Multi-residue analytical method for detecting pesticides, veterinary drugs, and mycotoxins in feed using liquid- and gas chromatography coupled with mass spectrometry.

Modified QuEChERS and triple quadrupole mass spectrometry (LC and GC-MS/MS) technology were used to sequentially analyze pesticides, veterinary drugs, and mycotoxins in feed. In order to analyze the harmful substances that may remain or occur in the feed, we performed optimization experiments for sample preparation and LC-MS/MS and GC-MS/MS conditions. Optimized sample preparation involves extracting 5 g of sample with 15 mL of 0.25 M EDTA and 10 mL of acetonitrile. And some extracts were diluted 10-fold with 100 mM ammonium formate aqueous solution and analyzed by LC-MS/MS, and some extracts were purified through 25 mg PSA and analyzed by GC-MS/MS by adding an analyte protectant. We confirmed the matrix effect of feed ingredients and compound feeds, and added a dilution process after extraction to increase on-site efficiency. Matrix-matched calibration was applied for quantification. Method validation was performed for 197 pesticides, 56 components for veterinary drugs, and 5 components for toxins. All the components showed good linearity (r2 ≥ 0.98) in the developed analytical method. For most compounds, the limit of quantitation was 0.05 mg/kg. The recovery rate experiment was repeated three times at three concentrations including LOQ in feed ingredient, compound feed for livestock, and compound feed for pets. The recovery rate was 70.09-119.76% and relative standard deviations were ≤ 18.91%. And the accuracy and precision were further verified through cross-validation between laboratories. The developed analytical method was used to monitor 414 domestically distributed and imported feeds.

Optimization of the QuEChERS-Based Analytical Method for Investigation of 11 Mycotoxin Residues in Feed Ingredients and Compound Feeds.

Mycotoxins are toxic substances naturally produced by various fungi, and these compounds not only inflict economic damage, but also pose risks to human and animal health. The goal of the present study was to optimize the QuEChERS-based extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of 11 mycotoxins, such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), T-2 toxin, HT-2 toxin, zearalenone (ZEN), and deoxynivalenol (DON), commonly found in feed. The QuEChERS method, characterized by being "quick, easy, cheap, effective, rugged, and safe", has become one of the most common extractions and clean-up procedures for mycotoxin analyses in food. Therefore, in this experiment, an optimal method for the analysis of 11 mycotoxins in feed was established by modifying the general QuEChERS method. In this process, it was confirmed that even if feed samples of different weights were extracted, the quantitative value of mycotoxins in the feed was not affected. To reduce matrix effects, 13C-labeled compounds and deuterium were used as internal standards. This optimized method was then applied in the determination of 11 mycotoxins in 736 feed ingredients and compound feeds obtained from South Korea. The results showed that the occurrence rates of FBs, ZEN, and DON were 59.4%, 38.0%, and 32.1%, respectively, and OTA, AFs, and T-2 toxin and HT-2 toxin were found in fewer than 1% of the 736 feeds. The mean concentration ranges of FBs, ZEN, and DON were 757-2387, 44-4552, and 248-9680 µg/kg, respectively. Among the samples in which DON and ZEN were detected, 10 and 12 samples exceeded the management recommendation standards presented by the Ministry of Agriculture, Food and Rural Affairs (MAFRA). However, when the detected concentrations of DON and ZEN were compared with guideline levels in foreign countries, such as the US, Japan, China, and the EU, the number of positive samples changed. In addition, the co-occurrence of mycotoxins in the feed was analyzed, and the results showed that 43.8% of the samples were contaminated with two or three mycotoxins, among which the co-occurrence rate of FBs, ZEN, and DON was the highest. In conclusion, these results suggest the need for stricter management standards for FBs, DON, and ZEN in South Korea, and emphasize the importance of the continuous monitoring of feeds.

Comparison of sample preparation methods for the recovery of foodborne pathogens from fresh produce.

Sample preparation methods (pummeling, pulsifying, sonication, and shaking by hand) were compared for achieving maximum recovery of foodborne pathogens from iceberg lettuce, perilla leaves, cucumber, green pepper, and cherry tomato. Antimicrobial and dehydration effects also were examined to investigate causes of poor recovery of pathogens. Each produce type was inoculated with Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus at 6.0 log CFU/cm(2), and samples were prepared using the four methods. Bacterial populations recovered from the five types of produce were significantly different (P < 0.05) according to sample preparation methods and produce type. The bacterial populations recovered from pummeled and pulsified samples were higher (P < 0.05) than those recovered from sonicated and hand-shaken samples, except for cherry tomato. The number of bacteria recovered from produce was reduced (P < 0.05) from that of the inoculum by 0.16 to 2.69 log CFU/cm(2). Although extracts of iceberg lettuce, perilla leaves, cucumber, and green pepper had no antimicrobial activity, the populations of E. coli O157:H7, Salmonella Typhimurium, B. cereus, and L. monocytogenes in cherry tomato extract were slightly reduced after these treatments (P < 0.05). The pathogen populations on perilla leaves and cherry tomatoes decreased by >2 log CFU/cm(2) after exposure to 40% relative humidity for 1 h. No reduction was observed when the five pathogens were exposed to 90% relative humidity. These data suggest that pummeling and pulsifying are optimal sample preparation methods for detection of microorganisms. Acidic produce such as cherry tomato should be treated with a method that does not cause sample breakdown so that acid stress on the bacteria can be minimized. Dehydration stress also affects recovery of pathogens from produce.

