Reaction of fumonisin with glucose prevents promotion of hepatocarcinogenesis in female F344/N rats while maintaining normal hepatic sphinganine/sphingosine ratios

The reaction of the primary amine of fumonisin B(1) (FB(1)) with glucose was hypothesized to detoxify this mycotoxin. Eighty 10-day-old female F344/N rats were injected intraperitoneally with diethylnitrosamine (DEN; 15 mg/kg of body weight). At 4 weeks of age, the weaned rats were randomly assigned to one of four treatment groups with 20 rats each. At 9 weeks of age, four rats from each treatment group were killed. At 12 weeks, another five rats from each group were killed. At 20 weeks of age, the remaining rats were killed. In comparison with the rats fed basal diet or FB(1)-glucose (containing 25 ppm of FB(1)), rats fed 8 ppm (residual amount of free FB(1) in the FB(1)-glucose mixture) or 25 ppm of FB(1) had greater alanine aminotransferase activity at 9 and 20 weeks of age (P < 0.001), greater endogenous hepatic prostaglandin E(2) production at 20 weeks of age (P < 0.05), and significantly lower plasma cholesterol at 20 weeks of age (P < 0.01). Placental glutathione S-transferase (PGST)-positive and gamma-glutamyltransferase (GGT)-positive altered hepatic foci (AHF) occurred only in rats fed 25 ppm of FB(1) at 20 weeks of age. Hepatic natural killer (NK) cell activities were similar among the four groups, but the percentage of total liver-associated mononuclear cells exhibiting the NKR-P1(bright) marker was significantly greater in rats fed FB(1)-glucose, FB(1) (8 ppm) and FB(1) (25 ppm) than in control rats at 9 weeks of age, and FB(1)-glucose-treated rats had significantly lower NKR-P1(bright) cells as a percentage of total liver-associated mononuclear cells than rats fed 25 ppm of FB(1) at 20 weeks of age (P < 0.05). PGST- or GGT-positive AHF were not detected in any treatment group at 9 or 12 weeks of age. At 20 weeks of age, half of the rats fed 25 ppm of FB(1) had PGST- and GGT-positive AHF. The sphinganine (Sa) concentration and the Sa/sphingosine (So) ratio were significantly greater in the rats fed 25 ppm of FB(1) diet as compared with the control groups at, respectively, 12 or 20 weeks of age. Therefore, modifying FB(1) with glucose seems to prevent FB(1)-induced hepatotoxicity and promotion of hepatocarcinogenesis. The Sa/So ratio was not the most sensitive biomarker of FB(1) toxicity.     

Dietary apigenin suppresses IgE and inflammatory cytokines production in C57BL/6N mice

Flavonoids ubiquitously exist in plants, vegetables, fruits, and teas. We evaluated the effect of dietary apigenin, one of the well-known flavonoids, on the immune system in C57BL/6N mice. Mice were fed experimental diets containing apigenin for 2 weeks. After the experimental period, there was no significant difference in body and organ weights between the control and the apigenin group. The total immunoglobulin (Ig) E levels in mice fed apigenin were significantly suppressed, whereas levels of IgG, IgM, and IgA were not affected. We also examined the effect of the apigenin diet on cytokine expression in mice sera using a cytokine array. The production of regulated upon activation normal T cell expressed and secreted (RANTES) and soluble tumor necrosis factor receptor I (sTNFRI) in mice sera was down-regulated by the apigenin diet. These results suggest that a diet containing apigenin can reduce serum IgE and inflammatory cytokines such as RANTES and sTNFRI in mice.     

Excretion of (14)C-fumonisin B(1), (14)C-hydrolyzed fumonisin B(1), and (14)C-fumonisin B(1)-fructose in rats.

