High-Performance Liquid Chromatographic Analysis of Dipyrone Metabolites to Study Their Formation in Human Liver Microsomes

Pharmaceutical Research -     

Assessment of Human Exposure to Deoxynivalenol,Ochratoxin A,Zearalenone and Their Metabolites Biomarker in Urine Samples Using LC-ESI-qTOF

Human are exposed to a wide range of mycotoxins through dietary food intake, including processed food. Even most of the mycotoxin exposure assessment studies are based on analysis of foodstuffs, and evaluation of dietary intake through food consumption patterns and human biomonitoring methods are rising as a reliable alternative to approach the individual exposures, overcoming the limitations of the indirect dietary assessment. In this study, human urine samples were analyzed, seeking the presence of deoxynivalenol (DON), ochratoxin A (OTA), zearalenone (ZEA), and their metabolites. For this purpose, 40 urine samples from female and male adult residents in the city of Valencia (Spain) were evaluated by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-ESI-qTOF) after salting-out liquid–liquid extraction. Analytical data showed that 72.5% of analyzed samples were contaminated by at least one mycotoxin at variable levels. The most prevalent mycotoxins were de-epoxy DON (DOM-1) (53%), ZEA (40%), and α-zearalenol (αZOL) (43%), while OTA was only detected in one sample. The mean concentrations in positive samples were DON (9.07 ng/mL), DOM-1 (20.28 ng/mL), ZEA (6.70 ng/mL), ZEA-14 glucoside (ZEA-14-Glc) (12.43 ng/mL), αZOL (27.44 ng/mL), αZOL-14 glucoside (αZOL-14-Glc) (12.84 ng/mL), and OTA (11.73 ng/mL). Finally, probable daily intakes (PDIs) were calculated and compared with the established tolerable daily intakes (TDIs) to estimate the potential risk of exposure to the studied mycotoxins. The calculated PDI was below the TDI value established for DON in both female and male adults, reaching a percentage up to 30%; however, this percentage increased up to 92% considering total DON (DON + DOM-1). On the other hand, the PDI obtained for ZEA and its metabolites were higher than the TDI value fixed, but the low urine excretion rate (10%) considered should be highlighted. Finally, the PDI calculated in the detected positive sample for OTA exceeded the TDI value. The findings of the present study confirm the presence of the studied mycotoxins and their metabolites as some of the most prevalent in urine.     

人尿中奥美拉唑代谢物的提取与精制

目的对奥美拉唑在人体内代谢后尿中2种代谢物进行提取与精制后推测其结构并测定其含量。方法24名健康受试者单剂量口服奥美拉唑肠溶胶囊40mg后,收集服药后12h内尿液,用乙醚提取和浓缩,以高效液相色谱法进行分离,将相对纯品进行质谱扫描测定,推测其结构并计算含量。结果经分离得到的2种代谢物推测为吡啶5’—或3’—甲基氧化生成的羟基砜型代谢物、吡啶环上5’—甲基羟化硫醚型代谢物,含量分别为96.54%、97.26%。结论本方法分离得到的奥美拉唑尿中代谢物质纯度较高。     

Study on Metabolites of Aconitum Alkaloids in Human Urine

TheaconitebelongstoplantsofgenusAconituminfamilyofRanunculaceaandhavenotableclinicalfunctionsintreatingrheumaticarthritis ,heartfailure ,etc .However,accidentsofaconitine(fromaconite)poisoningfrequentlyoccurtomanypeopletakingtheseherbs .Theplantsofthisfamilycontainaconitine ,mesaconitine ,hypaconitineandotheraco nitumalkaloids .Theyarehighlytoxic ,andtheirtreatmentdoseapproachestothetoxicdoseorlethaldose .Duringtheperiodfrom 1989- 1995 ,morethan 30casesofpoisoningcausedbyaconitineinsometradit…     

电子阴道镜对宫颈疾病的诊断价值

目的探讨电子阴道镜对宫颈疾病的诊断价值。方法回顾性分析广州市海珠区妇幼保健院妇科门诊2010年1月至2010年12月行阴道镜检查的1722例患者资料对其结果进行总结。结果在1722例患者中,发现异常宫颈图像并行宫颈活检病理检查428例。病理活检诊断慢性宫颈炎172例,宫颈上皮内瘤样病变(CIN)Ⅰ～Ⅲ236例,宫颈浸润癌8例,宫颈湿疣12例。阴道镜诊断CIN的灵敏度、特异度、阳性预测值和阴性预测值分别是97.88%、95.31%、96.25%和97.34%;阴道镜诊断宫颈浸润癌的灵敏度、特异度、阳性预测值和阴性预测值分别是75%、100%、100%和99.53%。结论电子阴道镜检查对诊断宫颈疾病,尤其对诊断CIN和宫颈癌有重要价值,结合组织病理学检查可以提高诊断的准确率。     

