Determination of raloxifene and its glucuronides in human urine by liquid chromatography-tandem mass spectrometry assay

A selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-β-glucuronide (M1), raloxifene-4'-β-glucuronide (M2), raloxifene-6,4'-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC-MS/MS. The method was linear in a wide range with high determination coefficient (r(2) > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.     

Development and validation of a liquid chromatography-tandem mass spectrometry assay for determination of raloxifene and its metabolites in human plasma

This paper describes the development and validation of a method for the detection of raloxifene (Ral) and its two glucuronide metabolites, raloxifene-6-glucuronide (M1) and raloxifene-4'-glucuronide (M2), in human plasma samples. Both glucuronides were synthesized enzymatically, purified and used as authentic standards. The assay involves a simple solid phase extraction (SPE) procedure of 0.5 mL of human plasma and subsequent analysis by LC-MS-MS. The recoveries were higher than 71% and chromatographic separation of all the analytes was accomplished in less than 7 min. Linear ranges (r(2)>0.99) were found from 0.200 to 340 microg/L, from 1.600 to 2720 microg/L and from 0.088 to 60.00 microg/L, for M1, M2 and Ral, respectively. The limits of detection achieved were 8, 11 and 6 ng/L for M1, M2 and Ral, respectively. The method presented was successfully applied to a genetic polymorphism study of 47 plasma samples from women taking Evista (raloxifene hydrochloride).     

Synthesis and Stereochemical Characterization of Hydroxy- and Epoxy-derivatives of Rotenone

One to four routes of synthesis are described for 8′-hydroxyrotenone, 5′-hydroxyrotenone, two epimers of 6′,7′-dihydro-6′,7′-dihydroxyrotenone, two epimers of 6′,7′-epoxyrotenone and the four rotenolones derived from each of these compounds. The stereochemical relationships are determined, in each case, by chemical interconversion, ORD and monochromatic rotation to assess the absolute configuration of the B/C ring juncture and by IR, MS and NMR for the cis- or trans-nature of this juncture. The new compounds described are useful standards for studies on the metabolites and photodecomposition products of rotenone insecticide chemical.     

Flavonoid glycosides and the chemosystematics of Eucalyptus camaldulensisis

Leaf flavonoid glycosides of Eucalyptus camaldulensis were identified as kaempferol 3-glucoside and 3-glucuronide; quercetin 3-glucoside, 3-glucuronide, 3-rhamnoside, 3-rutinoside and 7-glucoside, apigenin 7-glucuronide and luteolin 7-glucoside and 7-glucuronide. Two chemical races were observed based on the flavonoid glycosides. These races correspond to the northern and southern populations of species growing in Australia. The Middle Eastern species examined were found to belong to the southern Australian chemical race. The major glycosides of E. occidentalis proved to be quercetin and myricetin 3-glucuronide.     

Towards a high-yield bioconversion of ferulic acid to vanillin

Natural vanillin is of high interest in the flavor market. Microbial routes to vanillin have so far not been economical as the medium concentrations achieved have been well below 1 g l⁻¹. We have now screened microbial isolates from nature and known strains for their ability to convert eugenol or ferulic acid into vanillin. Ferulic acid, in contrast to the rather toxic eugenol, was found to be an excellent precursor for the conversion to vanillin, as doses of several g l⁻¹ could be fed. One of the isolated microbes, later identified as Pseudomonas putida, very efficiently converted ferulic acid to vanillic acid. As vanillin was oxidized faster than ferulic acid, accumulation of vanillin as an intermediate was not observed. A completely different metabolic flux was observed with Streptomyces setonii. During the metabolism of ferulic acid, this strain accumulated vanillic acid only to a level of around 200 mg l⁻¹ and then started to accumulate vanillin as the principal metabolic overflow product. In shake-flask experiments, vanillin concentrations of up to 6.4 g l⁻¹ were achieved with a molar yield of 68%. This high level now forms the basis for an economical microbial production of vanillin that can be used for flavoring purposes. Received: 15 October 1998 / Received revision: 13 January 1999 / Accepted: 18 January 1999     

Sudan-β-d-glucuronides and their use for the histochemical localization of β-glucuronidase activity in transgenic plants

