Intact Side Chain Research Articles

A Commercial Extract of Cyanotis arachnoidea Roots as a Source of Unusual Ecdysteroid Derivatives with Insect Hormone Receptor Binding Activity.

Ecdysteroids act as molting hormones in insects and as nonhormonal anabolic agents and adaptogens in mammals. A wide range of ecdysteroid-containing herbal extracts are available worldwide as food supplements. The aim of this work was to study such an extract as a possible industrial source of new bioactive ecdysteroids. A large-scale chromatographic isolation was performed from an extract of Cyanotis arachnoidea roots. Ten ecdysteroids (1–10) including eight new compounds were isolated and characterized by extensive nuclear magnetic resonance studies. Highly unusual structures were identified, including a H-14β (1, 2, 4, and 10) moiety, among which a 14β(H)17β(H) phytosteroid (1) is reported for the first time. Compounds with an intact side chain (4–10) and 11 other natural or semisynthetic ecdysteroids (11–21) were tested for insect ecdysteroid receptor (EcR) binding activity. Two new compounds, i.e., 14-deoxydacryhainansterone (5) and 22-oxodacryhainansterone (6), showed strong EcR binding activity (IC50 = 41.7 and 380 nM, respectively). Six compounds were identified as EcR agonists and another two as antagonists using a transgenic ecdysteroid reporter gene assay. The present results demonstrate that commercial C. arachnoidea extracts are rich in new, unusual bioactive ecdysteroids. Because of the lack of an authentic plant material, the truly biosynthetic or artifactual nature of these compounds cannot be confirmed.

Phase I metabolism of synthetic cannabinoid receptor agonist PX-1 (5F-APP-PICA) via incubation with human liver microsomes and UHPLC-HRMS.

Studies of the metabolic and pharmacological profiles of indole carboxamide synthetic cannabinoids (a prevalent class of new psychoactive substances) are critical in ensuring that their use can be detected through bioanalytical testing. We have determined the in vitro Phase I metabolism of one such compound, PX-1 (5F-APP-PICA), and appropriate markers to demonstrate human consumption. PX-1 was incubated with human liver microsomes, followed by analysis of the extracts via high-resolution mass spectrometry. A total of 10 metabolites were identified, with simultaneous defluorination and monohydroxylation of the pentyl side chain as the primary biotransformation product (M1). Additional metabolites formed were hydroxylation products of the indole and benzyl moieties, distal amide hydrolysis, N-desfluoropentyl, and carboxypentyl metabolites. Three monohydroxylated metabolites specific to PX-1 were identified and are reported for the first time in this study. The primary metabolite, M1, was further oxidized to M5, a carboxypentyl metabolite. M8 is PX-1 specific, possessing an intact fluoropentyl side chain. These three metabolites are the most suitable for implementation into bioanalytical assays for demonstrating PX-1 consumption. The findings of this study can be used by analytical scientists and medical professionals to determine PX-1 ingestion and predict the metabolites of synthetic cannabinoids sharing structural elements.

Cyano Metabolite as a Biomarker of Nitrofurazone in Channel Catfish

The use of nitrofuran drugs in food-producing animals continues to attract international concern as a food safety issue. Methods for monitoring nitrofuran residues have been directed to the intact side chain of tissue-bound metabolites. Semicarbazide, the side chain of nitrofurazone (NFZ), can enter food products from non-NFZ sources, suggesting the need for an alternative biomarker for confirmatory purposes. We characterized a cyano derivative as a major metabolite of NFZ in channel catfish (Ictalurus punctatus). The depletion of cyano metabolite was examined in the muscle of channel catfish after oral dosing (10 mg of NFZ/kg of body weight). Parent NFZ was rapidly eliminated in muscle, with a half-life of 6.3 h. The cyano metabolite was detected for up to 2 weeks, with an elimination half-life of 81 h. The cyano metabolite represents an alternative biomarker for confirming the use of NFZ in channel catfish.

