14 research outputs found
Mass spectrometric approaches for the analysis of phytosterols in biological samples.
Agriculture Development Fund grant, Ministry of Agriculture, Government of Saskatchewan, Canada.Peer ReviewedPlant sterols (phytosterols) are important structural components of plant cellular membranes and they play a major role during development and metabolism. They have health-associated benefits, especially in lowering blood cholesterol levels. Due to their many health claims, there is a growing interest in their analysis. Although various analytical strategies have been employed in analyzing phytosterols, chromatography linked to mass spectrometry (MS) is superior due to its sensitivity. Furthermore, specificity and selectivity are enhanced by utilizing tandem mass spectrometry (MS/MS). This article reviews the various mass spectrometric strategies used for the analysis of phytosterols. It highlights the applications and limitations associated with each MS strategy in various sample matrices such as plant, human, animal, food, and dietary supplements. GC-MS was historically the method of choice for analysis; however, the derivatization step rendered it tedious and time-consuming. On the other hand, liquid chromatography coupled to MS (LC-MS) simplifies the analysis. Many ionization techniques have been used namely electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). APCI showed superiority in terms of ion intensity and consistency in ion formation, primarily forming [M+H-H2O]+ ions rather than [M+H]+. In addition, matrix assisted laser desorption ionization (MALDI) as well as ambient mass spectrometry such as direct analysis in real time (DART) have also been evaluated
Fast Quantification Without Conventional Chromatography, The Growing Power of Mass Spectrometry
Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant.
Ministry of Agriculture, Government of Saskatchewan, Canada for an Agriculture Development Fund (ADF) grant.
German Federal Ministry of Education and Research (BMBF).Peer ReviewedMass spectrometry (MS) in hyphenated techniques is widely accepted as the gold standard quantitative tool in life sciences. However, MS possesses intrinsic analytical capabilities that allow it to be a stand-alone quantitative technique, particularly with current technological advancements. MS has a great potential for simplifying quantitative analysis without the need for tedious chromatographic separation. Its selectivity relies on multistage MS analysis (MSn), including tandem mass spectrometry (MS/MS), as well as the ever-growing advancements of high-resolution MS instruments. This perspective describes various analytical platforms that utilize MS as a stand-alone quantitative technique, namely, flow injection analysis (FIA), matrix assisted laser desorption ionization (MALDI), including MALDI-MS imaging and ion mobility, particularly high-field asymmetric waveform ion mobility spectrometry (FAIMS). When MS alone is not capable of providing reliable quantitative data, instead of conventional liquid chromatography (LC)-MS, the use of a guard column (i.e., fast chromatography) may be sufficient for quantification. Although the omission of chromatographic separation simplifies the analytical process, extra procedures may be needed during sample preparation and clean-up to address the issue of matrix effects. The discussion of this manuscript focuses on key parameters underlying the uniqueness of each technique for its application in quantitative analysis without the need for a chromatographic separation. In addition, the potential for each analytical strategy and its challenges are discussed as well as improvements needed to render them as mainstream quantitative analytical tools. Overcoming the hurdles for fully validating a quantitative method will allow MS alone to eventually become an indispensable quantitative tool for clinical and toxicological studies
Simple quantification of phytosterols and tocopherols using fast chromatography -Tandem mass spectrometry.
Tocopherols and Phytosterols are highly abundant compounds in waste products resulting from Canola Oil production. They have significant antioxidant and cholesterol lowering properties, respectively. Since Canola is a major crop product in Canada, effective extraction of these metabolites has economical impact. Hence, there is a need for the development of a fast and easy quantification method of these active metabolites.
Our analytical strategy relied on the use of fast chromatography -Tandem mass spectrometry (FC-MS/MS). A guard column was used to achieve fast separation and the method is compared to use of conventional C18 column. The mobile phase consisted of acetonitrile:methanol (99:1) with 0.1% acetic acid. The compounds were ionized in the positive ion mode using atmospheric pressure chemical ionization (APCI). The following parameters were employed: source temperature 380°C, curtain gas 40 psi, nebulizer current 2.5 µA and ion source gas 1 30 psi. 5α-Cholestan-3β-ol and Rac-tocol were used as internal standards for phytosterols and tocopherols, respectively.