14C-Fumonisin B(1) (FB(1)) was produced by Fusarium proliferatum M-5991 in modified Myro liquid medium and purified to >95% purity with a specific activity of 1.7 mCi/mmol. Nine male and nine female F344/N rats were each dosed by gavage with 0.69 micromol of (14)C-FB(1), (14)C-hydrolyzed FB(1), or (14)C-FB(1)-fructose/kg body weight. Urinary excretion of (14)C-FB(1) and (14)C-FB(1)-fructose was 0.5% and 4.4% of the total dose, respectively, and was similar between male and female rats. Urinary excretion of (14)C-hydrolyzed HFB(1) was significantly greater (P > 0.05) in female rats as compared with male rats (17.3% vs 12.8% of the total dose, respectively). There were no significant (P > 0.05) differences in biliary excretion of the three fumonisin compounds with a mean of 1. 4% of the dose excreted at 4 h after dosing. Lesser amounts continued to be excreted up to 9.25 h after dosing. Although biliary excretion of the (14)C-FB(1), (14)C-hydrolyzed FB(1), and (14)C-FB(1)-fructose was similar, increased urinary excretion of the (14)C-hydrolyzed FB(1) as compared to (14)C-FB(1) and (14)C-FB(1)-fructose indicated a greater absorption of the hydrolyzed form.     

Evaluating N/N and C/C isotope ratio analysis to investigate trophic relationships of elaterid larvae (Coleoptera: Elateridae)

Carbon and nitrogen isotope ratios in consumer tissues can be used to analyse the diet and trophic level of soil animals. However, life history traits may significantly influence stable isotope patterns. We evaluated in a series of experiments how stable isotope ratios of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) at natural abundance can be used to study the diet and trophic position of long-lived macro-invertebrates, elaterid larvae, which are major below-ground herbivores. Small, but significant differences in δ¹³C signatures were found between the larvaes’ anterior and posterior body segments, whereas exuvia reflected the body's overall isotopic composition. The species-specific trophic shift (±SE) in δ¹⁵N for Agriotes obscurus and Agriotes sputator (1.62±0.24‰ and 1.08±0.27‰, respectively) was significantly lower than “mean enrichment estimates” reported in the literature, showing the limited applicability of such generalised estimates in studies of invertebrate trophic ecology. To avoid false-positive assignments to two trophic levels due to variation in δ¹⁵N values, a minimum sample size of three and five individuals for A. obscurus and A. sputator, respectively, was needed to reduce this risk to below α=5%. Keeping elaterid larvae for up to 128 days without food did not affect their isotopic signatures, in contrast to previous studies on starving animals. Switching wireworms to isotopically different diets induced changes in their isotopic signatures within 2 weeks. Changes, however, were significant only when the isotopic difference between diets was large. We conclude that experimental studies evaluating how specific life history traits affect stable isotope signatures in consumers have to precede any interpretation of stable isotope data gathered in the field.     

Food matrix effects on bioactivity of broccoli-derived sulforaphane in liver and colon of F344 rats

Sulforaphane (SF) is considered to be the major anticarcinogenic component in broccoli. The effects of feeding rats purified SF (5 mmol/kg of diet), broccoli containing SF formed in situ during laboratory hydrolysis (broccoli-HP; 20% freeze-dried broccoli diet, 0.16 mmol of SF/kg of diet), and broccoli containing intact glucosinolates (broccoli-GS; 20% freeze-dried broccoli diet, 2.2 mmol of glucoraphanin/kg of diet) were compared. Rats (male F344 rats, five per group) were fed control (modified AIN-76 B-40), SF, broccoli-HP, or broccoli-GS for 5 days. In rats fed broccoli-GS, quinone reductase activities (QR) in the colon and liver were greater (4.5- and 1.4-fold over control, respectively) than in rats fed broccoli-HP (3.2- and 1.1-fold over control, respectively). Broccoli-GS and SF diets increased QR to the same extent, even though the broccoli-GS diet contained far less SF (as the unhydrolyzed glucosinolate, glucoraphanin) than the purified SF diet. In a second experiment, rats were fed one of six diets for 5 days: (1) control; (2) 20% broccoli-GS; (3) diet 2 + low SF (0.16 mmol/kg of diet); (4) diet 2 + high SF (5 mmol/kg of diet); (5) low SF (0.32 mmol/kg of diet); or (6) high SF (5.16 mmol/kg of diet). In both liver and colon, QR was increased most by high SF plus broccoli-GS; individually, high SF and broccoli-GS had similar effects, and adding the low-dose SF to broccoli-GS had either no effect or a negative effect. In both experiments, urinary SF-mercapturic acid correlated with QR activity, not with dietary intake. It was concluded that all diets were substantially more effective in the colon than in the liver and that broccoli-GS was more potent than SF or broccoli-HP.     