 Synthesis of five different Sudan-β-d-glucuronides (I, II, III, IV, and RedB) was performed by condensation of a set of red Sudan diazo dyes with methyl (1-deoxy-2,3,4-tri-O-acetyl-1-trichloroacetimidoyl-α-d-glucopyran)uronate. After the acid and alcohol groups had been deprotected, the resulting compounds were used for histochemical localization of β-glucuronidase (GUS) activity in transgenic plants (Petunia hybrida, Arabidopsis thaliana, and Nicotiana tabacum) that contained the GUS reporter system. Because the cleavage of the β-glucuronide results in the liberation of an insoluble Sudan dye, Sudan substrates gave no diffusion artifacts as described for the commonly used 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc). A comparison of assays with different Sudan glucuronides and X-gluc demonstrated that the SudanIV variant is a valuable glucuronide substrate for the precise histochemical localization of GUS activity in transgenic plants. Received: 9 December 1999 / Revision received: 25 January 2000 / Accepted: 26 January 2000     

Isolation and characterisation of a ropy Lactobacillus strain producing the exopolysaccharide kefiran

A capsular-polysaccharide-producing strain, LM-17, was isolated from kefir grains and was identified as a slime-forming, rod-shaped Lactobacillus. According to ¹H- and ¹³C-NMR spectral data, the exopolysaccharide produced by the isolated bacterial strain is identical to the glucogalactan extracted from kefir grains and therefore known as kefiran. The kefiran produced was characterised by means of viscosity, optical rotatory power, circular dichroism and IR spectral measurements. A batch procedure was set up for the culture and extraction of the exopolysaccharide in laboratory conditions, resulting in a yield of 2 g/l purified kefiran from the culture supernatant of the LM-17 strain. Received: 6 April 1999 / Received revision: 30 July 1999 / Accepted: 13 August 1999     

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high‐efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two‐stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion‐body stability is promoted by the addition of 15 mM 2‐hydroxyethyldisulfide (2‐HEDS), also known as oxidized β‐mercaptoethanol, to the permeabilization buffer (6 M urea + 3 mM ethylenediaminetetraacetate [EDTA]). 2‐HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion‐body fraction by centrifugation. In the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two‐stage process yielded an 81% (w/w) recovery of the recombinant protein Long‐R³‐IGF‐I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot‐scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 455–460, 1999.     

Properties of Quercetin Conjugates: Modulation of LDL Oxidation and Binding to Human Serum Albumin

Quercetin is an important dietary flavonoid with in vitro antioxidant activity. However, it is found in human plasma as conjugates with glucuronic acid, sulfate or methyl groups, with no significant amounts of free quercetin present. The antioxidant properties of the conjugates found in vivo and their binding to serum albumin are unknown, but essential for understanding possible actions of quercetin in vivo. We, therefore, tested the most abundant human plasma quercetin conjugates, quercetin-3-glucuronide, quercetin-3′-sulfate and isorhamnetin-3-glucuronide, for their ability to inhibit Cu(II)-induced oxidation of human low density lipoprotein and to bind to human albumin, in comparison to free flavonoids and other quercetin conjugates. LDL oxidation lag time was increased by up to four times by low (<2?μM) concentrations of quercetin-3-glucuronide, but was unaffected by equivalent concentrations of quercetin-3′-sulfate and isorhamnetin-3-glucuronide. In general, the compounds under study prolonged the lag time of copper-induced LDL oxidation in the order: quercetin-7-glucuronide>quercetin>quercetin-3-glucuronide=quercetin-3-glucoside>catechin>quercetin-4′-glucuronide>isorhamnetin-3-glucuronide>quercetin-3′-sulfate. Thus the proposed products of small intestine metabolism (quercetin-7-glucuronide, quercetin-3-glucuronide) are more efficient antioxidants than subsequent liver metabolites (isorhamnetin-3-glucuronide, quercetin-3′-sulfate). Albumin-bound conjugates retained their property of protecting LDL from oxidation, although the order of efficacy was altered (quercetin-3′-sulfate>quercetin-7-glucuronide>quercetin-3-glucuronide>quercetin-4′-glucuronide=isorahmnetin-3-glucuronide). K_q values (concentration required to achieve 50% quenching) for albumin binding, as assessed by fluorescence quenching of Trp214, were as follows: quercetin-3′-sulfate (～4?μM)=quercetin≥quercetin-7-glucuronide>quercetin-3-glucuronide=quercetin-3-glucoside>isorhamnetin-3-glucuronide>quercetin-4′-glucuronide (～20?μM). The data show that flavonoid intestinal and hepatic metabolism have profound effects on ability to inhibit LDL oxidation and a lesser but significant effect on binding to serum albumin.     

