retarded by the solubilizates and occurred at later dilution stage at higher aqueous phase contents (50 wt.% aqueous region for empty microemulsion and >60 wt.% for solubilized microemulsion). As a result, the bicontinuous isotropic region, in the presence of the guest molecules, becomes much broader. It seems that the main reason for such “guest‐induced structural transitions” is related to a significant flattening and enhanced rigidity of the interface. The guest molecules of the high molecular volume are occupying high volume fraction of the interface (when the solubilization is maximal).     

Novel analytical methods for the determination of steroid hormones in edible matrices

This paper reviews recently published multi-residue chromatographic methods for the determination of steroid hormones in edible matrices. After a brief introduction on steroid hormones and their use in animal fattening, the most relevant EU legislation regarding the residue control of these substances is presented. An overview of multi-residue analytical methods, covering sample extraction and purification as well as chromatographic separation and different detection methods, being in use for the determination of steroid hormones (estrogens, gestagens and androgens), is provided to illustrate common trends and method variability. Emphasis was laid on edible matrices and more specifically on meat, liver, kidney, fat and milk. Additionally, the possibilities of novel analytical approaches are discussed. The review also covers specific attention on the determination of natural steroids. Finally, the analytical possibilities for phytosterols, naturally occurring steroid analogues of vegetable origin and a specific group of steroid hormones with a hemi-endogenous status are highlighted.     

Simultaneous determination of β‐sitosterol,campesterol, and stigmasterol in rat plasma by using LC–APCI‐MS/MS: Application in a pharmacokinetic study of a titrated extract of the unsaponifiable fraction of Zea mays L.

A liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated to investigate the pharmacokinetic properties of β‐sitosterol, campesterol, and stigmasterol in rat plasma. Cholesterol‐d₆ was used as an internal standard. To avoid interference of the three phytosterols in rat plasma and minimize matrix effects, a small volume (10 μL) of 4% bovine serum albumin was used as a surrogate matrix for making calibrators and quality control samples. Rat plasma (10 μL) samples were extracted by liquid–liquid extraction with methyl tert‐butyl ether and separated on a Kinetex C₁₈ column. The detection was performed on a triple quadrupole tandem mass spectrometer in selected reaction monitoring mode using positive atmospheric pressure chemical ionization. This assay was linear over concentration ranges of 250–5000 ng/mL (β‐sitosterol), 250–5000 ng/mL (campesterol), and 50–2000 ng/mL (stigmasterol). Additionally, a second set of quality controls made in rat plasma was also evaluated against calibration curves made using the surrogate matrix. All the validation data, including the specificity, precision, accuracy, recovery, matrix effect, stability, and incurred sample reanalysis conformed to the acceptance requirements. Our method was successfully applied to study the pharmacokinetics of three phytosterols in rats.     

A single step solid-phase extraction method for complete separation of sterol oxidation products in food lipids

One of the crucial steps in determination of sterol oxidation products (SOPs) in foods is their enrichment and purifications by various preparative methods for further analysis by GC and GC–MS. Among the preparative methods, SPE of various adsorbents and solvent systems, are being used most widely. At present, no single step SPE method is suitable to completely separate the SOPs. In this study, a SPE (1 g silica) method, suitable for both transesterified and cold saponified oil samples, was developed to separate completely SOPs from other lipid components. This method resulted in high recovery from rapeseed oil of added 5β,6β-epoxycholestan-3β-ol (94-96%), cholest-5-en-3β-ol-7-one(94%), cholestane-3β,5α,6β-triol (88–91%), cholest-5-en-3β,7α-diol and 5α,6α-epoxycholestan-3β-ol (88–90%). The method has a high sample capacity of up to 1 g transesterified or cold-saponified oil sample. The method was tested and applied to different vegetable oils and to monitor the effects of refining processes on POPs in hazelnut oil.     

Analysis of phytosterols in extra-virgin olive oil by nano-liquid chromatography

In this work the applicability of nano-liquid chromatography (nano-LC) was evaluated for the determination of phytosterols in extra-virgin olive oil samples. These compounds represent a minor part of lipids in vegetable oils, but their quantification can be useful to establish oil origin and to reveal intentional adulterations. The analysis of five main sterols, specifically brassicasterol, stigmasterol, campesterol, cholesterol and β-sitosterol, was performed in a laboratory-assembled nano-LC system coupled with a UV detector. The separation of all compounds was obtained in about 20 min, employing a capillary column packed with a C18-RP (sub-2 μm particles) stationary phase for 15 cm. Methanol only was used as mobile phase. The simple method developed and optimized was validated in terms of repeatability, linearity, limit of detection and limit of quantification (0.78 and 1.56 μg/mL, respectively) achieving good results. After this, it was applied to the determination of phytosterols in extra-virgin olive oil samples. Isolation of phytosterols was obtained by solid-phase extraction, after saponification and liquid–liquid extraction of the unsaponified fraction with diethyl ether. Recovery tests were performed and values between 90% and 103%, with RSDs within 5%, were obtained. Moreover the nano-LC system was coupled with a mass spectrometer for an accurate identification of phytosterols.     

