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Measurements of a 2H-labeling of water in biological fluids are required for determining the rates of biochemical flux and for estimating body composition. We have been using the method which relies on the base-catalyzed exchange of hydrogen (deuterium) between water and acetone. 2H-labeling of acetone is then determined using GCMS. Although not noted in the original paper, when chloroform is used to extract the acetone there is slow but substantial back exchange between [2H]acetone and solvent (unpublished observations). We report herein on a refinement of the assay that utilizes headspace analysis, which minimizes the number of transfers and decreases sample preparation time and instrument run time.
The therapeutic effect of Cannabis largely depends on the content of its pharmacologically active secondary metabolites, mainly phytocannabinoids, flavonoids, and terpenoids. Recent studies suggest there are therapeutic effects of specific terpenoids as well as synergistic effects with other active compounds in the plant. Although Cannabis contains an overwhelming milieu of terpenoids, only a limited number are currently reported and used for metabolic analysis of Cannabis chemovars. In this study, we report the development and validation of a method for simultaneous quantification of 93 terpenoids in Cannabis air-dried inflorescences and extracts. This method employs the full evaporation technique via a static headspace sampler, followed by gas chromatography-mass spectrometry (SHS-GC/MS/MS). In the validation process, spiked terpenoids were quantified with acceptable repeatability, reproducibility, sensitivity, and accuracy. Three medical Cannabis chemovars were used to study the effect of sample preparation and extraction methods on terpenoid profiles. This method was further applied for studying the terpenoid profiles of 16 different chemovars acquired at different dates. Our results demonstrate that sample preparation methods may significantly impact the chemical fingerprint compared to the nontreated Cannabis. This emphasizes the importance of performing SHS extraction in order to study the natural terpenoid contents of chemovars. We also concluded that most inflorescences expressed relatively unique terpenoid profiles for the most pronounced terpenoids, even when sampled at different dates, although absolute concentrations may vary due to aging. The suggested method offers an ideal tool for terpenoid profiling of Cannabis and sets the scene for more comprehensive works in the future.
Abstract:In this study, a novel approach in headspace gas chromatographic analysis using the selective absorption of the gas extractant during concentration of the analytes was developed. The carbon dioxide used as the gas extractant was removed from the sample flow by passing it through a column packed with microdispersed sodium hydroxide granules. The analytical capabilities of the suggested method were illustrated by the determination of aliphatic and aromatic hydrocarbons in water. We established that this method allows the preconcentration of analytes in the gas phase to be increased proportionally to the volume ratios of the gas extractant before and after absorption, while the analyte limits of detection decrease 30-fold. For example, benzene can be detected in water at a concentration of 0.5 μg/L.Keywords: gas chromatography; aliphatic and aromatic hydrocarbons; headspace analysis; gas- phase extraction; carbon dioxide; gas-extractant absorption
19: Musshoff F, Junker H, Madea B. Simple determination of 22 organophosphorous pesticides in human blood using headspace solid-phase microextraction and gas chromatography with mass spectrometric detection. J Chromatogr Sci. 2002 Jan;40(1):29-34. doi: 10.1093/chromsci/40.1.29. PMID: 11866384.
Evidence suggests that a diet high in fruits and vegetables may decrease the risk of chronic diseases, such as cardiovascular disease and cancer, and phytochemicals including phenolics, flavonoids and carotenoids from fruits and vegetables may play a key role in reducing chronic disease risk. Apples are a widely consumed, rich source of phytochemicals, and epidemiological studies have linked the consumption of apples with reduced risk of some cancers, cardiovascular disease, asthma, and diabetes. In the laboratory, apples have been found to have very strong antioxidant activity, inhibit cancer cell proliferation, decrease lipid oxidation, and lower cholesterol. Apples contain a variety of phytochemicals, including quercetin, catechin, phloridzin and chlorogenic acid, all of which are strong antioxidants. The phytochemical composition of apples varies greatly between different varieties of apples, and there are also small changes in phytochemicals during the maturation and ripening of the fruit. Storage has little to no effect on apple phytochemicals, but processing can greatly affect apple phytochemicals. While extensive research exists, a literature review of the health benefits of apples and their phytochemicals has not been compiled to summarize this work. The purpose of this paper is to review the most recent literature regarding the health benefits of apples and their phytochemicals, phytochemical bioavailability and antioxidant behavior, and the effects of variety, ripening, storage and processing on apple phytochemicals.