Grafted megaporous materials as ion‐exchangers for bioproduct adsorption

Megaporous chromatographic materials were manufactured by a three‐step procedure, including backbone synthesis, chemical grafting, and introduction of ion‐exchange functionality. The backbone of the adsorbent cylindrical bodies was prepared by polymerization of methacrylic acid and poly(ethylene glycol) diacrylate at sub‐zero temperatures. Grafting was performed employing glycidyl methacrylate and a chemical initiator, cerium ammonium nitrate. The degree of grafting was adjusted by modifying the concentration of the initiator in the reaction mixture to a range of values (23, 39, 62, 89, and 105%). Further, the pendant epoxy‐groups generated by the previous step were reacted to cation‐ and anion‐exchanging moieties utilizing known chemical routes. Infrared spectroscopy studies confirmed the incorporation of epoxy and ion‐exchanger groups to the backbone material. Optimized materials were tested for chromatography applications with model proteins; the dynamic binding capacity, as recorded at 10% breakthrough and 2.0 × 10^?4 m/s superficial velocity, were 350 and 58 mg/g for the cation‐exchanger and the anion‐exchanger material, respectively. These results may indicate that long tentacle‐type polymer brushes were formed during grafting therefore increasing the ability of the megaporous body to efficiently capture macromolecules. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 386–393, 2013     

High-performance liquid chromatographic-electrospray mass spectrometric determination of morphine and its 3- and 6-glucuronides: application to pharmacokinetic studies

A rapid and selective assay of morphine and its 3- and 6-glucuronides in serum, based on high-performance liquid chromatography-electrospray mass spectrometry has been developed. The analytes and the internal standard, codeine or naltrexone, were subjected to solid-phase extraction, using ethyl solid-phase extraction columns, prior to chromatography. A reversed-phase column and a gradient mobile phase consisting of water and methanol were used. The mass spectrometer was operated in the selected-ion monitoring mode. The following ions were used: m/z 286 for morphine, m/z 300 for codeine, m/z 342 for naltrexone, and m/z 462 for morphine 3- and 6-glucuronides. The limit of quantitation observed with this method was 10 ng/ml morphine, 50 ng/ml morphine-6-glucuronide and 100 ng/ml morphine-3-glucuronide. The present method proved useful for the determination of serum levels of the parent drug and its metabolites in pain patients, heroin addicts and in morphine-treated mice.     

Metabolism of the ethanolamine-type antihistamine diphenhydramine (Benadryl)TM by the fungus Cunninghamella elegans

Two strains of the filamentous fungus Cunninghamella elegans (ATCC 9245 and ATCC 36112) were grown in Sabouraud dextrose broth and screened for the ability to metabolize the ethanolamine-type antihistamine diphenhydramine. Based on the amount of parent drug recovered after 7 days incubation, both C. elegans strains metabolized approximately 74% of the diphenhydramine, 58% of this being identified as organic extractable metabolites. The organic extractable metabolites were isolated by reversed-phase high-performance liquid chromatography and identified by analyzing their mass and nuclear magnetic resonance spectra. Desorption chemical ionization mass spectrometry (DCIMS) with deuterated ammonia was used to differentiate possible isobaric diphenhydramine metabolites and to probe the mechanisms of ion formation under ammonia DCIMS conditions. C. elegans transformed diphenhydramine by demethylation, oxidation, and N-acetylation. The major metabolites observed were diphenhydramine-N-oxide (3%), N-desmethyldiphenhydramine (30%), N-acetyldidesmethyldiphenhydramine (13%), and N-acetyl-N-desmethyldiphenhydramine (12%). These compounds are known mammalian metabolites of diphenhydramine and may be useful for further toxicological studies. Received: 24 June 1999 / Revision received: 23 August 1999 / Accepted: 24 September 1999     

CPMG sequences with enhanced sensitivity to chemical exchange

Improved relaxation-compensated Carr–Purcell–Meiboom-Gill pulse sequences are reported for studying chemical exchange of backbone ¹⁵N nuclei. In contrast to the original methods [J. P. Loria, M. Rance, and A. G. Palmer, J. Am. Chem. Soc. 121, 2331–2332 (1999)], phenomenological relaxation rate constants obtained using the new sequences do not contain contributions from ¹H-¹H dipole-dipole interactions. Consequently, detection and quantification of chemical exchange processes are facilitated because the relaxation rate constant in the limit of fast pulsing can be obtained independently from conventional ¹⁵N spin relaxation measurements. The advantages of the experiments are demonstrated using basic pancreatic trypsin inhibitor.     