Identification of Novel Glycosyltransferases Required for Assembly of the Pasteurella multocida A:1 Lipopolysaccharide and Their Involvement in Virulence

We previously determined the structure of the Pasteurella multocida Heddleston type 1 lipopolysaccharide (LPS) molecule and characterized some of the transferases essential for LPS biosynthesis. We also showed that P. multocida strains expressing truncated LPS display reduced virulence. Here, we have identified all of the remaining glycosyltransferases required for synthesis of the oligosaccharide extension of the P. multocida Heddleston type 1 LPS, including a novel alpha-1,6 glucosyltransferase, a beta-1,4 glucosyltransferase, a putative bifunctional galactosyltransferase, and two heptosyltransferases. In addition, we identified a novel oligosaccharide extension expressed only in a heptosyltransferase (hptE) mutant background. All of the analyzed mutants expressing LPS with a truncated main oligosaccharide extension displayed reduced virulence, but those expressing LPS with an intact heptose side chain were able to persist for long periods in muscle tissue. The hptC mutant, which expressed LPS with the shortest oligosaccharide extension and no heptose side chain, was unable to persist on the muscle or cause any disease. Furthermore, all of the mutants displayed increased sensitivity to the chicken antimicrobial peptide fowlicidin 1, with mutants expressing highly truncated LPS being the most sensitive.

Gas Release Characteristics of Various Species in Pyrolysis of Wood and Grass Biomass

This paper presents a new method for predicting the gas release characteristics of various species in pyrolysis of wood and grass biomass. The pyrolysis experiment of various biomass samples were carried out, and the gas release process in pyrolysis was measured. Large amount of CO2 and CO were released at first. CH4 was subsequently released. Additional CO was released at more than 700°C in the pyrolysis of lignin and lignin-rich biomass. Finally, H2 release was increased. This gas release Characteristics indicate that the pyrolysis mechanism of biomass was almost same with that of coal. Based on these experimental results, biomass-CPD model was modified to divide the reactions of intact bridge, side chain and aromatic cluster for predicting the gas release process of various species. In the first reaction, H2O, CO and CO2 were released from intact bridge and side chain. Next reaction was the release of CH4 from intact bridge and side chain. Further CO release from intact bridge and side chain occurred at high temperature only in lignin pyrolysis. Finally, H2 was released from intact bridge, side chain and aromatic cluster. Kinetic parameters of those reactions were determined from values of the conventional pyrolysis model both of coal and biomass. Consequently, the gas release process of various species was well predicted by using above mentioned new biomass-CPD model.

Three-dimensional structure of yellow fluorescent protein zYFP538 from Zoanthus sp. at the resolution 1.8 Å

The three-dimensional structure of yellow fluorescent proteins zYFP538 (zFP538) from the button polyp Zoanthus sp. was determined at a resolution of 1.8 angstrom by X-ray analysis. The monomer of zYFP538 adopts a structure characteristic of the green fluorescent protein (GFP) family, a beta-barrel formed from 11 antiparallel beta segments and one internal alpha helix with a chromophore embedded into it. Like the TurboGFP, the beta-barrel of zYFP538 contains a water-filled pore leading to the chromophore Tyr67 residue, which presumably provides access of molecular oxygen necessary for the maturation process. The post-translational modification of the chromophore-forming triad Lys66-Tyr67-Gly68 results in a tricyclic structure consisting of a five-membered imidazolinone ring, a phenol ring of the Tyr67 residue, and an additional six-membered tetrahydropyridine ring. The chromophore formation is completed by cleavage of the protein backbone at the Calpha-N bond of Lys66. It was suggested that the energy conflict between the buried positive charge of the intact Lys66 side chain in the hydrophobic pocket formed by the Ile44, Leu46, Phe65, Leu204 and Leu219 side chains is the most probable trigger that induces the transformation of the bicyclic green form to the tricyclic yellow form. A stereochemical analysis of the contacting surfaces at the intratetramer interfaces helped reveal a group of conserved key residues responsible for the oligomerization. Along with others, these residues should be taken into account in designing monomeric forms suitable for practical application as markers of proteins and cell organelles.

Serglycin Constitutively Secreted by Myeloma Plasma Cells Is a Potent Inhibitor of Bone Mineralization in Vitro

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n=2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.