90-day oral toxicity study of a grape seed extract (IH636) in rats

To assess the safety of grape seed extract with less than 5.5% catechin monomers (IH636), 4 groups of male and female Sprague-Dawley rats were provided grape seed extract in the diet at levels of 0 (control), 0.5, 1.0, or 2.0% for a period of 90 days. All animals survived the duration of the study, and no significant changes in clinical signs, hematological parameters, organ weights, ophthalmology evaluations, or histopathological findings were observed. A significant increase in food consumption was observed in male and female rats provided the grape seed extract diets compared to that of the control rats, especially in male rats consuming 2.0% grape seed extract. This effect was not accompanied by increases in body weight gains. Grape seed extract appeared to increase the insoluble fraction of the diet. Male rats in the high-dose group exhibited decreased serum iron levels and decreased serum iron/total iron binding capacity ratio compared to those of the controls, although all values were within historical ranges for Sprague-Dawley rats. In conclusion, administration of the grape seed extract IH636 to male and female Sprague-Dawley rats in the feed at levels of 0.5, 1.0, or 2.0% for 90 days did not induce any significant toxicological effects.     

Compartmentalization of the soil animal food web as indicated by dual analysis of stable isotope ratios (N/N and C/C)

The soil animal food web has become a focus of recent ecological research but trophic relationships still remain enigmatic for many taxa. Analysis of stable isotope ratios of N and C provides a powerful tool for disentangling food web structure. In this study, animals, roots, soil and litter material from a temperate deciduous forest were analysed. The combined measurement of δ¹⁵N and δ¹³C provided insights into the compartmentalization of the soil animal food web. Leaf litter feeders were separated from animals relying mainly on recent belowground carbon resources and from animals feeding on older carbon. The trophic pathway of leaf litter-feeding species appears to be a dead end, presumably because leaf litter feeders (mainly diplopods and oribatid mites) are unavailable to predators due to large size and/or strong sclerotization. Endogeic earthworms that rely on older carbon also appear to exist in predator-free space. The data suggest that the largest trophic compartment constitutes of ectomycorrhizal feeders and their predators. Additionally, there is a smaller trophic compartment consisting of predators likely feeding on enchytraeids and potentially nematodes.     

Physiological difference between free and triglyceride-type conjugated linoleic acid on the immune function of C57BL/6N mice

Previous studies have shown the physiological significance of dietary conjugated linoleic acid (CLA) in various experimental animals and in human beings. One of the important problems to better elucidate is the difference between triglyceride (TG) and free (FFA) dietary CLA. Here, using splenocytes, this study assesses how TG- and FFA-CLA modulate immunoglobulin and various cytokine productions. In this study, C57BL/6N mice were fed an experimental diet containing 0% CLA, 0.1 or 1% FFA-CLA, or 0.1 or 1% TG-CLA for 3 weeks. The production of immunoglobulin tended to be up-regulated by 1% FFA-CLA. As a result of protein array analysis using the supernatant from splenocytes cultured with no CLA, 1% FFA-CLA, and TG-CLA, some cytokine production was shown to be remarkably regulated by dietary FFA- and TG-CLA. A total of 32 cytokines were examined, and 11-14 produced cytokines that were 2-fold up-regulated as compared with control for FFA- or TG-CLA, respectively. Especially, the production of IL-9 and MCP-5 and other cytokines was remarkably up-regulated by both FFA- and TG-CLA. In addition, seven cytokines were 2-fold down-regulated by TG-CLA. These data show that there is a slight but significant difference between the functionalities of FFA- and TG-CLA.     

Endogenous formation of N'-nitrosonornicotine in F344 rats in the presence of some antioxidants and grape seed extract

