Development of a Quantitative Method for Detection of Multiclass Veterinary Drugs in Feed Using Modified QuPPe Extraction and LC-MS/MS.

A method was developed for the rapid and quantitative analysis of 30 veterinary drugs belonging to 17 classes (amphenicols (1), anthelmintics (1), cephalosporins (4), coccidiostats (1), lincosamides (1), macrolide (1), nitroimidazole (1), penicillins (3), phenylhydrazines (1), polypeptides (1), pyrethrins (1), quinolones (5), sulfonamides (3), tetracycline (3), neuroleptic agents (1), triazene trypanocidal agents (1), other. (1)) in feeds. The proposed method with a modified Quick Polar Pesticides (QuPPe) sample preparation was validated for the determination of 30 veterinary drugs in feed samples by liquid chromatography triple-quadrupole mass spectrometry (LC-MS/MS). The sample was extracted with methanol containing 1% acetic acid and purified by dispersive solid-phase extraction (d-SPE) with C18. Good linearity (r2 ≥ 0.98) was observed, and the LOQ values ranged from 10 to 200 µg/kg. Average recoveries ranged from 70.8 to 118.4%, and the relative standard deviation was ≤ 18.7%. This validated method was used in the determination of 30 veterinary drugs in 142 feed samples obtained from South Korea. The results show that lincomycin was present in only one of the tested feed samples, although it was detected at a value lower than the LOQ. In conclusion, this multi-residue method can be used for screening through the detection and quantitation of residual multiclass veterinary drugs in feed samples.

In vitro modulatory effects of ginsenoside compound K, 20(S)-protopanaxadiol and 20(S)-protopanaxatriol on uridine 5'-diphospho-glucuronosyltransferase activity and expression.

We explored the inhibitory effect of ginsenoside compound K (CK), 20(S)-protopanaxadiol (PPD), and 20(S)-protopanaxatriol (PPT) on six uridine 5'-diphospho-glucuronosyltransferase (UGT) enzyme (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) activities in human liver microsomes (HLMs) and 10 UGT enzyme (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17) activities in recombinant UGT isoforms.PPD was a potent inhibitor of UGT1A3 activity with half-maximal inhibitory concentration values of 5.62 and 3.38 µM in HLMs and recombinant UGT1A3, respectively. UGT1A3 inhibition by CK and PPD was competitive with inhibitory constant (Ki) values of 17.4 and 1.21 µM, respectively, and inhibition by PPT was non-competitive with a Ki value of 8.07 µM in HLMs. PPD exhibited more than 3.4-fold selectivity for UGT1A3 inhibition compared with other UGT isoforms inhibition, while CK and PPT showed more than 2.16- and 2.21-fold selectivity, respectively.PPD did not significantly increase the mRNA expression of UGT1A1, 1A3, 1A4, 1A9, and 2B7 in hepatocytes.Given the low plasma concentrations of PPD in healthy human subjects and the absence of induction potential on UGT isoforms, we conclude that PPD cause no pharmacokinetic interactions with other co-administered drugs metabolised by UGT1A3.

Ginsenoside Rc Is a New Selective UGT1A9 Inhibitor in Human Liver Microsomes and Recombinant Human UGT Isoforms.

Ginseng is known to have inhibitory effects on UGT1A9 activity. However, little is known about the inhibitory effects of ginsenosides, the major active compounds in ginseng, on UGT1A9 activity. In vitro investigation of UGT1A9 inhibition by ginsenosides was carried out using human liver microsomes (HLMs). Among 10 ginsenosides, ginsenoside Rc was the strongest inhibitor of UGT1A9-mediated mycophenolic acid glucuronidase activity. Further inhibition kinetic studies using HLMs suggested that ginsenoside Rc competitively and noncompetitively inhibited UGT1A9-mediated propofol and mycophenolic acid glucuronidation activities, with K i values of 2.83 and 3.31 µM, respectively. Next, to investigate whether the inhibitory effect of ginsenoside Rc is specific to the UGT1A9 isoform, we studied the inhibitory potency of ginsenoside Rc on nine human uridine diphospho-glucuronosyltransferase (UGT) activities using recombinant human UGT isoforms. Ginsenoside Rc exhibited a 12.9-fold selectivity (which was similar to niflumic acid at 12.5-fold) for UGT1A9 inhibition. Ginsenoside Rc at 50 µM also inhibited none of the other UGT isoform-specific activities above 12.0%, except for UGT1A9 (>91.5%) in HLMs, indicating that ginsenoside Rc might be used as a selective UGT1A9 inhibitor in reaction phenotyping studies of new chemical entities. Considering lower plasma concentrations (0.01 µM) of ginsenoside Rc in healthy subjects and no induction potential on UGT isoforms, ginsenoside Rc does not cause pharmacokinetic drug interactions with other coadministered drugs metabolized by UGT1A9. SIGNIFICANCE STATEMENT: Ginsenoside Rc selectively inhibited UGT1A9-mediated propofol and mycophenolic acid glucuronidation activities in human liver microsomes and recombinant uridine diphospho-glucuronosyltransferase (UGT) isoforms. It exhibited a 12.9-fold selectivity for UGT1A9 inhibition. Therefore, ginsenoside Rc might be used as a selective UGT1A9 inhibitor in reaction phenotyping studies of new chemical entities, such as niflumic acid.

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.

Effect of Korean Red Ginseng on Cholesterol Metabolites in Postmenopausal Women with Hypercholesterolemia: A Pilot Randomized Controlled Trial.