Synthesis and Biological Evaluation of Apicularen A Analogues

AbstractReactions between amide anions and propanal derivatives containing benzolactone components at their 3‐positions produced hemiaminals, which were dehydrated via the corresponding acetates to provide the apicularen A analogues 21, 29, 34, 38, and 41. Of these, 21 and 29 contain the intact enamide side chain of apicularen A but have modifications in the macrolactone core. On the other hand, compounds 34, 38, and 41 are characterized by the natural core structure but are modified in the enamide part. Biological studies showed that 21 and 29 are quite active but that the other three analogues show only minor activities. The 11‐deoxy analogue 21 turned out to be the most active compound against a mdr cell line. It can be concluded that the macrolactone part of apicularen A will tolerate modifications to some degree, whereas the enamide part is rather sensitive towards structural modifications. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Site-directed Mutagenesis, Proteolytic Cleavage, and Activation of Human Proheparanase

Heparanase is an endo-beta-D-glucuronidase that degrades heparan sulfate in the extracellular matrix and cell surfaces. Human proheparanase is produced as a latent 65-kDa polypeptide undergoing processing at two potential proteolytic cleavage sites, located at Glu109-Ser110 (site 1) and Gln157-Lys158 (site 2). Cleavage of proheparanase yields 8- and 50-kDa subunits that heterodimerize to form the active enzyme. The fate of the linker segment (Ser110-Gln157) residing between the two subunits, the mode of processing, and the protease(s) engaged in proheparanase processing are currently unknown. We applied multiple site-directed mutagenesis and deletions to study the nature of the potential cleavage sites and amino acids essential for processing of proheparanase in transfected human choriocarcinoma cells devoid of endogenous heparanase but possessing the enzymatic machinery for proper processing and activation of the proenzyme. Although mutagenesis at site 1 and its flanking sequences failed to identify critical residues for proteolytic cleavage, processing at site 2 required a bulky hydrophobic amino acid at position 156 (i.e. P2 of the cleavage site). Substitution of Tyr156 by Ala or Glu, but not Val, resulted in cleavage at an upstream site in the linker segment, yielding an improperly processed inactive enzyme. Processing of the latent 65-kDa proheparanase in transfected Jar cells was inhibited by a cell-permeable inhibitor of cathepsin L. Moreover, recombinant 65-kDa proheparanase was processed and activated by cathepsin L in a cell-free system. Altogether, these results suggest that proheparanase processing at site 2 is brought about by cathepsin L-like proteases. The involvement of other members of the cathepsin family with specificity to bulky hydrophobic residues cannot be excluded. Our results and a three-dimensional model of the enzyme are expected to accelerate the design of inhibitory molecules capable of suppressing heparanase-mediated enhancement of tumor angiogenesis and metastasis.

Influence of parameters on pyrolysis-GC/MS of lignin in the presence of tetramethylammonium hydroxide

Thermally assisted hydrolysis and methylation (THM) with tetramethylammonium hydroxide (TMAH) was performed on spruce milled wood lignin (MWL) in order to investigate THM parameters. The objective was the maximization of information on lignin structure. The main emphasis was laid on the formation of lignin monomer derivatives with intact propane side chain, as these fragments are thought to be indicators of favourable THM conditions. In the tested temperature range (310–710 °C) the lowest temperature of 310 °C was found to produce the highest yield of these monomers. The elution of unmethylated lignin monomers, such as phenols and methoxyphenols, was also minimized at 310 °C. In addition, a number of different TMAH to sample ratios were applied. The yield of the most lignin monomer derivatives was increased by using the highest tested TMAH concentration, whereas some unmethylated products, such as phenol, were unaffected. Methanol as solvent for TMAH was compared to an aqueous solution of TMAH of same concentration in respect of the influence on degradation product yields. The elution of 3,4-dimethoxybenzoic acid methyl ester increased and the yield of 3,4-dimethoxybenzaldehyde decreased when methanol was used. At last a prolonged incubation time of the TMAH–sample mixture was shown to be useful to enhance especially the yield of lignin monomer derivatives with intact propane side chain in case of application of a low TMAH to sample ratio.