N'-Nitrosonornicotine (NNN) is one of the most abundant strong carcinogens in unburned tobacco and cigarette smoke and is classified by the International Agency for Research on Cancer as carcinogenic to humans. Human exposure to NNN mainly occurs upon use of tobacco products. It is also possible that additional amounts of NNN are formed endogenously. The goal of this study was to evaluate the inhibitory effect of some antioxidants, including ascorbic acid and grape seed extract (GSE), on endogenous NNN formation in rats treated with nornicotine and sodium nitrite by gavage twice daily for 3 days. The study included four groups of rats: (1) negative control group A, to which no chemical was administered; (2) negative control group B, treated with nornicotine alone (2.5 micromol per gavage); (3) positive control group, to which both nornicotine (2.5 micromol per gavage) and sodium nitrite (7.5 micromol per gavage) were administered; and (4) rats treated with nornicotine (2.5 micromol per gavage), inhibitor (7.5 or 37.5 micromol per gavage), and sodium nitrite (7.5 micromol per gavage). The mean (+/-SD) total amount of NNN in the 3-day urine of rats treated with both nornicotine and sodium nitrite was 4.78 +/- 2.88 nmol. The order of inhibition of endogenous NNN formation in rats at the molar ratio [nitrite]:[inhibitor] 1:5 was as follows: ascorbic acid (91%) > dihydroxyfumaric acid (86%) approximately catechin (85%) > resveratrol (no inhibition). Treatment of rats with grape seed extract did not produce statistically significant inhibition of endogenous nornicotine nitrosation. This is the first study that demonstrates endogenous NNN formation in rats treated with nornicotine and sodium nitrite and effective inhibition of this process by ascorbic acid, dixydroxyfumaric acid, and catechin.     

Certification of B-group vitamins (B1, B2, B6, and B12) in four food reference materials

In 1989, the Community Bureau of Reference started a research program to improve the quality of vitamin analysis in food. To achieve this task, vitamin methodology was evaluated and tested by interlaboratory studies and the preparation of certified reference materials, which will be used for quality control of vitamin measurements. The main improvements in methodology were achieved by testing and standardizing the extraction condition and enzymatic hydrolysis procedures. Results for each individual material are derived from five replicate determinations using at least two independent methods: liquid chromatography (HPLC) and microbiological assay for vitamins B1, B2, and B6; and radioprotein binding and microbiological assays for vitamin B12. The certificate of analysis for four reference materials gives mass fraction values for water-soluble vitamins. These certified values were based on the acceptable statistical agreement of results from collaborating laboratories. Certified values with uncertainties (mg/kg dry matter) for each CRM are as follows: 4.63 (0.20) and 4.10 (0.51) for vitamins B1 and B6, respectively, in CRM 121 (wholemeal flour); 6.51 (0.24), 14.54 (0.3), 6.66 (0.43), and 0.034 (0.003) for vitamins B1, B2, B6, and B12, respectively, in CRM 421 (milk powder); 3.07 (0.17) and 4.80 (0.40) for vitamins B1 and B6, respectively, in CRM 485 (lyophilized mixed vegetables), and 8.58 (0.55), 106.8 (2.8), 19.3 1.5), and 1.12 (0.044) for vitamins B1. B2, B6, and B12, respectively, in CRM 487 (lyophilized pig liver).     

Conjugated linoleic acid-induced fatty liver can be attenuated by combination with docosahexaenoic acid in C57BL/6N mice

We investigated the effect of dietary combination of conjugated linoleic acid (CLA) and docosahexaenoic acid (DHA) to attenuate CLA-induced fatty liver in C57BL/6N mice. Mice were fed semisynthetic diets that contained either 6% high linoleic safflower oil (HL-SAF), 4% HL-SAF + 2% CLA, or 3.5% HL-SAF + 2% CLA + 0.5% DHA for 4 weeks. This 4 week feeding of CLA showed hepatic lipid accumulation concomitant with the decrease in adipose tissue weight in mice. However, 0.5% supplementation of DHA to the CLA diet could alleviate fatty liver without decreasing the antiobesity effect of CLA. The CLA diet promoted fatty acid synthesis in the liver, but DHA supplementation significantly attenuated the increase in enzyme activity induced by CLA. On the other hand, serum adipocytokines, leptin and adiponectin, were drastically decreased by CLA feeding, and DHA supplementation did not affect those levels. These results show that DHA supplementation to the CLA diet can attenuate CLA-induced fatty liver through the reduction of hepatic fatty acid synthesis without affecting adipocytokine production in C57BL/6N mice.     