Allelopathic activity of luteolin 7-O-β-glucuronide isolated from Chrysanthemum morifolium L.

Two flavones, luteolin 7-O-β-glucuronide and diosmetin 7-O-β-glucuronide, were isolated and identified from Chrysanthemum morifolium L. v. Ramat leaves. Identification techniques included HPLC DAD, MS, ¹H and ¹³C NMR spectroscopy. At concentrations of 0.2 and 2.0 mM, luteolin 7-O-β-glucuronide significantly reduced the frond number and chlorophyll content of Lemna gibba plants, but did not significantly affect dry weight. At a concentration of 0.2 mM diosmetin 7-O-β-glucuronide had no significant effect on frond number, dry weight or chlorophyll concentration of L. gibba. These results indicate that an ortho-3′,4′-dihydroxy arrangement of the B-flavonoid ring in the luteolin compound is probably responsible for allelopathic activity.     

Enzyme-assisted synthesis and structure characterization of glucuronic acid conjugates of losartan, candesartan, and zolarsartan

Three angiotensin II receptor antagonists—losartan, candesartan, and zolarsartan—were investigated. All the compounds, which are structural analogues, are metabolized via conjugation to glucuronic acid. Interestingly, both O- and N-glucuronidation take place, so that regioisomers are formed. One ether O-glucuronide, two acyl O-glucuronides, and five tetrazole-N-glucuronides were biosynthesized, in milligram scale, from the three sartan aglycones. Liver microsomes from bovine, moose, rat, and pig and recombinant human UDP-glucuronosyltransferases were used as catalysts. The synthesized compounds were identified as sartan glucuronides by mass spectrometry, while the sites of glucuronidation were determined by nuclear magnetic resonance spectroscopy. Drug metabolites are needed as standards for pharmaceutical research and, as the present study shows, they can easily be produced with enzymes as catalyst.     

牛种布鲁氏菌2308不同途径小鼠感染模型的建立与评估

[背景] 布鲁氏菌可经口、皮肤、黏膜和呼吸道感染人和动物。小鼠是布鲁氏菌研究中最常用的模型动物。[目的] 建立牛种布鲁氏菌2308不同途径和剂量感染BALB/c小鼠的模型,为布鲁氏菌小鼠感染试验提供参考。[方法] 用10¹-10⁵ CFU这5个不同感染剂量,分别经注射、口服和点眼方式感染BALB/c小鼠。在感染后不同时间点采集小鼠血清,检测IgG、IgM、IgA抗体含量、脾脏重量及脾脏含菌量,评价布鲁氏菌经不同途径感染BALB/c小鼠的效果。[结果] 10 CFU是注射感染BALB/c小鼠的最小感染剂量;10⁵ CFU是口服感染BALB/c小鼠的最小感染剂量。10¹-10⁵ CFU这5个不同感染剂量经点眼途径均未能成功感染BALB/c小鼠。在10⁵ CFU感染剂量下,口服与注射感染组小鼠每克脾脏平均含菌量分别为10^5.673 CFU/g和10^5.009 CFU/g,无显著差异（P>0.05）,但口服感染组小鼠脾脏平均重量为0.310 g,显著高于注射感染组0.165 g （P<0.01）。在试验期内,注射感染组和口服感染组小鼠体内IgG抗体的滴度均随感染时间延长而持续升高;整体上,口服感染组IgG抗体峰值显著高于注射感染组;2组IgM抗体变化趋势一致;口服感染组有2只小鼠在感染28 d后产生IgA抗体,注射感染组均未检测到IgA抗体。[结论] 建立了牛种布鲁氏菌2308通过不同途径感染BALB/c小鼠的模型。     

Phytochemical variability during vegetation of Chamerion angustifolium (L.) Holub genotypes derived from in vitro cultures