Use of the excluded protecting group (EPG) method for peptide synthesis

The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin-1, [Ala(19), Asn(22)]magainin-1. In the approach reported, the C-terminal amino acid is esterified to the 2-position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl-steroid as the Fmoc-pentafluorophenyl-ester. The Fmoc group is removed with Et(2)NH/DMF ( approximately 15% v/v) and, after evaporation to approximately 10 mL, the solution chromatographed on Sephadex LH-20 in DMF. The dipeptidyl-steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc-amino acid-pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide. On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn degrees /AcOH to yield the peptide with intact alpha-amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C-terminal amino acids in other EPG syntheses.

Identification of intermediates in the bile acid synthetic pathway as ligands for the farnesoid X receptor

Bile acid synthesis from cholesterol is tightly regulated via a feedback mechanism mediated by the farnesoid X receptor (FXR), a nuclear receptor activated by bile acids. Synthesis via the classic pathway is initiated by a series of cholesterol ring modifications and followed by the side chain cleavage. Several intermediates accumulate or are excreted as end products of the pathway in diseases involving defective bile acid biosynthesis. In this study, we investigated the ability of these intermediates to activate human FXR. In a cell-based reporter assay and coactivator recruitment assays in vitro, early intermediates possessing an intact cholesterol side chain were inactive, whereas 26- or 25-hydroxylated bile alcohols and C27 bile acids were highly efficacious ligands for FXR at a level comparable to that of the most potent physiological ligand, chenodeoxycholic acid. Treatment of HepG2 cells with these precursors repressed the rate-limiting cholesterol 7alpha-hydroxylase mRNA level and induced the small heterodimer partner and the bile salt export pump mRNA, indicating the ability to regulate bile acid synthesis and excretion. Because 26-hydroxylated bile alcohols and C27 bile acids are known to be evolutionary precursors of bile acids in mammals, our findings suggest that human FXR may have retained affinity to these precursors during evolution.

An aldol approach to the synthesis of the EF fragment of spongistatin 1.

A synthesis of the C29-C51 fragment of spongistatin 1, containing the E and F rings, has been completed. The approach relies on four diastereoselective aldol additions and an asymmetric glycolate alkylation to establish eight of the eleven stereogenic centers. The intact chlorodiene side chain was appended by a Lewis acid catalyzed addition of an allylstannane to an epoxy enol ether.

Gas-phase fragmentation of di- and tripeptides via ion–molecule reactions with ClPCl +

The reactivity of the phosphenium ion ClPCl + toward gaseous neutral di- and tripeptides was examined by Fourier transform ion cyclotron resonance mass spectrometry. ClPCl + was found to react with the peptides at collision rate. Extensive fragmentation of the peptides leads to only one (dipeptides) or two (tripeptides) dominant product ions. For each dipeptide, the major product ion has a molecular weight that is 34 u less than that of the dipeptide, i.e. the side chains are retained in the fragment ion. A substantial amount of effort was given to resolve the structure of this product ion, with a focus on the ion ( m/z 98) generated from reaction of Gly–Gly (MW 132) with ClPCl +. The molecular formula of the ion of m/z 98 was determined to be C 4H 6NO by exact mass measurements. The relative energies of a variety of isomeric ions with this elemental composition were examined by molecular orbital calculations in order to identify the most likely structures. Collision-activated dissociation experiments, mechanistic considerations, and comparison of calculated adiabatic recombination energies of isomeric ions to that determined experimentally for the unknown ion, led to the assignment of a six-membered, highly unsaturated ring structure to the product ion of m/z 98 of Gly–Gly. This assignment is supported by the finding that the experimentally determined acidity of the unknown ion is in excellent agreement with that calculated for the proposed six-membered structure. The most likely pathway for the formation of this product ion, as well as those obtained for the other dipeptides, involves an electrophilic attack of ClPCl + at the amide carbonyl oxygen of the dipeptide, followed by a hydride shift, proton transfer, and loss of H 2O and Cl 2PO ·. The very high exothermicity of the initial addition (69 kcal mol −1, AM1) allows the adduct to form high-energy intermediates (highest estimated to lie at 34 kcal mol −1 above the adduct) during the reaction that is exothermic by about 36 kcal mol −1 overall. An analogous mechanism readily rationalizes the formation of the two major product ions observed for tripeptides. These reactions involve the cleavage of each peptide bond, yielding product ions that contain the intact side chains of two adjacent amino acids. Similar observations were made for the BrPBr + reagent ion. The results presented here suggest that ClPCl + (and BrPBr +) may provide useful sequence information for peptides.