Seasonal and age-related changes in the stable isotope composition (15N/14N and 13C/12C) of millipedes and collembolans in a temperate forest soil

Seasonal changes in environmental conditions and biotic interactions are often ignored when using stable isotope analysis for reconstructing the trophic structure of soil communities in temperate ecosystems. In this study, we estimated seasonal and age-related changes in δ¹³C and δ¹⁵N values in three epigeic species of collembolans (Pogonognathellus longicornis, Orchesella flavescens and Isotoma viridis) and two litter-dwelling species of millipedes (Polydesmus denticulatus and Leptoiulus proximus) in deciduous and coniferous forest stands in central Russia. Age-related changes in δ¹³C or δ¹⁵N values were either absent or negligible (within 1‰) in L. proximus, but adult and subadult specimens of P. denticulatus were enriched in ¹⁵N compared to early larval stages. Since the adults of P. denticulatus were generally more enriched in ¹⁵N than adults of L. proximus, they presumably occupy more distinct trophic niches than juveniles do. Age-related changes in isotopic composition were small or absent in collembolans studied. Neither δ¹³C nor δ¹⁵N values of millipedes changed significantly during the vegetation season. In contrast, consistent seasonal changes in δ¹³C and δ¹⁵N values were found in collembolans. Increased δ¹³C values coincided with the period of minimum soil moisture and correlated with a decreased C/N ratio in collembolan tissues. These changes can largely be attributed to the depletion of lipid-rich storage tissues. Seasonal changes in δ¹⁵N values were similar among collembolan species, yet slightly varied between habitats. A general trend of increasing δ¹⁵N values from June to September–October may indicate either a reduced importance of non-vascular plants (algae and lichen) in collembolan diet or variation in the isotopic composition of these plants. Overall, our data show that seasonal variations should be taken into account when estimating the isotopic composition of epigeic collembolans in forest soils.     

Effect of fermentation on Sweetpotato (Ipomoea batatas) toxicity in mice

Unfortunate bovine fatalities occurring after ingestion of mold-damaged sweetpotatoes preclude the use of the culled tubers in livestock feed. In cattle, mold-damaged sweetpotatoes induce an acute respiratory distress syndrome resulting in asphyxiation. Because of this potential toxicity and the general abundance of culled sweetpotatoes, the detoxification efficacy of ensiling was explored since it is an easy and economically viable technique often applied to preserve livestock feed. Sweetpotato slices with or without mold damage were stored either frozen (to represent unfermented samples) or fermented for 6 weeks at room temperature. Following fermentation, organic extracts were generated for administration to mice. Thirty hours following administration of the extracts, mice were evaluated for gross and microscopic lesions affecting the lungs, liver, and kidneys. Fermentation of 6 weeks duration was observed to inadequately eliminate the lung, liver, and kidney toxicity caused by mold-damaged sweetpotatoes. In fact, fermentation exacerbated the hepatotoxicity of mold-damaged sweetpotatoes. This is also the first demonstration that sweetpotato regions lacking visible mold damage can induce lung and kidney injury, which, however, is preventable by fermentation.     

Amelioration of obesity and glucose intolerance in high-fat-fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas)

Much attention has been focused on food that may be beneficial in preventing diet-induced body fat accumulation and possibly reduce the risk of diabetes and heart disease. Cornelian cherries (Cornus mas) are used in the preparation of beverages in Europe and also to treat diabetes-related disorders in Asia. In this study, the most abundant bioactive compounds in C. mas fruits, the anthocyanins and ursolic acid, were purified, and their ability to ameliorate obesity and insulin resistance in C57BL/6 mice fed a high-fat diet was evaluated. Mice were initially fed a high-fat diet for 4 weeks and then switched to a high-fat diet containing anthocyanins (1 g/kg of high-fat diet) and ursolic acid (500 mg/kg of high-fat diet) for an additional 8 weeks. The high-fat diet induced glucose intolerance, and this was prevented by anthocyanins and ursolic acid. The anthocyanin-treated mice showed a 24% decrease in weight gain. These mice also showed decreased lipid accumulation in the liver, including a significant decrease in liver triacylglycerol concentration. Anthocyanin and ursolic acid treated mice exhibited extremely elevated insulin levels. Both treatments, however, showed preserved islet architecture and insulin staining. Overall, these data suggest that anthocyanins and ursolic acid purified from C. mas fruits have biological activities that improve certain metabolic parameters associated with diets high in saturated fats and obesity.     