Korean red ginseng (KRG) is known to exert beneficial effects on cardiovascular health. Meanwhile, reduced estrogen at menopause has been shown to have various adverse impacts on cardiovascular risk factors, including blood lipids. The aim of this pilot study was to investigate the effect of KRG on cholesterol metabolites, which are surrogate markers of cholesterol absorption and biosynthesis, in postmenopausal women with hypercholesterolemia. The present study is an exploratory study which used data from a 4-week, double-blinded, placebo-controlled clinical pilot study in 68 postmenopausal women with hypercholesterolemia. Patients received KRG (2 g) or placebo (2 g) once daily. The primary endpoints were changes in the levels of nine sterols. Serum sterols were analyzed using liquid chromatography-mass spectrometry (LC-MS)/MS analysis. Among the sterols, reduction in cholesterol level were significantly larger in the KRG group than in the placebo group (the changes: -148.3 ± 261.1 nmol/mL in the ginseng group vs. -23.0 ± 220.5 nmol/mL in the placebo group, p = 0.039). Additionally, changes in 7-hydroxycholesterol (7-OHC) were significantly larger in the KRG group than in the placebo group (the changes: -0.05 ± 0.09 nmol/mL in the ginseng group vs. -0.002 ± 0.1 nmol/mL in the placebo group, p = 0.047). Oxysterols, cholesterol derivates, have been known to play a role in chronic inflammation diseases such as cardiovascular diseases. KRG improves sterol metabolism by decreasing cholesterol and 7-OHC levels in postmenopausal women with hypercholesterolemia.

6,8-Diprenylorobol Induces Apoptosis in Human Hepatocellular Carcinoma Cells via Activation of FOXO3 and Inhibition of CYP2J2.

6,8-Diprenylorobol is a phytochemical derived from the roots of Glycyrrhiza uralensis Fisch. 6,8-Diprenylorobol exhibits several biological activities, but the effects of 6,8-diprenylorobol on cancers have been hardly investigated. This study is aimed at elucidating the anticancer effect and working mechanism of 6,8-diprenylorobol in HepG2 and Huh-7, two kinds of human hepatocellular carcinoma (HCC) cell lines. WST-1, cell counting, and colony formation assays and morphological change analysis showed that 6,8-diprenylorobol treatment decreased the cell viability and proliferation rate. Cell cycle analysis indicated that 6,8-diprenylorobol treatment increased the population of the G1/0 stage. Annexin V/PI double staining and TUNEL analysis showed that 6,8-diprenylorobol treatment increased the apoptotic cell population and DNA fragmentation. Western blot analysis showed that 6,8-diprenylorobol treatment increased the expression of cleaved PARP1, cleaved caspase-3, FOXO3, Bax, Bim, p21, and p27 but decreased the expression of Bcl2 and BclXL. Interestingly, 6,8-diprenylorobol inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activities with K i values of 9.46 and 2.61 µM, respectively. CYP2J2 siRNA transfection enhanced the anticancer effect of 6,8-diprenylorobol in HepG2 and Huh-7 cells through the downregulation of CYP2J2 protein expression and upregulation of FOXO3. Taken together, this study proposes that 6,8-diprenylorobol treatment may be a useful therapeutic option against HCC by targeting CYP2J2 and FOXO3.

Strong and Selective Inhibitory Effects of the Biflavonoid Selamariscina A against CYP2C8 and CYP2C9 Enzyme Activities in Human Liver Microsomes.

Like flavonoids, biflavonoids, dimeric flavonoids, and polyphenolic plant secondary metabolites have antioxidant, antibacterial, antiviral, anti-inflammatory, and anti-cancer properties. However, there is limited data on their effects on cytochrome P450 (P450) and uridine 5'-diphosphoglucuronosyl transferase (UGT) enzyme activities. In this study we evaluate the inhibitory potential of five biflavonoids against nine P450 activities (P450s1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A) in human liver microsomes (HLMs) using cocktail incubation and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most strongly inhibited P450 activity was CYP2C8-mediated amodiaquine N-dealkylation with IC50 ranges of 0.019~0.123 µM. In addition, the biflavonoids-selamariscina A, amentoflavone, robustaflavone, cupressuflavone, and taiwaniaflavone-noncompetitively inhibited CYP2C8 activity with respective Ki values of 0.018, 0.083, 0.084, 0.103, and 0.142 µM. As selamariscina A showed the strongest effects, we then evaluated it against six UGT isoforms, where it showed weaker inhibition (UGTs1A1, 1A3, 1A4, 1A6, 1A9, and 2B7, IC50 1.7 µM). Returning to the P450 activities, selamariscina A inhibited CYP2C9-mediated diclofenac hydroxylation and tolbutamide hydroxylation with respective Ki values of 0.032 and 0.065 µM in a competitive and noncompetitive manner. However, it only weakly inhibited CYP1A2, CYP2B6, and CYP3A with respective Ki values of 3.1, 7.9, and 4.5 µM. We conclude that selamariscina A has selective and strong inhibitory effects on the CYP2C8 and CYP2C9 isoforms. This information might be useful in predicting herb-drug interaction potential between biflavonoids and co-administered drugs mainly metabolized by CYP2C8 and CYP2C9. In addition, selamariscina A might be used as a strong CYP2C8 and CYP2C9 inhibitor in P450 reaction-phenotyping studies to identify drug-metabolizing enzymes responsible for the metabolism of new chemicals.