Sialylation of the Sialic Acid Binding Lectin Sialoadhesin Regulates Its Ability to Mediate Cell Adhesion

AbstractThe macrophage-specific cell surface receptor sialoadhesin, which is a member of the newly recognized family of sialic acid binding lectins called siglecs, binds glycoprotein and glycolipid ligands containing a2-3–linked sialic acid on the surface of several leukocyte subsets. Recently, the sialic acid binding activity of the siglec CD22 has been demonstrated to be regulated by sialylation of the CD22 receptor molecule. In the present work, we show that desialylation of in vivo macrophage sialylconjugates enhances sialoadhesin-mediated lectin activity. Herein, we show that receptor sialylation of soluble sialoadhesin inhibits its binding to Jurkat cell ligands, and that charge-dependent repulsion alone cannot explain this inhibition. Furthermore, we show that the inhibitory effect of sialic acid is partially dependent on the presence of an intact exocyclic side chain. These results, in conjunction with previous findings, suggest that sialylation of siglecs by specific glycosyltransferases may be a common mechanism by which siglec-mediated adhesion is regulated.

Sialylation of the Sialic Acid Binding Lectin Sialoadhesin Regulates Its Ability to Mediate Cell Adhesion

The macrophage-specific cell surface receptor sialoadhesin, which is a member of the newly recognized family of sialic acid binding lectins called siglecs, binds glycoprotein and glycolipid ligands containing a2-3–linked sialic acid on the surface of several leukocyte subsets. Recently, the sialic acid binding activity of the siglec CD22 has been demonstrated to be regulated by sialylation of the CD22 receptor molecule. In the present work, we show that desialylation of in vivo macrophage sialylconjugates enhances sialoadhesin-mediated lectin activity. Herein, we show that receptor sialylation of soluble sialoadhesin inhibits its binding to Jurkat cell ligands, and that charge-dependent repulsion alone cannot explain this inhibition. Furthermore, we show that the inhibitory effect of sialic acid is partially dependent on the presence of an intact exocyclic side chain. These results, in conjunction with previous findings, suggest that sialylation of siglecs by specific glycosyltransferases may be a common mechanism by which siglec-mediated adhesion is regulated.

Calicheamin-mediated DNA damage in a reconstituted nucleosome is not affected by histone acetylation: the role of drug structure in the target recognition process.

We have examined the role of drug structure and histone acetylation in DNA damage produced by the enediyne antibiotic calicheamicin gammaII in nucleosomes reconstituted onto the 5S rRNA gene of Xenopus borealis. Consistent with previous observations, calicheamicin damage at the 3'-end of a purine tract (positions -13 and -14) was enhanced in the nucleosome compared to the naked DNA while damage at other sites was somewhat reduced in the nucleosome. However, damage produced by esperamicin C, an analog of calicheamicin missing the terminal sugar-aromatic ring in the side chain, showed no enhancement at positions -13 and -14, and its sequence selectivity in naked DNA was markedly different from that of calicheamicin. This highlights the importance of the intact tetrasaccharide side chain in the recognition of the structural deformation occurring at the 3'-ends of purine tracts. Both drugs produced identical cleavage patterns in normal and hyperacetylated nucleosomes. Given the sensitivity of calicheamicin to local DNA conformation, this observation is consistent with other studies that suggest that histone acetylation alone does not significantly affect the local conformation of core DNA in the nucleosome.