There has been some concern that apple antioxidants do not directly inhibit tumor cell proliferation, but instead they indirectly inhibit cell proliferation by generating H2O2 in reaction with the cell culture media [34]. However, more recently it has been reported that apple extracts did not generate H2O2 formation in WME, DMEM, or DMEM/Ham F12 media, and H2O2 addition to culture medium did not inhibit Hep G2 cell proliferation or Caco-2 colon cancer cell proliferation [35]. Additionally, the addition of catalase did not block the antiproliferative activity of apple extracts.
Although apple juice typically contains less phenolics than whole apples, it is still a widely consumed source of dietary antioxidants. Pearson et al [37] examined the effects of six commercial apple juices and Red Delicious apples (whole apples, peels alone, and flesh alone) on human LDL oxidation in vitro. LDL oxidation was measured using headspace analysis of hexanal produced from copper-induced lipid oxidation in vitro. The dose of the apple juices and whole apple, apple peel and apple flesh, were standardized for gallic acid equivalents, and each LDL solution was treated with 5 μM gallic acid equivalents for each apple sample. LDL oxidation inhibition varied greatly between brands of fruit juice, ranging from 9 to 34% inhibition and whole apples inhibited LDL oxidation by 34%. Apple peels inhibited LDL oxidation by 34%, while the flesh alone showed significantly less inhibition (21%) [37].
The compounds most commonly found in apple peels consist of the procyanidins, catechin, epicatechin, chlorogenic acid, phloridzin, and the quercetin conjugates. In the apple flesh, there is some catechin, procyanidin, epicatechin, and phloridzin, but these compounds are found in much lower concentrations than in the peels. Quercetin conjugates are found exclusively in the peel of the apples. Chlorogenic acid tends to be higher in the flesh than in the peel [47].
In another study involving human subjects, quercetin bioavailability from apples was only 30% of the bioavailability of quercetin from onions [61]. In this study, quercetin levels reached a peak after 2.5 hours in the plasma, however the compounds were hydrolyzed prior to analysis, so the extent of quercetin conjugation in the plasma is unknown. The bioavailability differences between apples and onions most likely are from the differences in quercetin conjugates in the different foods. Onions contain more quercetin aglycone and more quercetin glucosides, whereas apples tend to contain more quercetin monoglycosides and quercetin rutinoside, which may be less bioavailable. Our lab has examined the bioavailability of both quercetin and quercetin-3-glucoside from apple peel extracts and onion extracts in Caco-2 cells. Apple peel extracts contained no free quercetin, and no quercetin accumulation was seen in the Caco-2 cells following incubation with apple peel extract. Low amounts of quercetin-3-glucoside were absorbed by the cells (4%). However, onions contain some free quercetin and greater amounts of quercetin glucosides, and the absorption of quercetin into the Caco-2 cells from onion extracts was much greater than from apple extracts.
The above results can be explained by recent research examining quercetin and quercetin glycoside bioavailability. In a study by Walle et al. [62], it was found that, in the ileostomy fluid, quercetin primarily existed as the aglycone form. The group hypothesized that β-glucosidases hydrolyzed quercetin glucosides to quercetin, which could be then passively transported [62]. In support of this theory, Day et al. [63] determined that quercetin glycosides were mainly deglycosylated by lactase phlorizin hydrolase before the aglycone then passed into the cell. Some intact glycoside transport by SGLT1 occurred and the glucosides were deglycosylated within the cell by cytosolic β-glucosidase. Quercetin-3-glucoside appeared to utilize only the lactase phlorizin hydrolase pathway, not the SLGT1 transporter, but quercetin-4-glucoside used both pathways [63]. Apples contain some quercetin-3-glucoside that, following hydrolysis by LPH, would be available for uptake by intestinal cells. However, apples also contain other conjugates such as quercetin rhamnosides, quercetin xylosides, and quercetin galactosides that are not easily hydrolyzed by lactase phlorizin hydrolase, and most likely are not readily absorbed by small intestinal cells. In comparison, the quercetin in onions is almost all in the form of quercetin glucosides and free quercetin, making it more bioavailable to small intestinal cells. 153554b96e
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