Mold occurrence and aflatoxin B(1) and fumonisin B(1) determination in corn samples in Venezuela

Fumonisins are mycotoxins produced mainly by Fusarium moniliforme and Fusarium proliferatum, which have been associated with several animal and human diseases. Aflatoxins are hepatotoxic, mutagenic, and teratogenic metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Both have been reported at high levels in corn. This study was pursued to determine mold, aflatoxin B(1) (AFTB(1)), and fumonisin B(1) (FB(1)) levels in white and yellow corn. Mold levels were determined using potato dextrose agar and identification of the main genus of molds present in corn, AFTB(1) levels by immunoaffinity chromatography, and FB(1) levels by a Bond-Elut SAX cartridge and HPLC. AFTB(1) an     

Simultaneous determination of fumonisin B(1) and hydrolyzed fumonisin B(1) in corn products by liquid chromatography/electrospray ionization mass spectrometry

A method for the simultaneous determination of fumonisin B(1) (FB(1)) and its major hydrolysis product (HFB(1)), which is known to be formed during alkaline treatment of fumonisin-containing corn meal, was devised to analyze the levels of these mycotoxins in corn products available on the German market. Liquid chromatography/electrospray mass spectrometry in combination with selected ion monitoring (SIM) was used for unambiguous detection of FB(1) and HFB(1) after extraction of samples with acetonitrile/methanol/water (25:25:50) and solid-phase C18 cleanup. Quantitation was carried out using labeled fumonisin FB(1)-D(6) as an internal standard. The detection limits achieved with this method were 8 ng/g for HFB(1) (signal-noise ratio = 5:1) and 5 ng/g for FB(1) (s/n = 5:1) using the protonated molecule signals m/z 406 and 722 in the SIM mode. A screening of several corn-containing foodstuffs, among them extrusion products and alkali-processed corn food such as tortilla chips, showed HFB(1) and FB(1) contamination with levels of 8-80 and 5-450 ng/g, respectively.     

Differential effects of powdered whole soy milk and its hydrolysate on antiobesity and antihyperlipidemic response to high-fat treatment in C57BL/6N mice

This study was performed to investigate the beneficial effects of powdered whole soy milk and its hydrolysate, compared to the processed soy milk and its hydrolysate, on the alteration of lipid metabolism and their possible effects on antiobesity in C57BL/6N mice fed a high-fat and -cholesterol diet. The mice were divided into a control group (20% casein) and four test groups for 5 weeks: soy milk (SM, 20% soy milk protein), soy milk hydrolysate (SMH, 20% hydrolyzed soy milk protein), whole soy milk (WSM, 20% whole soy milk protein), and whole soy milk hydrolysate (WSMH, 20% whole soy milk hydrolysate protein). The body weight and adipose tissue weights were significantly lowered in SMH, WSM, and WSMH groups compared to the control group despite providing an isoenergetic diet. Plasma lipid concentrations and hepatic fatty acid synthase (FAS) and glucose-6-phosphate dehydrogenase (G6PD) activities were significantly lowered in all soy milk groups; however, the hepatic lipid contents and malic enzyme (ME) activity were only significantly lowered in the WSM and WSMH groups, compared to the control group. Data suggest that powdered WSM or WSMH appears to be more beneficial than SM or SMH in overall antiobesity and antihyperlipidemic properties following in the order WSMH/WSM, SMH, SM, and casein.     

Determination of vitamin B(6) in cooked sausages

A reverse-phase high-performance liquid chromatography (HPLC) method has been described for the determination of various active forms of vitamin B(6) in meat products. Different extracting agents were tested to solubilize fully the analyte for quantification. The best data were obtained by extracting the samples with 5% (w/v) metaphosphoric acid. Separation by HPLC was performed with fluorescence detection (excitation, 290 nm; emission, 395 nm), on a 10 cm x 0.46 cm i.d. Hypersil BDS C(18) 5 microm column using a mixture of 50 mM phosphate buffer (pH 3.2) and acetonitrile (99:1, v/v) as mobile phase. Precision of the method was 0.5% (within a day) and 4.3% (between days). The detection limits were 0.020 mg/100 g for pyridoxal and pyridoxamine, 0.017 mg/100 g for pyridoxamine phosphate, 0.500 mg/100 g for pyridoxal phosphate, and 0.033 mg/100 g for pyridoxol, with a signal-to-noise ratio of 3. The recovery ranged from 92.0 to 100.0%.