Quantification of Three Lidocaine Metabolites and Their Conjugates

A method has been developed to quantify three lidocaine metabolites, N-ethylglycyl-2,6-xylidide (MEGX), glycyl-2,6-xylidide (GX), and 4-hydroxy-2,6-xylidine (4-OH-XY), and their conjugates in pooled human urine using enzymic hydrolysis. The commonly used enzymes, pure -glucuronidase, sulfatase, and a mixture of the two, were tested for their efficiencies in hydrolyzing the conjugates. Initially, it was found that 4-OH-XY was highly unstable after it was released from conjugates by -glucuronidase and the enzyme mixture. This problem was corrected by purging the sample with nitrogen prior to incubation. It has been determined that 4-OH-XY is present in human urine exclusively as its glucuronide. The percentage of MEGX in free and in conjugated forms (glucuronide, sulfate, and others) are 44.9 ± 6.8,16.6 ± 4.5, 6.6 ± 1.8, and 31.9 ± 4.4, respectively. GX was present mostly in the free form (90.6 ± 10.5%).     

Analysis of Diclofenac and Four of Its Metabolites in Human Urine by HPLC

An HPLC method for the determination of diclofenac (DCF) and four of its metabolites (3-hydroxydiclofenac, 4-hydroxydiclofenac, 5-hydroxydiclofenac, and 3-hydroxy-4-methoxydiclofenac) in human urine is described. Following base hydrolysis, the samples were neutralized and extracted. Evaporated extracts were reconstituted in mobile phase containing ascorbic acid, and chromatographed, using flow-rate programming, on a reversed-phase column. Absolute recovery (average), was at least 78% for diclofenac and ranged from 75 to 85% for the four metabolites. Standard curves showed linearity over the range of concentrations of 0.2 to 40 ug/mL, using 0.25 mL of urine. Specificity was demonstrated by examining chromatograms of extracts of blank urine from 8 volunteers and 24 study subjects. Good accuracy was observed for all compounds over the concentration range of 0.2 to 40 ug/mL using 0.25 mL of urine. Based on accuracy and precision criteria, the limit of quantitation for all 5 analytes was 0.4 ug/mL, using 0.25 mL of urine. Analysis of urine from subjects with normal and reduced renal function who received diclofenac orally demonstrated that total diclofenac and metabolites excreted in the urine represented approximately 31% and 4% of an oral dose of diclofenac, respectively.     

Human Biomonitoring of T-2 Toxin,T-2 Toxin-3-Glucoside and Their Metabolites in Urine through High-Resolution Mass Spectrometry

The metabolic profile of T-2 toxin (T-2) and its modified form T-2-3-glucoside (T-2-3-Glc) remain unexplored in human samples. Therefore, the present study aimed to investigate the presence of T-2, T-2-3-Glc and their respective major metabolites in human urine samples (n = 300) collected in South Italy through an ultra-high performance liquid chromatography (UHPLC) coupled to Q-Orbitrap-HRMS methodology. T-2 was quantified in 21% of samples at a mean concentration of 1.34 ng/mg Crea (range: 0.22–6.54 ng/mg Crea). Almost all the major T-2 metabolites previously characterized in vitro were tentatively found, remarking the occurrence of 3′-OH-T-2 (99.7%), T-2 triol (56%) and HT-2 (30%). Regarding T-2-3-Glc, a low prevalence of the parent mycotoxin (1%) and its metabolites were observed, with HT-2-3-Glc (17%) being the most prevalent compound, although hydroxylated products were also detected. Attending to the large number of testing positive for T-2 or its metabolites, this study found a frequent exposure in Italian population.     