Analysis of ceramide metabolites in differentiating epidermal keratinocytes treated with calcium or vitamin C.

Ceramides (Cer) comprise the major constituent of sphingolipids in the epidermis and are known to play diverse roles in the outermost layers of the skin including water retention and provision of a physical barrier. In addition, they can be hydrolyzed into free sphingoid bases such as C(18) sphingosine (SO) and C(18) sphinganine (SA) or can be further metabolized to C(18) So-1-phosphate (S1P) and C(18) Sa-1-phosphate (Sa1P) in keratinocytes. The significance of ceramide metabolites emerged from studies reporting altered levels of SO and SA in skin disorders and the role of S1P and Sa1P as signaling lipids. However, the overall metabolism of sphingoid bases and their phosphates during keratinocyte differentiation remains not fully understood. Therefore, in this study, we analyzed these Cer metabolites in the process of keratinocyte differentiation. Three distinct keratinocyte differentiation stages were prepared using 0.07 mM calcium (Ca(2+)) (proliferation stage), 1.2 mM Ca(2+) (early differentiation stage) in serum-free medium, or serum-containing medium with vitamin C (50 µL/mL) (late differentiation stage). Serum-containing medium was also used to determine whether vitamin C increases the concentrations of sphingoid bases and their phosphates. The production of sphingoid bases and their phosphates after hydrolysis by alkaline phosphatase was determined using high-performance liquid chromatography. Compared to cells treated with 0.07 mM Ca(2+), levels of SO, SA, S1P, and SA1P were not altered after treatment with 1.2 mM Ca(2+). However, in keratinocytes cultured in serum-containing medium with vitamin C, levels of SO, SA, S1P, and SA1P were dramatically higher than those in 0.07- and 1.2-mM Ca(2+)-treated cells; however, compared to serum-containing medium alone, vitamin C did not significantly enhance their production. Taken together, we demonstrate that late differentiation induced by vitamin C and serum was accompanied by dramatic increases in the concentration of sphingoid bases and their phosphates, although vitamin C alone had no effect on their production.

The effects of magnesium on the course of atrial fibrillation and coagulation in patients with atrial fibrillation undergoing mitral valve annuloplasty.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Magnesium has been reported to be effective in reducing the incidence or prophylaxis of AF. Magnesium is also an essential constituent of many enzyme systems and plays a physiological role in coagulation regulation. The aim of the present study was to examine the effects of magnesium, whether magnesium infusion might decrease the incidence of AF and induce hypocoagulable state in patients with AF, who were undergoing mitral valve annuloplasty. METHODS: This prospective laboratory study was performed using blood from patients with AF undergoing mitral valve annuloplasty. The radial artery was punctured with a 20 gauge catheter and used for monitoring continuous arterial pressure and blood sampling. After anesthesia induction, 4 g of magnesium was mixed with 100 ml normal saline and infused for 5 minutes. Magnesium, calcium, activated clotting time (ACT) and thromboelastographic parameters were checked before and 60 minutes after the magnesium infusion. The electrocardiography changes after magnesium infusion were also checked before commencing cardiopulmonary bypass. RESULTS: After magnesium infusion, the serum level of magnesium increased significantly but serum calcium did not change significantly. ACT did not change significantly before or after magnesium infusion. The thromboelastographic parameters showed no significant changes before or after magnesium infusion. None of the patients converted to sinus rhythm from AF after the magnesium infusion. CONCLUSIONS: A magnesium infusion did not influence the course of AF and coagulation in patients during prebypass period with AF undergoing mitral valve annuloplasty.

Effect of atmospheric pressure plasma on inactivation of pathogens inoculated onto bacon using two different gas compositions.