Four phytosterols and four tocopherols, namely Stigmasterol, β-Sitosterol, Brassicasterol, Campesterol, Alpha-tocopherol, Gamma-tocopherol, Beta-tocopherol and Delta-tocopherol were determined using FC-Multiple-Reaction-Monitoring (MRM). The run time was 2 minutes only, compare to 6.5 minutes with the column injection. Beta-tocopherol and Gamma-tocopherol couldn’t be resolved on the guard column nor on the C18 column. The FC-MS/MS methods addressed the issue of cross talks among the target analytes. For example, stigmasterol and β-sitosterol precursor ions observed as [M+H-H20]+ has the same m/z values for the ion designated as [M+H-4H]+ for campesterol and brassicasterol. In fact, such interferences prevented the full removal of the column (i.e. loop injection). Calibration curves were established and a good linearity was achieved (0.25-10 µg/ml) with R2 of 0.996 and 0.997 for tocopherols and phytosterols, respectively.
In conclusion, a fast and simple FC-MS/MS method for the simultaneous quantitation of phytosterols and tocopherols was successfully developed
Influence of acid hydrolysis, saponification and sample clean-up on the measurement of phytosterols in dairy cattle feed using GC–MS and GC with flame ionization detection
The fortification of processed foods including dairy products is increasingly commonplace with phytosterols among many compounds used to improve the nutritional value of food products. It is also increasingly common practice for some dairy cattle feeds to be fortified for their potential to increase phytosterol levels in milk. In this study, a combined, streamlined protocol using acid hydrolysis, saponification and sample clean-up was developed to enable the rapid and reliable measurement of phytosterols. The method was developed with focus on streamlining the overall technique to make it suitable for commercial laboratories, to reduce labor and consumable costs, while maintaining accuracy. A total of 12 different feed types commonly used in the dairy industry were analyzed with the highest and lowest sterol contents found in cotton seed oil and tannin with average phytosterol contents of 256 and 0.99, the method was validated for milk to enable feed comparison studies with respect to the total phytosterol content in raw milk
Application of Supercritical Carbon Dioxide in Agricultural Products Processing
Extraction is indispensable for chemical processing and plays a crucial role for both qualitative and quantitative analysis. Conventional extraction techniques employ the use of organic solvents which are known to be toxic, flammable, expensive, and have negative environmental impact. The use of alternate solvents that are safe, renewable, and produce high quality extracts can be used to address these shortcomings. Supercritical carbon dioxide (SCO2) is generally regarded as safe (GRAS) and it has mild critical point values (73 atm, 31 °C) which are easily achievable. It is relatively inert, making it ideal for chemically and thermally labile compounds. It possesses dual characteristics, i.e. it dissolves like a liquid and diffuses like a gas, which is easily fine-tuned by changing temperature and pressure values. Besides, carbon dioxide is ubiquitous, cheap, recyclable, and it leaves no residue on the extract. Supercritical fluid extraction (SFE) using carbon dioxide as an alternate solvent was explored in processing of selected agricultural products (beeswax, distillers dried grains with solubles, and waste sheep wool) to produce value-added products. Beeswax is composed of esters of fatty acids and long-chain aliphatic alcohols with major application in pharmaceutical and cosmetic industries. Conventional extraction methods employ the use of organic solvents such as heptanes and hexanes. SCO2utilization was explored in beeswax refinement from honeycomb, slumgum, filter cake, and cappings. Vital parameters on extraction efficiency, pressure, temperature, and time, were investigated. Optimum extraction conditions for a 1.0000 g sample were found to be 10,000 psi, 70 °C, and dynamic extraction time of 30 min, with a 1:1 ratio of wax to activated carbon for complete bleaching. The flow was maintained at 0.5-1.0 mL/min of the condensed gas. Fatty acids, alcohols, and aliphatic hydrocarbons with carbon-chain lengths of C16-C30, C24-C30, and C23-C31 respectively were identified using GC-MS. This was in agreement when compared with the standard wax and, therefore, simultaneous extraction and bleaching of beeswax wax using carbon dioxide maintained its quality and SCO2 can serve as an ideal alternate solvent. Fourier-transform infrared spectroscopy (FTIR) showed similar absorbance bands when compared to commercial beeswax, while differential scanning calorimetry (DSC) confirmed the melting point to be approximately 62 °C which falls within the literature range (62-65 °C). Distillers dried grain with solubles (DDGS) is a byproduct during the ethanol production process from corn that is exclusively used as a livestock feed. It contains low starch and high levels of protein, fiber, minerals, and vitamins, making it ideal as a diet to treat medical conditions like diabetes and celiac disease in human beings. The presence of fatty acids creates an off flavor, while the dark color affects the appearance of baked products limiting its utilization. Bleaching of corn distillers dried grain with solubles (DDGS) to produce an upgraded food ingredient using SCO2as an alternate to ethanol washing was investigated. With a sample size of 5.0000 g, pressure, temperature and time