血清CA125、CA199、CEA和AFP检测对肝癌和卵巢癌的诊断评估

目的探讨CA125、CA199、CEA和AFP四种肿瘤标志物在肿瘤性疾病诊断中的临床意义和应用价值。方法采用放射免疫分析方法（RIA）检测。结果①在肝脏良、恶性两组疾病中，血清CA125、CA199和AFP水平均高于正常对照组，其中肝癌组AFP又明显高于良性病变组（P〈0．01）；对于单检的诊断效率，AFP高于其他指标；另外，CA199、CEA和AFP联检在敏感性、特异性及准确性上均显著高于单检组；②在卵巢癌和良性病变组，血清CA125和CA199水平均高于正常对照组，其中卵巢癌组CA125又明显高于良性病变组（P〈0．01）；对于单检的诊断效率，CA125高于其他指标；另外，CA199、CEA和CA125联检在敏感性、特异性及准确性上均显著高于单检组。结论应用多项指标联检可以提高肝癌和卵巢癌的诊断效率。     

肿瘤标志物的应用及其筛选技术研究进展

肿瘤标志物是肿瘤细胞通过基因表达而合成、分泌,或是机体对肿瘤反应而异常产生或表达水平异常的一类物质．在肿瘤早期发现、诊断及预后中均起到重要作用。随着生物技术的发展,新型肿瘤标志物不断被发现,肿瘤标志物的筛选方法也发生了根本性的变革。对新型肿瘤标志物及其筛选技术进行汇总和介绍,旨在为肿瘤标志物的临床应用与深入研究提供参考。     

A Preliminary Nuclear Magnetic Resonance Metabolomics Study Identifies Metabolites that Could Serve as Diagnostic Markers of Major Depressive Disorder

Background: The evaluation of metabolites that are directly involved in the physiological process, few steps short of phenotypical manifestation, remains vital for unravelling the biological moieties involved in the development of the (MDD) and in predicting its treatment outcome.Methodology: Eight (8) urine and serum samples each obtained from consenting healthy controls (HC), twenty-five (25) urine and serum samples each from first episode treatment naïve MDD (TNMDD) patients, and twenty (22) urine and serum samples each s from treatment naïve MDD patients 2 weeks after SSRI treatment (TWMDD) were analysed for metabolites using proton nuclear magnetic resonance (1HNMR) spectroscopy. The evaluation of patients’ samples was carried out using Partial Least Squares Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Square-Discriminant Analysis (OPLSDA) models.Results: In the serum, decreased levels of lactate, glucose, glutamine, creatinine, acetate, valine, alanine, and fatty acid and an increased level of acetone and choline in TNMDD or TWMDD irrespective of whether an OPLSDA or PLSDA evaluation was used were identified. A test for statistical validations of these models was successful.Conclusion: Only some changes in serum metabolite levels between HC and TNMDD identified in this study have potential values in the diagnosis of MDD. These changes included decreased levels of lactate, glutamine, creatinine, valine, alanine, and fatty acid, as well as an increased level of acetone and choline in TNMDD. The diagnostic value of these changes in metabolites was maintained in samples from TWMDD patients, thus reaffirming the diagnostic nature of these metabolites for MDD.     

Unique/Major Human Metabolites: Why,How, and When to Test for Safety in Animals

A Draft Guidance for Industry on “Safety Testing of Drug Metabolites” was released by FDA in 2005. According to these recommendations, there may be instances when the safety profile of human metabolites may mandate their direct safety testing in animals prior to registration and approval of new molecular entity. In response to this evolving regulatory environment, pragmatic and scientifically driven approaches should be used to assess which (if any) metabolites may require direct safety testing in animals. A specific Lilly case study highlights a strategic approach for evaluation of unique and major human metabolites of a drug in Phase 2 development.     

深海来源真菌Aspergillus sp .SCSIOW3抗A-beta多肽聚集活性成分的研究

摘要：目的 对一株南海深海来源真菌（Aspergillus sp .SCSIOW3）的抗Aβ聚集活性成分进行了分离、鉴定。方法 利用ThT荧光模型活性追踪分离 Aspergillus sp .SCSIOW3发酵产物中抗Aβ42多肽聚集活性物质,结合理化性质、波谱数据并参阅文献确定活性化合物的结构。结果 从Aspergillus sp .SCSIOW3中分离鉴定了2个具有抗Aβ42多肽聚集活性的桔霉素类衍生物：phenol A acid（1）和Penicitrinone A（2）,其中化合物2在100 uM表现出与阳性对照EGCG同等程度的活性。结论 phenol A acid（1）和Penicitrinone A（2）为橘霉素衍生物,这是首次报导该类化合物的抗Aβ多肽聚集活性,具有潜在的理论和实际应用研究价值。     

肠道菌群代谢产物与宿主疾病

研究发现人体中的肠道菌群在各种疾病的发生和发展中扮演着重要的角色,被认为是人体的“第9大系统”“第2基因组”。肠道菌群及其代谢产物直接影响着人体的健康。本文综述肠道菌群代谢产物与糖尿病、高血脂、免疫系统、结肠癌及心血管疾病的相关性及其潜在的作用机制。     