Atmospheric pressure plasma (APP) is an emerging non-thermal pasteurization method for the enhancement of food safety. In this study, the effect of APP on the inactivation of pathogens inoculated onto bacon was observed. Sliced bacon was inoculated with Listeria monocytogenes (KCTC 3596), Escherichia coli (KCTC 1682), and Salmonella Typhimurium (KCTC 1925). The samples were treated with APP at 75, 100, and 125 W of input power for 60 and 90 s. Two gases, helium (10 lpm) or a mixture of helium and oxygen, (10 lpm and 10 sccm, respectively) were used for the plasma generation. Plasma with helium could only reduce the number of inoculated pathogens by about 1-2 Log cycles. On the other hand, the helium/oxygen gas mixture was able to achieve microbial reduction of about 2-3 Log cycles. The number of total aerobic bacteria showed 1.89 and 4.58 decimal reductions after plasma treatment with helium and the helium/oxygen mixture, respectively. Microscopic observation of the bacon after plasma treatment did not find any significant changes, except that the L∗-value of the bacon surface was increased. These results clearly indicate that APP treatment is effective for the inactivation of the three pathogens used in this study, although further investigation is needed for elucidating quality changes after treatment.

Royal jelly enhances migration of human dermal fibroblasts and alters the levels of cholesterol and sphinganine in an in vitro wound healing model.

Oral administration of royal jelly (RJ) promotes wound healing in diabetic mice. Concerns have arisen regarding the efficacy of RJ on the wound healing process of normal skin cells. In this study, a wound was created by scratching normal human dermal fibroblasts, one of the major cells involved in the wound healing process. The area was promptly treated with RJ at varying concentrations of 0.1, 1.0, or 5 mg/ml for up to 48 hrs and migration was analyzed by evaluating closure of the wound margins. Furthermore, altered levels of lipids, which were recently reported to participate in the wound healing process, were analyzed by HPTLC and HPLC. Migration of fibroblasts peaked at 24 hrs after wounding. RJ treatment significantly accelerated the migration of fibroblasts in a dose-dependent manner at 8 hrs. Although RJ also accelerated the migration of fibroblasts at both 20 hrs and 24 hrs after wounding, the efficacy was less potent than at 8 hrs. Among various lipid classes within fibroblasts, the level of cholesterol was significantly decreased at 8 hrs following administration of both 0.1 ug/ml and 5 mg/ml RJ. Despite a dose-dependent increase in sphinganines, the levels of sphingosines, ceramides, and glucosylceramides were not altered with any concentration of RJ. We demonstrated that RJ enhances the migration of fibroblasts and alters the levels of various lipids involved in the wound healing process.

Effect of dietary mixture of gallic acid and linoleic acid on antioxidative potential and quality of breast meat from broilers.

The effect of dietary mixture of gallic acid and linoleic acid (MGL) on the antioxidative potential and quality of breast meat from broilers was investigated. Broilers during the 22-36days on trial received 3 dietary treatments: 1) control (commercial finisher diet), 2) 0.5% MGL (gallic acid:linoleic acid=1M:1M), and 3) 1.0% MGL. The feed efficiency, DPPH radical scavenging activity, ABTS(+) reducing activity, reducing power, TBARS, and total phenolic content in the breast from the broilers improved significantly by 1.0% MGL dietary treatment. Arachidonic and docosahexaenoic acids were higher in the broilers fed both levels of MGL diets. In addition, water holding capacity of the breast was enhanced by the 1.0% dietary MGL treatment and was accompanied by a slight antimicrobial activity (1 decimal reduction) during storage. In conclusion, 1.0% dietary supplementation with MGL can improve the antioxidative potential, and nutritional and functional qualities of broiler breast meat.

Gliotoxin enhances radiotherapy via inhibition of radiation-induced GADD45a, p38, and NFkappaB activation.

The purpose of the study was to elucidate the mechanism underlying the enhancement of radiosensitivity to 60Co gamma-irradiation in human hepatoma cell line HepG2 pretreated with gliotoxin. Enhancement of radiotherapy by gliotoxin was investigated in vitro with human hepatoma HepG2 cell line. Apoptosis related proteins were evaluated by Western blotting. Annexin V/PI and reactive oxygen species (ROS) were quantified by Flow Cytometric (FACS) analysis. Gliotoxin (200 ng/ml) combined with radiation (4 Gy) treated cells induced apoptosis. Cells treated with gliotoxin (200 ng/ml) prior to irradiation at 4 Gy induced the expression of bax and nitric oxide (NO). The gliotoxin-irradiated cells also increased caspase-3 activation and ROS. Gadd45a, p38, and nuclear factor kappa B (NFkappaB) activated in irradiated cells was inhibited by Gliotoxin. Specific inhibitors of p38 kinase, SB203580, significantly inhibited NFkappaB activation and increased the cytotoxicity effect in cells exposed to gliotoxin combined with irradiation. However, SB203580 did not suppress the activation of Gadd45a in irradiated cells. Gliotoxin inhibited anti-apoptotic signal pathway involving the activation of Gadd45a-p38-NFkappaB mediated survival pathway that prevent radiation-induced cell death. Therefore, gliotoxin, blocking inflammation pathway and enhancing irradiation-induced apoptosis, is a promising agent to increase the radiotherapy of tumor cells.