of 5,000 psi, 50 °C, and 20 min respectively were found to be optimal at a flow of 1.5-2.0 mL/min of the condensed gas. Using a Hunter colorimeter, the residual DDGS color values of L*, a*, and b* were 81.47 ± 0.28, 1.05 ± 0.05, and 22.83 ± 0.61 respectively while palmitic acid, stearic acid, oleic acid, and linoleic acid were the major fatty acids present in the extract. Crude protein content of the processed DDGS was found to be 33.50 ± 0.20% w/w which was in agreement with the DDGS obtained via the ethanolwashing method. Waste sheep wool possesses no economic value and requires laborious and costly methods for its refinement. However, it contains lanolin, a high-value product in the cosmetic and pharmaceutical industries because of its emollient features. Supercritical carbon dioxide was employed to selectively isolate lanolin without any prior clean-up process. Response-surface methodology was applied to optimize the processing parameters using face-centered central-composite design. The independent variables were pressure (5,000-10,000 psi), temperature (60-100 °C), and time (20-80 min). Optimized conditions that gave the highest recovery were identified as 10,000 psi pressure, 100 °C temperature, and 66 min dynamic extraction time. The sample size was approximately 1.0000 g and the flow was maintained at 1.5-2.0 mL/min of the condensed gas. Using these optimized conditions, wool grease was extracted and refined from tags, greasy fleece, and choice fine wool. Comparative characterization using GC-MS was conducted for qualitative analysis. Branched fatty acids (iso or anteiso) ranging from C13-C30, alcohols ranging from C16-C30, and sterols formed the major composition of the extracted lanolin. DSC thermograms showed similar behavior between our refined lanolin and commercial lanolin. Overall, the results compared well with commercial lanolin and florisil at a ratio of 1:1 with wool grease showed the best results for bleaching and deodorization
Development of a Fast Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for Simultaneous Determination of Phytosterols and Tocopherols
Analytical Strategies to Analyze the Oxidation Products of Phytosterols, and Formulation-Based Approaches to Reduce Their Generation
Phytosterols are a class of lipid molecules present in plants that are structurally similar to cholesterol and have been widely utilized as cholesterol-lowering agents. However, the susceptibility of phytosterols to oxidation has led to concerns regarding their safety and tolerability. Phytosterol oxidation products (POPs) present in a variety of enriched and non-enriched foods can show pro-atherogenic and pro-inflammatory properties. Therefore, it is crucial to screen and analyze various phytosterol-containing products for the presence of POPs and ultimately design or modify phytosterols in such a way that prevents the generation of POPs and yet maintains their pharmacological activity. The main approaches for the analysis of POPs include the use of mass spectrometry (MS) linked to a suitable separation technique, notably gas chromatography (GC). However, liquid chromatography (LC)-MS has the potential to simplify the analysis due to the elimination of any derivatization step, usually required for GC-MS. To reduce the transformation of phytosterols to their oxidized counterparts, formulation strategies can theoretically be adopted, including the use of microemulsions, microcapsules, micelles, nanoparticles, and liposomes. In addition, co-formulation with antioxidants, such as tocopherols, may prove useful in substantially preventing POP generation. The main objectives of this review article are to evaluate the various analytical strategies that have been adopted for analyzing them. In addition, formulation approaches that can prevent the generation of these oxidation products are proposed.Medicine, Faculty ofNon UBCReviewedFacult
Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives
Phytosterols are plant sterols recommended as adjuvant therapy for hypercholesterolemia and tocopherols are well-established anti-oxidants. However, thermo-sensitivity, lipophilicity and formulation-dependent efficacy bring challenges in the development of functional foods, enriched with phytosterols and tocopherols. To address this, we developed liposomes containing brassicasterol, campesterol and β-sitosterol obtained from canola oil deodorizer distillate, along with alpha, gamma and delta tocopherol. Three approaches; thin film hydration-homogenization, thin film hydration-ultrasonication and Mozafari method were used for formulation. Validated liquid chromatographic tandem mass spectrometry (LC-MS/MS) was utilized to determine the entrapment efficiency of bioactives. Stability studies of liposomal formulations were conducted before and after pasteurization using high temperature short time (HTST) technique for a month. Vesicle size after homogenization and ultrasonication (<200 nm) was significantly lower than by Mozafari method (>200 nm). However, zeta potential (−9 to −14 mV) was comparable which was adequate for colloidal stability. Entrapment efficiencies were greater than 89% for all the phytosterols and tocopherols formulated by all three methods. Liposomes with optimum particle size and zeta potential were incorporated in model orange juice, showing adequate stability after pasteurization (72 °C for 15 s) for a month. Liposomes containing phytosterols obtained from canola waste along with tocopherols were developed and successfully applied as a food additive using model orange juice