The purpose of the study was to evaluate Chamerion angustifolium (L.) Holub genotypes for preliminary selection and further breeding programs aimed at obtaining a suitable industrial form for the pharmaceutical applications. Clonally propagated plants representing 10 genotypes of Ch. angustifolium were regenerated under in vitro conditions, hardened and planted in the field. Studies included an evaluation of shoot proliferation, phytochemical assessment of in vitro and ex vitro plants as well as investigations of intraspecies variability regarding four phenological stages: vegetative, beginning of blooming, full blooming, and green fruit phases. Quantitative and qualitative analyses of bioactive compounds were performed using high-performance liquid chromatography coupled with diode array detector and tandem mass spectrometer (HPLC–DAD–MS/MS) and high-performance liquid chromatography (HPLC) methods. The efficiency of shoot multiplication varied between genotypes from 8.12 to 21.48 shoots per explant. A high reproduction rate (>?20 shoots per explant) was recorded for four lines (PL_45, PL_44, PL_58, DE_2). Plants grown in vitro synthesized oenothein B (11.2–22.3 mg g^?1 DW) and caffeic acid derivatives. Plants harvested from field contained the full spectrum of polyphenols characteristic for this species, and oenothein B and quercetin 3-O-glucuronide were the most abundant. The maximal content of oenothein B was determined in the vegetative phase of fireweed, while some flavonoids were found in the highest amount in full blooming phase. The results of analysis of variance indicated significant differences among genotypes in oenothein B, 3-O-caffeoylquinic acid and flavonoids accumulation in four phenological phases. PL_44 plants were characterized by high content of oenothein B and quercetin 3-O-glucuronide as well as a relatively high level of other flavonoids. Based on our phytochemical and micropropagation studies, PL_44 genotype was the best candidate for early selection and further breeding programs.

     

Novel naphthoquinonyl derivatives: Potential structural components for the synthesis of cytotoxic peptides

Summary Syntheses and physicochemical properties are described of several novel naphthoquinonyl amino acid derivatives, which are potential components in cytotoxic peptide conjugates. These compounds include N - and N -naphthoquinonyl derivatives of lysine as well as N-naphthoquinonyl-carboxylic derivatives. The former class of compounds can be employed as building blocks in a stepwise peptide synthesis, whereas the latter two are adequate for postpeptide chain-assembly modifications. The ability of the naphthoquinonyl derivatives to produce semiquinone radicals and hydroxyl radicals (OH), using chemical (i.e. NaBH₄) and enzymatic (i.e. NADPH-cytochrome P-450 reductase) routes, respectively, was evaluated employing electron spin resonance spectroscopy.     

Species differences in the metabolism of sulphadimethoxine

1. The fate of sulphadimethoxine (2,4-dimethoxy-6-sulphanilamidopyrimidine) was studied in man, rhesus monkey, dog, rat, guinea pig and rabbit. 2. About 20–46% of the dose (0·1g./kg.) of the drug is excreted in the urine in 24hr. in these species, except the rat, in which only 13% is excreted. 3. In man and the monkey sulphadimethoxine N¹-glucuronide is the major metabolite in the urine. In the rabbit and guinea pig N⁴-acetylsulphadimethoxine is the main metabolite. In the dog the drug is excreted mainly unchanged. In the rat equal amounts of the unchanged drug and its N⁴-acetyl derivative are the main products. 4. Small amounts of sulphadimethoxine N⁴-glucuronide are found in the urine of all the species. Sulphadimethoxine N¹-glucuronide occurs in small amounts in the urine of rat, dog and guinea pig; none is found in rabbit urine. 5. Sulphadimethoxine N⁴-sulphate was synthesized and found to occur in small amounts in rat urine. 6. Monkey liver homogenates fortified with UDP-glucuronic acid are able to synthesize sulphadimethoxine N¹-glucuronide with the drug as substrate. Rat liver has also this ability to a slight extent, but rabbit liver is unable to do so. 7. Sulphadimethoxine N⁴-glucuronide is formed spontaneously when the drug is added to human urine. 8. The biliary excretion of the drug and its metabolites was examined in rats. The drug is excreted in rat bile mainly as the N¹-glucuronide. The N¹- and N⁴-glucuronides administered as such are extensively excreted in the bile by rats.