Extraction optimization by response surface methodology: Purification and characterization of phytosterol from sugarcane (Saccharum officinarum L.) rind

A green, simple, and effective method for the extraction of sugarcane lipids from sugarcane rind was investigated by response surface methodology. The optimum conditions of technological progress obtained through response surface methodology were as follows: liquid‐to‐solid ratio 7.94: 1 mL/g, extraction temperature 50°C and extraction time 5.98 h. The practical sugarcane lipids extraction yield was 6.55 ± 0.28%, which was in good consistence with the predicted extraction yield of 6.47%. The results showed that the sugarcane lipids extraction yield obtained in optimum conditions increased by 1.16～7.28‐fold compared to the yields obtained in single‐factor experiments. After saponification and SPE steps, the nonsaponifiable fraction of sugarcane lipids was analyzed by gas chromatography with mass spectrometry and high‐performance liquid chromatography. β‐Sitosterol, stigmasterol, and campesterol were the prevailing phytosterols in the sample, while fucosterol, gramisterol, stigmast‐7‐en‐3‐ol, (3β,5α,24S)‐, stigmasta‐4,6,22‐trien‐3α‐ol, and cholest‐8(14)‐en‐3β‐ol acetate were also identified as minor steroids. Furthermore, the content of β‐sitosterol and a mixture of campesterol and stigmasterol (quantified by high‐performance liquid chromatography) was 44.18 mg/100 g dry weight and 43.20 mg stigmasterol/100 g dry weight, respectively. Our results indicate that sugarcane rind is a good source of phytosterol.     

Simultaneous extraction and determination of free and conjugated phytosterols in tobacco

Acid hydrolysis and alkaline saponification were incorporated into a microwave‐assisted extraction process for the simultaneous extraction of free and conjugated phytosterols from tobacco. The crude extract of the microwave‐assisted extraction was purified by C₁₈ solid‐phase extraction and then determined by high‐performance liquid chromatography. Phytosterols of cholesterol, ergosterol, stigmasterol, campesterol, and β‐sitosterol were determined by chromatographic quantification. The multiple parameters of microwave‐assisted extraction were optimized by a uniform design method. The optimal ratio of extraction ethanol solvent to tobacco mass was 30 mL/g. The microwave‐assisted extraction acid hydrolysis was carried out in sulfuric acid medium by heating for 10 min at 55°C. The microwave‐assisted extraction alkaline saponification was performed after adding excessive sodium hydroxide by heating another 10 min. The repeatability of the proposed method was acceptable with recoveries from 69.68 to 88.17% for the phytosterols. Five target phytosterols were all found in the tobacco samples, and the contents were significantly different in samples from different producing areas.     

Development of saw palmetto (Serenoa repens) fruit and extract standard reference materials

As part of a collaboration with the National Institutes of Health’s Office of Dietary Supplements and the Food and Drug Administration’s Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed two standard reference materials (SRMs) representing different forms of saw palmetto (Serenoa repens), SRM 3250 Serenoa repens fruit and SRM 3251 Serenoa repens extract. Both of these SRMs have been characterized for their fatty acid and phytosterol content. The fatty acid concentration values are based on results from gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS) analysis while the sterol concentration values are based on results from GC-FID and liquid chromatography with mass spectrometry analysis. In addition, SRM 3250 has been characterized for lead content, and SRM 3251 has been characterized for the content of β-carotene and tocopherols. SRM 3250 (fruit) has certified concentration values for three phytosterols, 14 fatty acids as triglycerides, and lead along with reference concentration values for four fatty acids as triglycerides and 16 free fatty acids. SRM 3251 (extract) has certified concentration values for three phytosterols, 17 fatty acids as triglycerides, β-carotene, and γ-tocopherol along with reference concentration values for three fatty acids as triglycerides, 17 fatty acids as free fatty acids, β-carotene isomers, and δ-tocopherol and information values for two phytosterols. These SRMs will complement other reference materials currently available with concentrations for similar analytes and are part of a series of SRMs being developed for dietary supplements. Contribution of the US Government; not subject to copyright     

Analysis of phytosterols and phytostanols in enriched dairy products by Fast gas chromatography with mass spectrometry

A Fast gas chromatography and mass spectrometry method for plant sterols/stanols analysis was developed, using a short capillary gas chromatography column (10 m × 0.1 mm internal diameter × 0.1 μm film thickness) coated with 5% diphenyl‐polysiloxane. A silylated mixture of the main plant sterols/stanols standards (β‐sitosterol, campesterol, stigmasterol, campestanol, sitostanol) was well separated in 1.5 min, with a good peak resolution (>1.4, determined on a critical chromatographic peak pair (β‐sitosterol and sitostanol)), repeatability (<13%), and sensitivity (<0.017 ng/mL). The suitability of this Fast chromatography method was tested on plant sterols/stanols‐enriched dairy products (yogurt and milk), which were subjected to lipid extraction, cold saponification, and silylation prior to injection. The analytical performance (sensitivity < 0.256 ng/mL and repeatability < 10.36%) and significant reduction of the analysis time and consumables demonstrate that Fast gas chromatography‐mass spectrometry method could be also employed for the plant sterols/stanols analysis in functional dairy products.