Isolation and Characterization of Major Phase I and II Metabolites of Ibuprofen

Pharmaceutical Research -     

Urinary Elimination of Nitrazepam and its Main Metabolites

Abstract Nitrazepam and its main metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, free and conjugated, were determined in the human urine after a single oral dose of 5 mg. The determinations were performed by GLC method using ⁶³Ni-EC-detector for unchanged nitrazepam and nitrogen selective detector for the metabolites. Unchanged nitrazepam was poorly eliminated through the kidneys (about 1 per cent of the dose). The interindividual variation of total excreted urinary metabolites was large ranging between 848-4933 pg (17-99 per cent of the dose during 7 days). Of this amount conjugated metabolites made up 57 per cent. The urinary half-lives of free and conjugated 7-aminonitrazepam were (mean and ranges) 44 (23-65) and 46 (25-69) hrs, and of 7-acetamidonitrazepam 12 (5-31) and 18 (5-46) hrs, respectively. The half-lives of the excreted amounts of the metabolites did not correlate with any pharmacokinetic parameter of unchanged nitrazepam in serum.     

The Pharmacokinetics of Antipyrine and Three of Its Metabolites in the Rabbit: Intravenous Administration of Pure Metabolites

Antipyrine (AP) is a commonly used probe of oxidative metabolism. Indirect evidence demonstrates formation rate limited disposition of its metabolites. Kinetic studies using antipyrine and its major metabolites 3-hydroxymethylantipyrine (HMA), norantipyrine (NORA), and 4-hydroxyantipyrine (OHA) were completed to investigate the metabolic fate of preformed antipyrine metabolite and to demonstrate directly formation rate-limited metabolite disposition in vivo. Bolus injections of antipyrine and preformed metabolites (40-50 mg/kg) were administered to male, New Zealand white rabbits. Plasma and urine were analyzed using HPLC. These studies demonstrate that HMA, NORA, and OHA are formation rate limited in the rabbit. NORA appears to undergo further extensive oxidative and conjugative metabolism. Unknown additional peaks were detected in urine after NORA dosing but not after HMA or OHA administration. Mass spectroscopy of the unknown HPLC eluents identified potential structures of these NORA metabolites.     

多胺在恶性肿瘤诊断及疗效观察中的应用

近年来研究表明,多胺在癌症患者体内合成与积累增多,可作为一种肿瘤标志物,但其临床应用价值还有待商榷。本文分析目前已发表的临床对照试验,包括乳腺癌、宫颈癌、白血病患者与健康受试者或良性疾病患者体液中的多胺成分或比例变化的情况,对多胺的临床应用进展做一综述。     

外泌体作为潜在肿瘤标志物的定量检测方法研究进展

外泌体是一种直径大小为40～150 nm 的细胞外囊泡,分布于几乎所有体液中。作为肿瘤液体活检的新靶标,癌细胞来源的外泌体包含多种肿瘤生物信息,对肿瘤早期诊断和预后评价有重要的应用价值。对外泌体蛋白的定量检测方法包括荧光法、比色法、电化学法、下一代测序、表面增强拉曼光谱法、表面等离子体共振法的研究进展进行综述。     

HPLC法测定生物样品中呋苄西林和其代谢物的浓度

目的 :测定血清、尿样和胆汁中呋苄西林和其代谢物的含量。方法 :高效液相色谱法 ,AlltimaC18(4 6mm× 15 0mm ,5 μm) ;流动相为 pH 3 0的 0 0 6mol·L-1磷酸二氢钠 (磷酸调 pH 3 0 ,含磷酸四丁基铵离子对试剂 ) -甲醇 -乙腈(5 8∶2 5∶17) ;检测波长 :2 70nm。结果 :血清样品经过甲醇沉淀离心 ,尿样和胆汁样品经过离心、稀释、滤过后即可在HPLC色谱柱上获得良好的分离并同时测定其浓度。对呋苄西林和其代谢物 ,其浓度分别在 0 5～ 12 8μg·mL-1的范围内 ,与峰面积呈线性关系 (r>0 999)。平均回收率在 96 %以上 ,最低检测浓度为 0 0 5或 0 1μg·mL-1,日内、日间精密度RSD小于 7%。结论 :方法简便、快速、准确 ,可以作为呋苄西林和其代谢物在人和动物体内的测定方法。