Identification of Metabolites from the Biological Transformation of the Nonionic Surfactant Residue Octylphenoxyacetic Acid and Its Brominated Analog

The aerobic biological transformation of octylphenoxyacetic acid (OP1EC) and its brominated analog (BrOP1EC) by groundwater enrichment cultures was studied, and persistent metabolites were identified by GC/MS. OP1EC is a representative of the class of alkylphenol ethoxycarboxylates (APEC), formed from alkylphenol polyethoxylate nonionic surfactants during sewage treatment. BrOP1EC is a byproduct formed during chlorine disinfection in the presence of bromide. The metabolite 2,4,4-trimethyl-2-pentanol was detected in stoichiometric quantities in OP1EC-metabolizing enrichment cultures, representing the intact alkyl side chain as a tertiary alcohol. BrOP1EC was transformed by the OP1EC-utilizing cultures only if OP1EC was simultaneously metabolized, suggesting a cometabolic mechanism of transformation. Brominated intermediates were also detected: brominated octylphenol and a compound tentatively identified as 2-aminomethoxy-3-bromo-5-(1,1,3,3-tetra- methylbutyl)phenol.

Glypican (heparan sulfate proteoglycan) is palmitoylated, deglycanated and reglycanated during recycling in skin fibroblasts.

Skin fibroblasts treated with brefeldin A produce a recycling variant of glypican (a glycosylphosphatidylinositolanchored heparan-sulfate proteoglycan) that is resistant to inositol-specific phospholipase C and incorporates sulfate and glucosamine into heparan sulfate chains (Fransson, L.-A. et al., Glycobiology, 5, 407-415, 1995). We have now investigated structural modifications of recycling glypican, such as fatty acylation from [3H]palmitate, and degradation and assembly of heparan sulfate side chains. Most of the 3H-radioactivity was recovered as lipid-like material after de-esterification. To distinguish between formation of heparan sulfate at vacant sites, elongation of existing chains or degradation followed by re-elongation of chain remnants, cells were pulse-labeled with [3H]glucosamine and then chase-labeled with [14C]glucosamine. Material isolated from the cells during the chase consisted of proteoglycan and mostly [3H]-labeled heparan-sulfate degradation products (molecular mass, 20-80 kDa) showing that the side chains were degraded during recycling. The degradation products were initially glucuronate-rich, but became more iduronate-rich with time. The glypican proteoglycan formed during the chase was degraded either with alkali to release intact side chains or with heparinase to generate distally located chain fragments that were separated from the core protein, containing the proximally located, covalently attached chain remnants. All of the [14C]-radioactivity incorporated during the pulse was found in peripheral chain fragments, and the chains formed were not significantly longer than the original ones. We therefore conclude that newly made heparan-sulfate chains were neither made on vacant sites, nor by extension of existing chains but rather by re-elongation of degraded chain remnants. The remodeled chains made during recycling appeared to be more extensively modified than the original ones.

Analysis of Protein-bound metabolites of Furazolidone and furaltadone in pig liver by high-performance liquid chromatography and liquid chromatography–mass spectrometry

Studies undertaken using radiolabelled furazolidone have demonstrated the covalent binding of residues of the drug to cellular protein in vivo. A portion of these bound residues and those formed by furaltadone, a related nitrofuran drug, possess intact side-chains, 3-amino-2-oxazolidinone (AOZ) and 5-morpholino-methyl-3-amino-2-oxazolidinone (AMOZ), respectively. These side-chains have molecular characteristics in common with the parent compounds and may be released from liver tissue under mild acidic conditions. Derivatization with 2-nitrobenzaldehyde (NBA) serves to isolate the released side-chains and the derivatives NPAOZ and NPAMOZ are chromophoric, thereby permitting UV detection. This paper reports the introduction of an extract clean-up step to the existing procedure which eliminates or decreases interference from NBA in the HPLC-UV determination of NPAOZ. The modified procedure was also applied to the determination of AMOZ. The development of an LC-MS method for the quantitative and confirmatory determination of AOZ and AMOZ extracted and derivatized according to the same procedure as that for HPLC-UV is described. The methods were validated for AOZ and AMOZ in fortified (intra- and inter-assay studies) and incurred (inter-assay studies) pig liver samples. The limit of determination for fortified control liver samples was 5 ng AOZ g-1 and 10 ng AMOZ g-1 by HPLC-UV and 10 ng AOZ or AMOZ g-1 by LC-MS. In addition, a study to determine the ratio of released AOZ to the total bound residues present in incurred liver samples from pigs treated with furazolidone is described.